From owner-7tms_r@net.bio.net Thu Jun 01 23:00:00 1995 Path: biosci!CBRC.MGH.HARVARD.EDU!Ethan_Lerner From: Ethan_Lerner@CBRC.MGH.HARVARD.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: receptor localization Date: 2 Jun 1995 03:08:43 -0700 Organization: CBRC, Massachusetts General Hospital Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <1995Jun02.060733.2884918053@cbrc.mgh.harvard.edu> NNTP-Posting-Host: net.bio.net Subject: Time: 4:57 AM OFFICE MEMO receptor localization Date: 6/2/95 Is anyone aware of literature describing immunohistochemical localization of 7tm receptors in either cells or tissue sections using the ligand as the receptor probe followed by detection with antibody/peroxidase conjugates? Has cross-linking using iodinated ligand, either cold or hot, and light been reported for 7tms? I am aware of epitope-tagged receptors, antibodies to receptors, radioligand binding etc. but not the approaches noted above. With immunohistochemistry, is the problem that the ligand appears to fall off or become internalized? Ethan Lerner ethan_lerner@cbrc.mgh.harvard.edu From owner-7tms_r@net.bio.net Thu Jun 01 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Better name for Inverse agonists? Date: 2 Jun 1995 13:12:10 GMT Organization: University of Michigan Lines: 14 Message-ID: <3qn2na$o42@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Windows; I; 16bit) I just was talking to a cardiology group about the role of constitutively activated 7tm receptors in human disease and brought up the concept of "inverse agonists" or drugs that produce the opposite conformational changes from agonists. I really dislike the term "inverse agonist". Does anybody else have a better one? I don't particularly like negative antagonists or super antagonists either but they at least give the impression that you are blocking responses. _________________________________________________________ Rick Neubig RNeubig@umich.edu University of Michigan Phone (313) 763-3650 http://www.umich.edu/~rneubig FAX (313) 763-4450 From owner-7tms_r@net.bio.net Fri Jun 02 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: yahud@aol.com (Yahud) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: fungal g-proteins Date: 4 Jun 1995 00:43:34 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 2 Sender: root@newsbf02.news.aol.com Message-ID: <3qrdlm$o9f@newsbf02.news.aol.com> References: <3q0fi7$rmn@service1.uky.edu> Reply-To: yahud@aol.com (Yahud) NNTP-Posting-Host: newsbf02.mail.aol.com I know that Prof. Jeremy Thorner at UC Berkeley was doing this kind of work some years back. From owner-7tms_r@net.bio.net Sun Jun 04 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsserver.jvnc.net!newsserver2.jvnc.net!netnews.upenn.edu!elmo.tju.edu!stubbs1.tjh.tju.edu!user From: yeagerm@jeflin.tju.edu (Mark Yeager) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Better name for Inverse agonists? Date: Mon, 05 Jun 1995 13:43:20 -0400 Organization: Thomas Jefferson University Lines: 33 Message-ID: References: <3qn2na$o42@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: stubbs1.tjh.tju.edu In article <3qn2na$o42@lastactionhero.rs.itd.umich.edu>, Rick Neubig wrote: > I just was talking to a cardiology group about the role of constitutively activated > 7tm receptors in human disease and brought up the concept of "inverse agonists" or > drugs that produce the opposite conformational changes from agonists. > > I really dislike the term "inverse agonist". Does anybody else have a better one? > I don't particularly like negative antagonists or super antagonists either but they > at least give the impression that you are blocking responses. > > _________________________________________________________ > Rick Neubig RNeubig@umich.edu How about: contragonists retroagonists (not bad) counteragonists alteragonists (kind of like that) annulagonists ? obstinagonists ?! (sorry) diametragonists (my favorite) What do I win if one gets picked? -- -.-- Mark Yeager -> yeagerm@jeflin.tju.edu Dept. Pathology and Cell Biology, Thomas Jefferson Univ., Phila., PA From owner-7tms_r@net.bio.net Sun Jun 04 23:00:00 1995 Path: biosci!PO.CWRU.EDU!pre From: pre@PO.CWRU.EDU (Paul.Ernsberger@slc5.INS.CWRU.Edu, "Ph.D.") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Better name for Inverse agonists? Date: 5 Jun 1995 17:12:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506060011.UAA00110@slc5.INS.CWRU.Edu> NNTP-Posting-Host: net.bio.net > I just was talking to a cardiology group about the role of constitutively activated > 7tm receptors in human disease and brought up the concept of "inverse agonists" or > drugs that produce the opposite conformational changes from agonists. > > I really dislike the term "inverse agonist". Does anybody else have a better one? > I don't particularly like negative antagonists or super antagonists either but they > at least give the impression that you are blocking responses. > A negative antagonist sounds like a double negative, as though it might actually be a regular agonist. I don't have the answer, but I do think that the concept of "negative efficacy" makes sense. We started teaching the medical students about inverse agonists this year and there was a lot of confusion. We had to hand out an extra syllabus page to straighten things out. So I agree with Rick that it's a real problem. From owner-7tms_r@net.bio.net Mon Jun 05 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!winx03!wrzx15!phak004 From: phak004@wrzx15.rz.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: help Date: 2 Jun 1995 14:22:40 GMT Organization: University of Wuerzburg, Germany Lines: 22 Distribution: world Message-ID: <3qn6rg$lrg@winx03.informatik.uni-wuerzburg.de> References: NNTP-Posting-Host: wrzx15.rz.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] C Wood (bsscw@bath.ac.uk) wrote: > By any chance does any one know the current location of Huber et al > (1988). Robert Huber is at the Max-Planck-Institut for Biochemistry Am Klopferspitz 18a D-82152 Martinsried Germany Rudolf Ladenstein has moved a few years ago to Sweden (?). I don't know his current address but I suppose Prof. Huber will be able to give it to you. --Cornelius. -- /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */ /* Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game you play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Mon Jun 05 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!msunews!netnews.upenn.edu!cronkite.ocis.temple.edu!VM.TEMPLE.EDU!SHICKLEY From: SHICKLEY@VM.TEMPLE.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Anti rho g protein antibody Date: Tue, 06 Jun 95 10:18:25 EDT Organization: Temple University Lines: 18 Message-ID: <173B590F4S86.SHICKLEY@VM.TEMPLE.EDU> References: <3qfgh1$3bq@dux.dundee.ac.uk> NNTP-Posting-Host: vm.temple.edu In article <3qfgh1$3bq@dux.dundee.ac.uk> a.mehta@dundee.ac.uk (Dr. Anil Mehta) writes: > >I need a clean antibody capable of detecting and immunoprecipitating this G protein in lung membranes. >Anyone got any ideas about a good reliable source? > >Anil Mehta, internet novice I know Dr. Gary Luthin at Hahnemann/MCP in Philadelphia has some anti-G antibodies. His email is: luthing@hal.hahnemann.edu Hope this helps. TIM SHICKLEY SHICKLEY@VM.TEMPLE.EDU TSHICKLEY@HAL.DENTAL.TEMPLE.EDU -----------------------STANDARD DISCLAIMERS APPLY----------------- From owner-7tms_r@net.bio.net Mon Jun 05 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!cisun2000.unil.ch!ulys.unil.ch!msautel From: msautel@ulys.unil.ch Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: receptor localization Date: 6 Jun 95 14:02:29 CET Organization: University of Lausanne - CH (Switzerland) Lines: 14 Distribution: world Message-ID: <1995Jun6.140229@ulys.unil.ch> References: <1995Jun02.060733.2884918053@cbrc.mgh.harvard.edu> NNTP-Posting-Host: ul9000.unil.ch In article <1995Jun02.060733.2884918053@cbrc.mgh.harvard.edu>, Ethan_Lerner@CBRC.MGH.HARVARD.EDU writes: > Subject: Time: 4:57 AM > OFFICE MEMO receptor localization Date: 6/2/95 > Is anyone aware of literature describing immunohistochemical localization > of 7tm receptors in either cells or tissue sections We have inserted a Flag epitope in Cterm of our receptor and we can then detect the presence of the receptor using an antibody coupled to peroxidase or FITC. (Walker et al 1994, JBC 269, 2863-2869) I hope this can help you Bye Martine Sautel From owner-7tms_r@net.bio.net Mon Jun 05 23:00:00 1995 Path: biosci!VIR.COM!m_dennis From: m_dennis@VIR.COM (Michael Dennis) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Better name for Inverse agonists? Date: 6 Jun 1995 17:10:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 37 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net In article <3qn2na$o42@lastactionhero.rs.itd.umich.edu> Rick Neubig writes: >To: 7tms_r@net.bio.net >From: Rick Neubig >Subject: Better name for Inverse agonists? >Date: 2 Jun 1995 13:12:10 GMT >I just was talking to a cardiology group about the role of constitutively >activated >7tm receptors in human disease and brought up the concept of "inverse agonists" >or >drugs that produce the opposite conformational changes from agonists. > >I really dislike the term "inverse agonist". Does anybody else have a better >one? >I don't particularly like negative antagonists or super antagonists either but >they >at least give the impression that you are blocking responses. > >_________________________________________________________ > I would propose that the best name for antagonists which inhibit spontaneous or basal receptor activity is, unimaginatively, antagonists. The growing body of data indicates that the great majority of receptor antagonists do possess negative (or inverse) efficacy; indeed, this would be expected based on the two-state (R-R*) equilibrium model. Thus, agonists would increase [R*] and antagonists would decrease [R*]. (I am rather uncomfortable with the idea that ligands "produce" conformational changes, however). Under this scheme, a small minority of ligands would exhibit the "classical" antagonist profile: i.e. they block responses without having any effect on their own. How would we name these "neutral ligands"? Neutragonists?? This is probably also a misnomer, since the effect of a drug on receptor activity (increase, decrease or no effect) will probably depend on the "state" of the receptor (cell type, recent history) at the time of study. Michael Dennis m_dennis@vir.com From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!novo.dk!byw From: byw@novo.dk (Robert Bywater) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 7 Jun 1995 03:16:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506071014.AA02892@ligand.novo.dk> NNTP-Posting-Host: net.bio.net Dan Donnelly has written to inform me that : >I think you'e got R and R* mixed up in your message on the net. >I think the active form is R* (stablized by agonist + G protein) >while the R form predominates in the absence of ligand or in the >presence of antagonists/inverse agonists. - He is of course quite right. I am glad there is at least someone who reads what gets disseminated on the read, but doesn't accept it without question. ****************************** * * * Robert Bywater * * * * Biostructure Department * * NOVO NORDISK A/S * * * * DK-2880 BAGSVAERD Denmark * * * * email byw@novo.dk * * fax :: +45 4442 1400 * ****************************** From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!novo.dk!byw From: byw@novo.dk (Robert Bywater) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 7 Jun 1995 06:05:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506071303.AA03196@ligand.novo.dk> NNTP-Posting-Host: net.bio.net Dan Donnelly has written to inform me that : >I think you'e got R and R* mixed up in your message on the net. >I think the active form is R* (stablized by agonist + G protein) >while the R form predominates in the absence of ligand or in the >presence of antagonists/inverse agonists. - He is of course quite right. I am glad there is at least someone who reads what gets disseminated on the net, but doesn't accept it without question. ****************************** * * * Robert Bywater * * * * Biostructure Department * * NOVO NORDISK A/S * * * * DK-2880 BAGSVAERD Denmark * * * * email byw@novo.dk * * fax :: +45 4442 1400 * ****************************** From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!novo.dk!byw From: byw@novo.dk (Robert Bywater) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 7 Jun 1995 00:00:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 51 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506070658.AA02313@ligand.novo.dk> NNTP-Posting-Host: net.bio.net Michael Dennis writes : >............................ Thus, agonists would increase [R*] and >antagonists would decrease [R*]. (I am rather uncomfortable with the idea that >ligands "produce" conformational changes, however). Of course, these changes are not 'produced' by the ligands. It simply means that : deltaG-0 R.Ag ^ | | deltaG-0 | v deltaG-2 R* + Ant <========> R*.Ant In the absence of any ligand, R* would predominate, in the presence of Ag, R would become complexed to Ag and the receptor population would be successively depleted of free R, and more R* would convert to the R form. In the presence of Ant, the reverse would occur. ****************************** * * * Robert Bywater * * * * Biostructure Department * * NOVO NORDISK A/S * * * * DK-2880 BAGSVAERD Denmark * * * * email byw@novo.dk * * fax :: +45 4442 1400 * ****************************** From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!VAX100.FUKUI-MED.AC.JP!ksakai From: ksakai@VAX100.FUKUI-MED.AC.JP Newsgroups: bionet.molbio.proteins.7tms_r Subject: test Date: 7 Jun 1995 00:55:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506070752.AA02287@fms101.fukui-med.ac.jp> NNTP-Posting-Host: net.bio.net help From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!agate!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Better name for Inverse agonists? Date: 7 Jun 1995 13:53:43 GMT Organization: University of Michigan Lines: 59 Message-ID: <3r4b17$gjq@lastactionhero.rs.itd.umich.edu> References: NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Windows; I; 16bit) To: m_dennis@VIR.COM m_dennis@VIR.COM (Michael Dennis) wrote: >In article <3qn2na$o42@lastactionhero.rs.itd.umich.edu> Rick Neubig writes: (stuff deleted) >>I really dislike the term "inverse agonist". Does anybody else have a better >>one? >I would propose that the best name for antagonists which inhibit spontaneous >or basal receptor activity is, unimaginatively, antagonists. The growing body >of data indicates that the great majority of receptor antagonists do possess >negative (or inverse) efficacy; indeed, this would be expected based on the >two-state (R-R*) equilibrium model. Thus, agonists would increase [R*] and >antagonists would decrease [R*]. (I am rather uncomfortable with the idea that >ligands "produce" conformational changes, however). > >Under this scheme, a small minority of ligands would exhibit the "classical" >antagonist profile: i.e. they block responses without having any effect on >their own. How would we name these "neutral ligands"? Neutragonists?? This is >probably also a misnomer, since the effect of a drug on receptor activity >(increase, decrease or no effect) will probably depend on the "state" of the >receptor (cell type, recent history) at the time of study. > >Michael Dennis >m_dennis@vir.com Thanks to all who sent private responses to me to follow up on this question. I'll summarize them when I get a minute or two. It shows how many "closet" readers we have who read but don't post. Some time people should post a "Hi" just so that we know who's listening out there! Michael, You are right that most antagonists probably do have negative efficacy and that we didn't recognize it until we could greatly overexpress receptors and raise basal activity. I think the problem with just plain "antagonists" is that the term is already taken for ligands with a totally neutral effect (your so-called neutragonists). Since generations of pharmacologists have learned that antagonists don't do anything some kind of new term seems warranted to express that. Also, the drug companies will need a new marketing gimmick ;) (it actually may be an important distinction as we learn more about receptors in pathophysiology). Also, when we get to in vivo experiments it will be harder to know if that "basal activity" is just the receptor or if it is due to those low circulating agonist concentrations. How about "active antagonists"? Keep those cards and letters coming! Rick _________________________________________________________ Rick Neubig RNeubig@umich.edu University of Michigan Phone (313) 763-3650 http://www.umich.edu/~rneubig FAX (313) 763-4450 From owner-7tms_r@net.bio.net Tue Jun 06 23:00:00 1995 Path: biosci!RECEPTOR.MGH.HARVARD.EDU!lfk From: lfk@RECEPTOR.MGH.HARVARD.EDU (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: R/R*, Agonist/Antagonist, dG (i.e. receptor thermodynamics) Date: 7 Jun 1995 08:08:41 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 68 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506071500.AA05206@receptor> References: <9506070658.AA02313@ligand.novo.dk> NNTP-Posting-Host: net.bio.net Bob Bywater writes: >Michael Dennis writes : >>Thus, agonists would increase [R*] and antagonists would decrease >>[R*]. (I am rather uncomfortable with the idea that ligands >>"produce" conformational changes, however). >Of course, these changes are not 'produced' by the ligands. >It simply means that : > deltaG-0 deltaG-1 < 0 > deltaG-2 < 0 > (Note R and R* are transposed) >where the deltaG's are defined by : > > deltaG-1 > R + Ag <========> R.Ag > > ^ > | > | deltaG-0 > | > v > deltaG-2 > R* + Ant <========> R*.Ant > This is a nice scheme, but is really very far from reality. How many states are there between R and R* and from R* back to R. Do these multiple states have differential responses, affinities, agonist/antagonist properties? Do these states indicate the multiple conformations of the complex ( ligand * receptor * G-protein). Do pre-coupled receptors have differential responses to "inverse agonists" than receptors which are not pre-coupled? This is not to point out flaws in the model, just to say that many drugs have partial properties. Are these partials examples of drugs which lock the receptor in a transition state? Rhodopsin is a good example of these states. THere are 6 or more spectroscopic states prior to Meta-I. So the pathway R --> Meta-I --> Meta-II --> Opsin But the meta-I and Meta-II states are in an equilibrium which is affected by several factors (pH, NaCl, etc). A recent JBC paper by Barry Knox and colleagues, show that the apo-protein opsin is also able to activate transducin. What does this imply about the these complex species that we generically refer to as R and R*. Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-355-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!serra.unipi.it!usenet From: buccione@cmns.mnegri.it (Roberto Buccione) Newsgroups: bionet.molbio.proteins.7tms_r Subject: "Orphan receptors" Date: 8 Jun 1995 09:12:33 GMT Organization: Consorzio Mario Negri Sud Lines: 19 Sender: -Not-Authenticated-[2686] Message-ID: <3r6eu1$stu@serra.unipi.it> NNTP-Posting-Host: neurobio03.cmns.mnegri.it X-Posted-From: InterNews 1.0.7@neurobio03.cmns.mnegri.it Xdisclaimer: No attempt was made to authenticate the sender's name. Hello to all! We study the regulation of intracellular membrane traffic and constitutive exocytosis. One of our main interests is the emerging concept of interorganelle signalling via "canonical" (or less canonical) transduction/signalling pathways. For instance via 7TM receptors. We were wondering whether some of you out there might be able to help us find a comprehensive list (if there exists one) of DNA and/or protein sequences of the so-called "orphan receptors", or, in alternative, a way to track them down on DNA/protein sequence data banks. We would also very much appreciate it if anyone is willing to share unpublished sequence data on such receptors. Please feel free to contact me directly at my E-mail address. Thanks for any help you might be able to give us. Roberto Buccione Laboratorio Di Neurobiologia Molecolare Consorzio Mario Negri Sud S. Maria Imbaro (Ch) From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!FISHMAILSERVER.NEURO.MSSM.EDU!stuart_sealfon From: stuart_sealfon@FISHMAILSERVER.NEURO.MSSM.EDU ("Stuart Sealfon") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: receptor localization Date: 8 Jun 1995 06:10:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 85 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Reply to: RE>>receptor localization Ethan_Lerner@CBRC.MGH.HARVARD.EDU wrote: > Is anyone aware of literature describing immunohistochemical localization > of 7tm receptors in either cells or tissue sections using the ligand as the > receptor probe followed by detection with antibody/peroxidase conjugates? Cornelius Krasel wrote >You think of proteinaceous ligands, right? I would assume that small >molecules (such as epinephrine) just get lost during the fixation >process. I know of one example of using an antibody to a small ligand. Martinelli, G.P., G.R. Holstein, P. Pasik, and B. Cohen, Monoclonal antibodies for ultrastructural visualization of L-baclofen-sensitive GABAB receptor sites. Neuroscience, 1992. 46: p. 23-33.Monoclonal antibodies were raised against the L-enantiomer of baclofen conjugated by glutaraldehyde to keyhole limpet hemocyanin. Hybridoma clones were selected for their stability and their production of high titers of antibodies directed against the p-chlorophenyl moiety of the L-baclofen molecule. The chosen antibody showed no cross-reactivity with conjugates of GABA and other neurotransmitters to human or bovine serum albumin. Specificity was further confirmed by the ability of L-baclofen-HCl to inhibit the binding of the antibody to L-baclofen-bovine serum albumin conjugate. Immunocytochemical studies were conducted on brain tissue from rats and monkeys injected with baclofen to localize baclofen-sensitive GABAB receptor sites. In these animals, the molecular layer of cerebellar cortex was clearly immunostained and the granular layer showed only some pale immunoreactivity. Ultrastructural observations were conducted in cerebellar cortex, as well as in the substantia nigra and the vestibular nuclei. Discrete labeling of neuronal profiles was observed in these structures, and both immunoperoxidase and colloidal gold methods were employed successfully. Material from saline-injected control animals showed no immunoreactivity at both light and electron microscopic levels. We conclude that the anti-L-baclofen antibody preferentially recognizes the p-chlorophenyl moiety of the baclofen molecule. Antibodies of such specificity are useful tools for the ultrastructural localization of baclofen-sensitive GABAB receptor sites. In general, antibodies directed against accessible moieties of specific neuroactive substances may serve as valuable markers for their sites of action. --Stuart Sealfon -------------------------------------- Date: 6/8/95 6:19 AM To: Stuart Sealfon From: Cornelius Krasel Ethan_Lerner@CBRC.MGH.HARVARD.EDU wrote: > Is anyone aware of literature describing immunohistochemical localization > of 7tm receptors in either cells or tissue sections using the ligand as the > receptor probe followed by detection with antibody/peroxidase conjugates? You think of proteinaceous ligands, right? I would assume that small molecules (such as epinephrine) just get lost during the fixation process. AFAIK it is not known what happens to the receptor ligand after the receptor has been internalized. I would be interested in papers on this issue. --Cornelius. -- /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */ /* Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game you play with God to find out what His rules are." */ ------------------ RFC822 Header Follows ------------------ Received: by fishmailserver.neuro.mssm.edu with SMTP;8 Jun 1995 06:16:49 -0500 Received: (from daemon@localhost) by net.bio.net (8.6.11/8.6.6) id AAA03132 for 7tms_r-list; Thu, 8 Jun 1995 00:26:47 -0700 Received: (from news@localhost) by net.bio.net (8.6.11/8.6.6) id AAA03127 for 7tms_r-arpanet; Thu, 8 Jun 1995 00:26:45 -0700 To: 7tms_r@net.bio.net From: phak004@wrzx15.rz.uni-wuerzburg.de (Cornelius Krasel) Subject: Re: receptor localization Date: 7 Jun 1995 20:37:34 GMT Message-ID: <3r52me$lh2@winx03.informatik.uni-wuerzburg.de> NNTP-Posting-Host: wrzx15.rz.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!psgrain!nntp.teleport.com!usenet From: Kim Neve Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: receptor localization Date: 8 Jun 1995 18:17:59 GMT Organization: Oregon Health Sciences University Lines: 11 Message-ID: <3r7esn$fn0@maureen.teleport.com> References: <3r7eo0$fa6@maureen.teleport.com> NNTP-Posting-Host: ip-pdx1-34.teleport.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) >receptor is being degraded, at some point the ligand becomes "unburied" >and would be accessible to the antagonist. ^^^^^^^^^^ I mean antibody, of course. ^^^^^^^^ Kim Neve From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!ucsvc.ucs.unimelb.edu.au!U5636655 From: U5636655@ucsvc.ucs.unimelb.edu.au Newsgroups: bionet.molbio.proteins.7tms_r Subject: "inverse agonists" Date: 8 Jun 1995 01:35:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 85 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HRH08FR30I8WX8QK@ucsvc.ucs.unimelb.edu.au> NNTP-Posting-Host: net.bio.net In response to Robert Bywater: What you describe is the process of conformational selection with agonists stabilising the R* state and antagonists stabilising the R state with consequent shifts in available free R* or R based on mass action. In this schema there is no conformational induction. That, however, does not preclude the potential for "conformational induction" where the agonist would bind to R and that binding to R would then allow/promote the formation of R*, presumably through reduction of an energy barrier. Although there is now a lot of evidence to support the occurence of conformational selection, I don't think that we can totally discard the possibility of conformational induction (no matter how much easier this may make life). In regard to Rick Neubigs' question of what do we call "inverse agonists": I agree that the term antagonist is inappropriate. The term antagonist is used to refer to something which acts in opposition to another compound, thus to prevent this action. In the case of competitive antagonists this is through prevention of the binding of the agonist. But the term is also used in other contexts eg. physiological antagonist (here again to describe the opposition of another action). My other reason is that when we describe the action of a compound to antagonise the effect of another compound, we are not describing the same action when refering to the "inverse agonist" property of the compound. The former (in a simplistic two-state conformational selection model) is dependent upon the absolute affinity for the inactive state of the receptor, while the later is more dependent upon the ratio of affinities for the inactive and active states of the receptor and the pre-existing equilibrium of the system (in terms of R and R*). This later interaction is essentially the same that we are describing when dealing with other agonists which favour R*. The relative efficacies will depend upon the absolute affinity for either R or R*. But whether they act as agonists, partial agonists, "inverse agonists" or partial "inverse agonists" is dependent upon the relative affinities for the different receptor states and the pre-existing equilibrium of the receptor system under study. I think that the important thing to consider is that we are not defining compounds but actions. As far as I'm concerned - when describing the "inverse agonist" action of a compound the term agonist is appropriate. The problem is in inappropriate use of the term antagonist to define a compound rather than the action of a compound and the realisation that the action of a compound is systems dependent. We do not appear to have a conceptual problem in viewing compounds that close down ion channels or through receptor modulation of Gi inhibit cAMP production, as agonists - I don't see that it is really such a big step to view a compound that directly switches off coupling to G-protein as an agonist in a system where the constitutively active state predominates. It is a direct action of the compound on the system independent of the presence of another ligand. In a different vein I would like to talk about receptor-G protein coupling, in particular pre-coupling. There is now considerable evidence to support the existence of pre-coupling of receptor to G-protein in the absence of external ligand. In most models (eg. extended terary complex model), this would imply that the system studied should exhibit constitutive activity as most models bear the underlying assumption that coupling of G-protein to the receptor is dependent upon the receptor being in the activated state ( R*). However, to my knowledge, the evidence supporting constitutive activity where receptors are pre-coupled is less prominent. While it may be the case that "we haven't seen it because we haven't looked", my suspicion is that many of the systems under study are not constitutively active. Thus the pre-coupled receptor must therefore be in the inactive state. Indeed, there is some experimental evidence to support this where co-incubation of antagonists does not alter the equilibrium between coupled and uncoupled receptors despite antagonism of ligand-induced signalling. What is the prevailing view of people working more closely in this area ? In relation to this I would draw attention to the cubic ternary complex model of Terry Kenakin and colleagues. This model is a thermodynamic extension (completion) of the extended ternary complex model and allows for both interaction of R with G as well as for the concept of conformational induction to be included. Patrick M. Sexton Neurobiology Unit, St. Vincent's Institute of Medical Research, From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!psgrain!nntp.teleport.com!usenet From: Kim Neve Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: receptor localization Date: 8 Jun 1995 18:15:28 GMT Organization: Oregon Health Sciences University Lines: 29 Message-ID: <3r7eo0$fa6@maureen.teleport.com> References: NNTP-Posting-Host: ip-pdx1-34.teleport.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) stuart_sealfon@FISHMAILSERVER.NEURO.MSSM.EDU ("Stuart Sealfon") wrote: > Reply to: RE>>receptor localization > >Ethan_Lerner@CBRC.MGH.HARVARD.EDU wrote: >> Is anyone aware of literature describing immunohistochemical localization >> of 7tm receptors in either cells or tissue sections using the ligand as the >> receptor probe followed by detection with antibody/peroxidase conjugates? > > >I know of one example of using an antibody to a small ligand. > >Martinelli, G.P., G.R. Holstein, P. Pasik, and B. Cohen, Monoclonal >antibodies for ultrastructural visualization of L-baclofen-sensitive GABAB >receptor sites. Neuroscience, 1992. 46: p. 23-33. A second example is a paper by Norman and Nathanson, J. Neurochem. 49 (1987) 939-943. They used the muscarinic alkylating antagonist propylbenzilycholine mustard to affinity label the receptors, then immunoprecipitated a proteolytic fragment of the receptor using an anti-PrBCM antibody. However, the antibody couldn't recognize the ligand when bound to the native, intact receptor, presumably because the antigenic determinants are buried within the receptor. They did not look at receptor localization, although you might imagine that when the receptor is being degraded, at some point the ligand becomes "unburied" and would be accessible to the antagonist. Kim Neve, Oregon Health Sciences University From owner-7tms_r@net.bio.net Wed Jun 07 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!winx03!wrzx15!phak004 From: phak004@wrzx15.rz.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: receptor localization Date: 7 Jun 1995 20:37:34 GMT Organization: University of Wuerzburg, Germany Lines: 19 Distribution: world Message-ID: <3r52me$lh2@winx03.informatik.uni-wuerzburg.de> References: <1995Jun02.060733.2884918053@cbrc.mgh.harvard.edu> NNTP-Posting-Host: wrzx15.rz.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Ethan_Lerner@CBRC.MGH.HARVARD.EDU wrote: > Is anyone aware of literature describing immunohistochemical localization > of 7tm receptors in either cells or tissue sections using the ligand as the > receptor probe followed by detection with antibody/peroxidase conjugates? You think of proteinaceous ligands, right? I would assume that small molecules (such as epinephrine) just get lost during the fixation process. AFAIK it is not known what happens to the receptor ligand after the receptor has been internalized. I would be interested in papers on this issue. --Cornelius. -- /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */ /* Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game you play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Thu Jun 08 23:00:00 1995 Path: biosci!hubrecht.nl!markv From: markv@hubrecht.nl Newsgroups: bionet.molbio.proteins.7tms_r Subject: conference Date: 9 Jun 1995 00:59:54 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506090759.AAA03800@net.bio.net> NNTP-Posting-Host: net.bio.net Can somebody tell me when and where on Earth a conference/symposium/workshopconcerning 'G-proteins & second messengers' takes place ?! A fax or emailnr for information would be nice too. Thanks Mark Verheijen Hubrecht Laboratory Utrecht, the Netherlands MarkV@hubrecht.nl From owner-7tms_r@net.bio.net Thu Jun 08 23:00:00 1995 Path: biosci!mcmail.vanderbilt.edu!lee.limbird From: lee.limbird@mcmail.vanderbilt.edu Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: conference Date: 9 Jun 1995 03:10:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <9505098026.AA802699305@in2.mcmail.vanderbilt.edu> NNTP-Posting-Host: net.bio.net IT WILL BEGIN OCTOBER 28,1995 IN NASHVILLE,TN, USA ON THE CAMPUS OF VANDERBILT UNIVERSITY, PROBABLY SELECTED AS A LOCATION AS IT IS THE INSTITUTION WHERE EARL SUTHERLAND COMPLETED HIS SCIENTIFIC CAREER. AS I AM AT VANDERBILT, I HAVE MADE A COPY OF YOUR EMAIL ADDRESS, AND HAVE ASKED JACKIE CORBIN AND SHARRON FRANCIS, ORGANIZERS, TO SEND A PACKET OF MATERIALS. ABSTRACTS WERE DUE 5/26/95, BUT REGISTRATION CAN STILL OCCUR. YOU ALSO WILL RECEIVE A FAX WITH SOME INFO JUNE 9 Can somebody tell me when and where on Earth a conference/symposium/workshopconcerning 'G-proteins & second messengers' takes place ?! A fax or emailnr for information would be nice too. Thanks Mark Verheijen Hubrecht Laboratory Utrecht, the Netherlands MarkV@hubrecht.nl From owner-7tms_r@net.bio.net Mon Jun 12 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!pipex!sunic!sunic.sunet.se!trane.uninett.no!daresbury!not-for-mail From: RMCMAH93@IRLEARN.UCD.IE Newsgroups: bionet.molbio.proteins.7tms_r Subject: consensus phosphorylation sites? Date: 13 Jun 1995 12:39:01 +0100 Lines: 5 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3rjtcl$t0p@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.AC.UK I was wondering if anyone knows of a site on the WWW which can determine potential phosphorylation sites in the protein sequence of a 7TM receptor. Thanks, Ruth. From owner-7tms_r@net.bio.net Mon Jun 12 23:00:00 1995 Newsgroups: bionet.molbio.proteins.7tms_r Path: biosci!daresbury!hgmp.mrc.ac.uk!ebi.ac.uk!EMBL-EBI.ac.uk!apweiler From: apweiler@EMBL-EBI.ac.uk (Rolf Apweiler) Subject: Re: consensus phosphorylation sites? Sender: news@ebi.ac.uk (Mr news) Message-ID: Date: Tue, 13 Jun 1995 13:18:28 GMT Distribution: bionet Lines: 25 Reply-To: apweiler@EMBL-EBI.ac.uk (Rolf Apweiler) References: <3rjtcl$t0p@mserv1.dl.ac.uk> Organization: European Bioinformatics Institute (EMBL) - UK X-Newsreader: mxrn 6.18-32 In article <3rjtcl$t0p@mserv1.dl.ac.uk>, RMCMAH93@IRLEARN.UCD.IE writes: > >I was wondering if anyone knows of a site on the WWW which can determine >potential phosphorylation sites in the protein sequence of a 7TM >receptor. >Thanks, Ruth. > Try URL: http://www.ebi.ac.uk/searches/prosite.html Cheers Rolf ============================================================ Rolf Apweiler Tel:+44-(0)1223 494435 EMBL Outstation Fax:+44-(0)1223 494468 The European Bioinformatics Institute Hinxton Hall Hinxton Cambridge CB10 1RQ UK Email:apweiler@ebi.ac.uk ============================================================ From owner-7tms_r@net.bio.net Mon Jun 12 23:00:00 1995 Newsgroups: bionet.molbio.proteins.7tms_r Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.starnet.net!wupost!news.utdallas.edu!corpgate!bcarh189.bnr.ca!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!bi575 From: bi575@FreeNet.Carleton.CA (Francesco Lipari) Subject: urea PAGE Message-ID: Sender: bi575@freenet.carleton.ca (Francesco Lipari) Organization: The National Capital FreeNet, Ottawa, Ontario, Canada Date: Tue, 13 Jun 1995 19:44:09 GMT Lines: 5 iHi, I am looking for a method to run acid urea polyacrylamide gels. Thanks Frank e-mail: lipari@medcor.mcgill.ca From owner-7tms_r@net.bio.net Wed Jun 14 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!pipex!oleane!jussieu.fr!univ-lyon1.fr!serra.unipi.it!usenet From: buccione@cmns.mnegri.it (Roberto Buccione) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Looking for Orphan Receptors!!!! Date: 15 Jun 1995 15:27:30 GMT Organization: Consorzio Mario Negri Sud Lines: 11 Sender: -Not-Authenticated-[2686] Message-ID: <3rpjh2$kte@serra.unipi.it> NNTP-Posting-Host: neurobio03.cmns.mnegri.it X-Posted-From: InterNews 1.0.7@neurobio03.cmns.mnegri.it Xdisclaimer: No attempt was made to authenticate the sender's name. We are strongly interested in examining any unpublished AA sequence of 7TMD receptors to look for particular consensus sequences. If there are persons willing to share their unpublished data with us, we will be more than happy to give more details on our specific aims and eventually collaborate. You can also reply directly to my E-mail address. Roberto Buccione Molecular Neurobiology Laboratory Consorzio Mario Negri Sud S. Maria Imbaro (Ch) From owner-7tms_r@net.bio.net Wed Jun 14 23:00:00 1995 Path: biosci!BIOMED.MED.YALE.EDU!KARNE From: KARNE@BIOMED.MED.YALE.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 15 Jun 1995 05:59:25 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HRQ9NAZEW2000HIW@BIOMED.MED.YALE.EDU> NNTP-Posting-Host: net.bio.net unsubscribe From owner-7tms_r@net.bio.net Wed Jun 14 23:00:00 1995 Path: biosci!rutgers!uwm.edu!news.moneng.mei.com!howland.reston.ans.net!EU.net!Belgium.EU.net!chaos.kulnet.kuleuven.ac.be!news.belnet.be!news.vub.ac.be!ucmb.ulb.ac.be!dimitri From: dimitri@vandijck.ulb.ac.be (Dimitri Gilis) Newsgroups: bionet.molbio.proteins.7tms_r Subject: protein G mutants Date: 15 Jun 1995 07:56:04 GMT Organization: UCMB @ Brussels Free University, Belgium Lines: 17 Sender: dimitri@vandijck (Dimitri Gilis) Distribution: world Message-ID: <3rop2k$ebf@rc1.vub.ac.be> NNTP-Posting-Host: ucmbsgi9.ulb.ac.be. Hello, I am looking for (DD)G values for mutations in protein G B1 domain (pdb code 2GB1 or 1PGB). Some references are welcome. Thanks in advance. -- ================================================================= Dimitri Gilis Unite de Conformation des Macromolecules Biologiques ULB (Belgium) tel: +32 2 648 52 00 fax: +32 2 648 89 54 e-mail : dimitri@ucmb.ulb.ac.be ================================================================= From owner-7tms_r@net.bio.net Wed Jun 14 23:00:00 1995 Path: biosci!WELCHLINK.WELCH.JHU.EDU!sblack From: sblack@WELCHLINK.WELCH.JHU.EDU (Seth Blackshaw) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Looking for Orphan Receptors!!!! Date: 15 Jun 1995 13:06:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <3rpjh2$kte@serra.unipi.it> NNTP-Posting-Host: net.bio.net I have cloned many different 7TMD receptors from catfish via degenerate PCR in the course of trying to find taste receptors. I have complete sequence of at least 7 different PCR products, and partial sequence of nearly 30. I would be delighted to share data with you. Please write back if you are interested. On 15 Jun 1995, Roberto Buccione wrote: > We are strongly interested in examining any unpublished AA sequence of > 7TMD receptors to look for particular consensus sequences. If there are > persons willing to share their unpublished data with us, we will be > more than happy to give more details on our specific aims and > eventually collaborate. You can also reply directly to my E-mail > address. > > Roberto Buccione > Molecular Neurobiology Laboratory > Consorzio Mario Negri Sud > S. Maria Imbaro (Ch) > > From owner-7tms_r@net.bio.net Fri Jun 16 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!europa.chnt.gtegsc.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Gq coupled receptor affinity changes Date: 17 Jun 1995 10:35:15 GMT Organization: University of Michigan Lines: 16 Message-ID: <3rub53$atb@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Windows; I; 16bit) I just started thinking again about the differences between Gi and Gs coupled receptors and Gq coupled receptors vis a viz high affinity agonist binding. Why is it that Gi and Gs coupled receptors show large agonist affinity changes on binding to G proteins while Gq coupled receptors generally have such punky "GTP-shifts"? If the energy used to drive the conformational change involved in G activation is related to affinity differences why aren't there larger differences in Kd for Gq coupled receptors with and without GTP? They clearly can activate the G protein just fine. _________________________________________________________ Rick Neubig RNeubig@umich.edu University of Michigan Phone (313) 763-3650 http://www.umich.edu/~rneubig FAX (313) 763-4450 From owner-7tms_r@net.bio.net Fri Jun 16 23:00:00 1995 Path: biosci!MIZAR.USC.EDU!cminkin From: cminkin@MIZAR.USC.EDU (Cedric Minkin) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 17 Jun 1995 11:21:16 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506171821.LAA27546@mizar.usc.edu> NNTP-Posting-Host: net.bio.net unsubscribe From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsserver.jvnc.net!netnews.sb.com!news From: Steven_Mcclue-1%notes@sb.com (Steve Mcclue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Gq coupled receptor affinity changes Date: 19 Jun 1995 08:06:58 GMT Organization: Smithkline Beecham Pharmaceuticals Lines: 16 Message-ID: <3s3b72$t8@phunn1.sb.com> References: <3rub53$atb@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: had542.uk.sb.com Mime-Version: 1.0 X-Newsreader: WinVN 0.99.2 In article <3rub53$atb@lastactionhero.rs.itd.umich.edu>, RNeubig@umich.edu says... > >I just started thinking again about the differences between Gi and Gs >coupled receptors and Gq coupled receptors vis a viz high affinity >agonist binding. Why is it that Gi and Gs coupled receptors show large >agonist affinity changes on binding to G proteins while Gq coupled >receptors generally have such punky "GTP-shifts"? This is a very good question, and I've never really come across a convincing explanation. Is it a coincidence that Gi and Gs have relatively high rates of GTPase activity, while Gq requires a GAP (ie PLC) to kick ass? I think we should be told! Steve From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!cs.utexas.edu!howland.reston.ans.net!europa.chnt.gtegsc.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Gq coupled receptor affinity changes Date: 19 Jun 1995 09:23:47 GMT Organization: University of Michigan Lines: 28 Message-ID: <3s3fn3$80m@lastactionhero.rs.itd.umich.edu> References: <3rub53$atb@lastactionhero.rs.itd.umich.edu> <3s3b72$t8@phunn1.sb.com> NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Windows; I; 16bit) Steven_Mcclue-1%notes@sb.com (Steve Mcclue) wrote: >In article <3rub53$atb@lastactionhero.rs.itd.umich.edu>, RNeubig@umich.edu says... > >> Why is it that Gi and Gs coupled receptors show large >>agonist affinity changes on binding to G proteins while Gq coupled >>receptors generally have such punky "GTP-shifts"? > >This is a very good question, and I've never really come across a convincing explanation. Is it >a coincidence that Gi and Gs have relatively high rates of GTPase activity, while Gq requires a >GAP (ie PLC) to kick ass? I think we should be told! > I'd wonder if it was due to slow GDP dissociation from Gq. Maybe if GDP can't get off you can never fully populate the R* state. I don't quite know how to tie in a slow GTPase rate with a requirement for GAP activity since there should be GAP in the membrane preps where binding is being done. These are just off-the-cuff thoughts. Anybody else have a clearer picture of this? Rick _________________________________________________________ Rick Neubig RNeubig@umich.edu University of Michigan Phone (313) 763-3650 http://www.umich.edu/~rneubig FAX (313) 763-4450 From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!elroy.jpl.nasa.gov!swrinde!cs.utexas.edu!news.sprintlink.net!europa.chnt.gtegsc.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Gq coupled receptor affinity changes - Ernsberger/Donnally Date: 19 Jun 1995 09:29:18 GMT Organization: University of Michigan Lines: 49 Message-ID: <3s3g1e$80m@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: warbler.med.umich.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1b3 (Windows; I; 16bit) Two personal replies from people who disagreed but were too polite to post to the newsgroup ;) From: Date: 19 Jun 1995 02:08:18 GMT Subject: Re: Gq coupled receptor affinity changes > I just started thinking again about the differences between Gi and Gs > coupled receptors and Gq coupled receptors vis a viz high affinity > agonist binding. Why is it that Gi and Gs coupled receptors show large > agonist affinity changes on binding to G proteins while Gq coupled > receptors generally have such punky "GTP-shifts"? Rick, I'm not so certain that's true. Consider the petite shifts seen for alpha2-adrenergics in our labs (Gi-coupled) compared to the drastic shifts seen for NK-1 receptors (Gq-coupled). It does seem to be true, however, that Gs-coupled receptors show the largest shifts. Something that has puzzled me is why different concentrations of GTP are required to shift different receptor types (applies to alpha2 vs. NK-1 comparison above). It can't be differences in GTP'ase activity, because non-hydrolyzable GTP analogs give the same discrepancy. --Paul ----------------------------------------------------------------------- From: BMB6HDD@vax.bio.leeds.ac.uk> Date: Mon, 19 Jun 95 10:05 GMT Dear Rick, Do you have a few examples of the range of GTP-shifts for different receptors. The receptor I work on (human NK2) is Gq linked and ha a GTP-shift of 2-3 orders of magnitude (nM --> 0.1/1uM). Secondly, should there be a relationship between the size of the GTP-shift and the extent of activation? I would think that the affinity of the R form of the receptor for the agonist is a `red herring' since (like antagonist binding) it is physiologically irrelevant. What is important is that the agonist shifts the equilibrium towards the R* form. What do you think? Best Regards, Dan Donnelly From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!musc.edu!laniersm From: laniersm@musc.edu ("Stephen M. Lanier") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Fall conferences Date: 19 Jun 1995 05:23:40 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 46 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Two meetings of note this fall. I. 9th International Conference on Second Messengers and Phosphoprotein (Signal Transduction in Health and Disease) October 27-November 1, 1995 Nashville, TN USA Organizers: Dr. Jackie Corbin and Dr. Sharon Francis Department of Molecular Physiology and Biophysics 702 Light Hall Vanderbilt University School of Medicine Nashville, TN 37232-7067 Telephone 615-322-7067 Fax 615-343-3794 email: corbin@LHMRBA.HH.Vanderbilt.edu ------- II. ASPET Colloquium: "Alpha2-adrenergic receptors: Structure, Function and Therapeutic Implications" Satellite Symposium of the 9th International Conference on Second Messengers and Phosphoproteins October 25-27, 1995 Holiday Inn - Vanderbilt Nashville, TN USA Organizers: Lee E. Limbird, Ph.D. Department of Pharmacology Vanderbilt University Telephone 615-322-2207 Fax 615-343-1084 email: lee.limbird@mcmail.vanderbilt.edu Stephen M. Lanier, Ph.D. Department of Pharmacology Medical University of South Carolina Telephone 803-792-2574 fax 803-792-2475 email: laniersm@musc.edu The ASPET colloquium is intended to bring together academic and industrial scientists to discuss various aspects of signalling, regulation and trafficking of alpha2-adrenergic receptors and their use as experimental templates of the superfamily of G-protein coupled receptors for understanding the above issues and defining novel therapeutic targets. Participation of students and young investigators is strongly encouraged. Abstract deadline: August 1, 1995 Contact S Lanier for registration form and program. From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: secretin GPCR family mutants etc Date: 19 Jun 1995 04:54:38 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <37A0D3AC_001E8428.009921C9F618F729$25_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net Does anybody out there collect references for mutagenesis / protein chemistry studies (etc etc) for the secretin GPCR family (family B). There doesn't seem to be much in the literature. Is there any kind of general picture developing about ligand binding sites etc? Is there evidence that the R/R* ideas apply? Are there any non-peptidic antagonists? Dan Donnelly From owner-7tms_r@net.bio.net Sun Jun 18 23:00:00 1995 Path: biosci!NEURON1.BERKELEY.EDU!garywed From: garywed@NEURON1.BERKELEY.EDU (Gary Wedemeyer) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 19 Jun 1995 04:29:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506191132.AA14608@neuron1.berkeley.edu> NNTP-Posting-Host: net.bio.net unsubscribe From owner-7tms_r@net.bio.net Tue Jun 20 23:00:00 1995 Path: biosci!hubrecht.nl!markv From: markv@hubrecht.nl Newsgroups: bionet.molbio.proteins.7tms_r Subject: Gp-IP antibodies? Date: 21 Jun 1995 07:11:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506211411.HAA11850@net.bio.net> NNTP-Posting-Host: net.bio.net Hi there! Does anyone know if there are somewhere, Gs-, Gq- or G11-immunoprecipitating antibodies available? *No? -thanks for reading this mail. *Yes? -where? Mark Verheijen Markv@hubrecht.nl From owner-7tms_r@net.bio.net Tue Jun 20 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!news.onramp.net!news.tcst.com!dildog.lgc.com!news.sesqui.net!oitnews.harvard.edu!bloch.nmr.mgh.harvard.edu!nntp.mgh.harvard.edu!lfk From: lfk@receptor.mgh.harvard.edu (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Excerpted JBC TOC 6/9-6/16 Date: 21 Jun 1995 14:18:26 GMT Organization: Mass. General Hospital, Renal Unit Lines: 158 Distribution: world Message-ID: NNTP-Posting-Host: receptor.mgh.harvard.edu AU Borkowski-J-A. Ransom-R-W. Seabrook-G-R. Trumbauer-M. Chen-H. Hill-R-G. Strader-C-D. Hess-J-F. TI Targeted disruption of a B2 bradykinin receptor gene in mice eliminates bradykinin action in smooth muscle and neurons SO J-Biol-Chem. 1995 June 9. 270(23). p 13706. AU Bouaboula-M. Bourrie-B. Rinaldi-Carmona-M. Shire-D. Fur-G-L. Casellas-P. TI Stimulation of cannabinoid receptor CB1 induces krox-24 expression in human astrocytoma cells SO J-Biol-Chem. 1995 June 9. 270(23). p 13973. AU Kim-J. Wess-J. Rhee-A-M-. Schoneberg-T. Jacobson-K-A. TI Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor SO J-Biol-Chem. 1995 June 9. 270(23). p 13987. AU Yamano-Y. Ohyama-K. Kikyo-M. Sano-T. Nakagomi-Y. Inoue-Y. Nakamura-N. Morishima-I. Guo-D-F. Hamakubo-T. Inagami-T. TI Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1) SO J-Biol-Chem. 1995 June 9. 270(23). p 14024. AU Chen-H. Kendall-D-A. TI Artificial transmembrane segments. Requirements for stop transfer and polypeptide orientation. SO J-Biol-Chem. 1995 June 9. 270(23). p 14115. AU Pang-J-H-S. Hung-R-Y. Wu-C-J. Fang-Y-Y. Chau-L-Y. TI Functional characterization of the promoter region of the platelet-activating factor receptor gene. Identification of an initiator element essential for gene expression in myeloid cells. SO J-Biol-Chem. 1995 June 9. 270(23). p 14123. AU Pumiglia-K-M. LeVine-H. Haske-T. Habib-T. Jove-R. Decker-S-J. TI A direct interaction between G-protein {beta}{gamma} subunits and the Raf-1 protein kinase SO J-Biol-Chem. 1995 June 16. 270(24). p 14251. AU Ohguro-H. Hooser-J-P-V. Milam-A-H. Palczewski-K. TI Rhodopsin phosphorylation and dephosphorylation in vivo SO J-Biol-Chem. 1995 June 16. 270(24). p 14259. AU Slepak-V-Z. Artemyev-N-O. Zhu-Y. Dumke-C-L. Sabacan-L. Sondek-J. Hamm-H-E. Bownds-M-D. Arshavsky-V-Y. TI An effector site that stimulates G-protein GTPase in photoreceptors SO J-Biol-Chem. 1995 June 16. 270(24). p 14319. AU Rathouz-M-M. Vijayaraghavan-S. Berg-D-K. TI Acetylcholine differentially affects intracellular calcium via nicotinic and muscarinic receptors on the same population of neurons SO J-Biol-Chem. 1995 June 16. 270(24). p 14366. AU Holtmann-M-H. Hadac-E-M. Miller-L-J. TI Critical contributions of amino-terminal extracellular domains in agonist binding and activation of secretin and vasoactive intestinal polypeptide receptors. Studies of chimeric receptors. SO J-Biol-Chem. 1995 June 16. 270(24). p 14394. AU Abrams-C-S. Wu-H. Zhao-W. Belmonte-E. White-D. Brass-L-F. TI Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. SO J-Biol-Chem. 1995 June 16. 270(24). p 14485. AU Philipson-L-H. Kuznetsov-A. Toth-P-T. Murphy-J-F. Szabo-G. Ma-G-H. Miller-R-J. TI Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1) SO J-Biol-Chem. 1995 June 16. 270(24). p 14604. AU Koumenis-C. Nunez-Regueiro-M. Raju-U. Cook-R. Eskin-A. TI Identification of three proteins in the eye of Aplysia, whose synthesis is altered by serotonin (5-HT). Possible involvement of these proteins in the ocular circadian system. SO J-Biol-Chem. 1995 June 16. 270(24). p 14619. AU Kimura-K. White-B-H. Sidhu-A. TI Coupling of human D-1 dopamine receptors to different guanine nucleotide binding proteins. Evidence that D-1 dopamine receptors can couple to both Gs and Go. SO J-Biol-Chem. 1995 June 16. 270(24). p 14672. AU Kramer-R-M. Roberts-E-F. Hyslop-P-A. Utterback-B-G. Hui-K-Y. Jakubowski-J-A. TI Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2. SO J-Biol-Chem. 1995 June 16. 270(24). p 14816. AU Kalman-V-K. Erdman-R-A. Maltese-W-A. Robishaw-J-D. TI Regions outside of the CAAX motif influence the specificity of prenylation of G protein {gamma} subunits SO J-Biol-Chem. 1995 June 16. 270(24). p 14835. AU Kravchenko-V-V. Pan-Z. Han-J. Herbert-J-M. Ulevitch-R-J. Ye-R-D. TI Platelet-activating factor induces NF-{kappa}B activation through a G protein-coupled pathway SO J-Biol-Chem. 1995 June 23. 270(25). p 14928. AU Brown-H-A. Gutowski-S. Kahn-R-A. Sternweis-P-C. TI Partial purification and characterization of Arf-sensitive phospholipase D from porcine brain SO J-Biol-Chem. 1995 June 23. 270(25). p 14935. AU Singer-W-D. Brown-H-A. Bokoch-G-M. Sternweis-P-C. TI Resolved phospholipase D activity is modulated by cytosolic factors other than Arf SO J-Biol-Chem. 1995 June 23. 270(25). p 14944. AU Schwenk-U. Schroder-J-M. TI 5-Oxo-eicosanoids are potent eosinophil chemotactic factors. Functional characterization and structural requirements. SO J-Biol-Chem. 1995 June 23. 270(25). p 15029. AU Rossier-M-F. Aptel-H-B-C. Python-C-P. Burnay-M-M. Vallotton-M-B. Capponi-A-M. TI Inhibition of low threshold calcium channels by angiotensin II in adrenal glomerulosa cells through activation of protein kinase C SO J-Biol-Chem. 1995 June 23. 270(25). p 15137. AU Offermanns-S. Simon-M-I. TI G{alpha}15 and G{alpha}16 couple a wide variety of receptors to phospholipase C SO J-Biol-Chem. 1995 June 23. 270(25). p 15175. AU Marx-U-C. Austermann-S. Bayer-P. Adermann-K. Ejchart-A. Sticht-H. Walter-S. Schmid-F-X. Jaenicke-R. Forssmann-W-G. Rosch-P. TI Structure of human parathyroid hormone 1-37 in solution SO J-Biol-Chem. 1995 June 23. 270(25). p 15194. AU Emoto-N. Yanagisawa-M. TI Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum SO J-Biol-Chem. 1995 June 23. 270(25). p 15262. AU Sato-M. Kataoka-R. Dingus-J. Wilcox-M. Hildebrandt-J-D. Lanier-S-M. TI Factors determining specificity of signal transduction by G-protein-coupled receptors. Regulation of signal transfer from receptor to G-protein. SO J-Biol-Chem. 1995 June 23. 270(25). p 15269. AU Palczewski-K. Ohguro-H. Premont-R-T. Inglese-J. TI Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations. SO J-Biol-Chem. 1995 June 23. 270(25). p 15294. -- Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-355-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Tue Jun 20 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!news.sprintlink.net!news.onramp.net!news.tcst.com!dildog.lgc.com!news.sesqui.net!oitnews.harvard.edu!bloch.nmr.mgh.harvard.edu!nntp.mgh.harvard.edu!lfk From: lfk@receptor.mgh.harvard.edu (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins.7tms_r Subject: AT1R -> JAK/STAT Date: 21 Jun 1995 14:23:19 GMT Organization: Mass. General Hospital, Renal Unit Lines: 21 Distribution: world Message-ID: NNTP-Posting-Host: receptor.mgh.harvard.edu Last month in Nature K. E. Bernstein and colleagues published data showing that stimulation of the AT1R in RASM cells promoted the phosphorylation of JAK/STAT family members. They then showed that with a polyclonal against the C-term of the AT1R, they were able to immunoprecipitate STAT. They then concluded that the AT1R talked directly to STAT without a G-protein. I believe that the AT receptors also show poor GTP shifts. Might this be because thay don't talk well to alphas? -- Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-355-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Tue Jun 20 23:00:00 1995 Path: biosci!SHRSYS.HSLC.ORG!wthomas From: wthomas@SHRSYS.HSLC.ORG Newsgroups: bionet.molbio.proteins.7tms_r Subject: constitutive internalization? Date: 21 Jun 1995 20:55:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <009923BA.9F55B15F.2@shrsys.hslc.org> NNTP-Posting-Host: net.bio.net Hi to all, As I'm sure most of this group would be aware, many of the 7TM receptors display a rapid agonist-stimulated endocytosis. I'm very interested in this process especially with respect to angiotensin receptors. Given a number of recent postings regarding constitutively active receptors, I was wondering: Do these receptors display constitutive internalization? and Is it even possibleto measure constitutive endocytosis in native (non-epitope tagged) receptors? Receptor internalization is classically measured by binding close to saturating concentrations of labelled agonist to intact cells at 4oC (low temperature prevents internalization). The cells are then washed and transferred to 37oC toinitiate internalization. At various time points, cells are rapidly cooled and extensively washed; cell surface receptors can be stripped with an acid wash, while internalized receptors are resistant to this treatment. The ratio of cpm associated with the acid washed cells to the total binding (acid wash + cells) gives an index of endocytosis. Typically, about 70-80% of receptors internalize within 10 min. So, back to my second question. If a receptor can be altered to promote constitutive endocytosis, the above methodology would appear inappropriate, because as soon as a receptor was in the plasma membrane it would immediately internalize. In fact, it may the case that the majority of the receptors would be located intracellularly (and targetted for degradation?). If anyone has any thoughts on a possible strategy, or any literature pertaining to constitutive internalization, I would appreciate your input. Thanks Wally Thomas Weis Center for Research Geisinger Clinic Danville, PA wthomas@shrsys.hslc.org From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!edi.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!imt950.imt-mrs.fr!taloa.unice.fr!biochim2-31.unice.fr!mckenzie From: Fergus R. McKenzie Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: AT1R -> JAK/STAT Date: 22 Jun 1995 15:50:10 GMT Organization: cnrs Lines: 55 Distribution: world Message-ID: <3sc3fi$bcd@taloa.unice.fr> References: NNTP-Posting-Host: biochim2-31.unice.fr X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Thu, 22 Jun 1995 17:46:08 GMT In article Lee F., lfk@receptor.mgh.harvard.edu writes: > >Last month in Nature K. E. Bernstein and colleagues published data >showing that stimulation of the AT1R in RASM cells promoted the >phosphorylation of JAK/STAT family members. > >They then showed that with a polyclonal against the C-term of the AT1R, >they were able to immunoprecipitate STAT. > >They then concluded that the AT1R talked directly to STAT without >a G-protein. I believe that the AT receptors also show poor GTP >shifts. Might this be because thay don't talk well to alphas? > Dear Frank, If I can continue Rick Neubig's original question on G-protein linked receptors which show poor changes in affinity in the presence of G-protein activating GTP analogues etc... My recollection of the AII receptor signalling field is that like most Gq-linked GPCR, the type 1 AII receptor does not show massive affinity changes in response to G-protein activation state. Prior to cloning of the type 11 AII receptor, many groups believed that this receptor was not G-protein coupled for exactly the reason mentioned above. However after cloning by two independent groups, the receptor appears to be a classic 7TM. This receptor seems to belong to a 'new' class of GPCR in that the binding data suggest that the receptors are NOT G-protein coupled. Other members of this class include the Somatostatin type 1 Receptor (SSTR1). (Now the tie in to your comment, Frank!). It is possible to co-purify SSTR1 and the tyrosine phosphatase, PTP1C, which appears to be functionaly linked to SSTR1 receptor activation. Hence, there are now several examples in the litterature of G-protein coupled receptors which may directly interact with enzymes other than G-proteins. Can we say that these receptors can talk to other proteins because they don't talk well to G-protein alphas? I don't know it it is related, however for receptors which can talk to both Gi and Gq family members (I'm basing this on pertussis toxin sensitivity of cyclase inhibition and insensitivity of PLC stimulation) you activate the two separate arms with differing agonist concentrations. I'm thinking here of the thrombin and some endothelin receptor subclasses. In general you need a one log higher concentration of agonist to get the Gq-like pathway going, compared to the Gi pathway. Is this because there is a difference in the intrinsic capacity of such receptors to activate different members of the G-protein family (specificity) or a mass action effect because a given G-protein alpha subunit is predominately expressed compared to the others (selectivity). I hope I haven't strayed too far away from the original thread! - look forward to some friendly fire. Fergus. From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!BIOCSERVER.BIOC.CWRU.EDU!roth From: roth@BIOCSERVER.BIOC.CWRU.EDU (Bryan Roth) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: constitutive internalization? Date: 22 Jun 1995 07:24:41 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506221422.KAA03068@biocserver.BIOC.CWRU.Edu> NNTP-Posting-Host: net.bio.net > >Hi to all, > >As I'm sure most of this group would be aware, many of the 7TM receptors >display a rapid agonist-stimulated endocytosis. Although it has been assumed that most 7TM receptors undergo endocytosis, there is scant information which is unambiguous. The best to date (to my mind) are the studies performed by Von Zastrow and Kobilka on the adrenergic receptors as well as one on substance P receptors in the latest issue of Science. We are currently using receptor antibodies as well as fluorescently tagged ligands to study this at 5-HT2A receptors. It turns out that in some cell lines we can find no evidence for endocytosis (using binding assays and confocal microscopy) whereas in other cell lines endocytosis occurs secondary to agonist (Roth et al submitted; Roth et al in press). More later ============================================================= Bryan L.Roth "The Tao that can be named is Department of Biochemistry not the true Tao" Room W438 CWRU Medical School Cleveland, OH 44106-4935 roth@biocserver.cwru.edu 216-368-2730 (Office) 216-368-4544 (FAX) From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!musc.edu!laniersm From: laniersm@musc.edu ("Stephen M. Lanier") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Fall conferences (fwd) Date: 22 Jun 1995 06:11:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 56 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net ---------- Forwarded message ---------- Date: Mon, 19 Jun 1995 08:22:21 -0400 (EDT) From: Stephen M. Lanier To: 7tms_r@net.bio.net Subject: Fall conferences Two meetings of note this fall. I. 9th International Conference on Second Messengers and Phosphoprotein (Signal Transduction in Health and Disease) October 27-November 1, 1995 Nashville, TN USA Organizers: Dr. Jackie Corbin and Dr. Sharon Francis Department of Molecular Physiology and Biophysics 702 Light Hall Vanderbilt University School of Medicine Nashville, TN 37232-7067 Telephone 615-322-7067 Fax 615-343-3794 email: corbin@LHMRBA.HH.Vanderbilt.edu ------- II. ASPET Colloquium: "Alpha2-adrenergic receptors: Structure, Function and Therapeutic Implications" Satellite Symposium of the 9th International Conference on Second Messengers and Phosphoproteins October 25-27, 1995 Holiday Inn - Vanderbilt Nashville, TN USA Organizers: Lee E. Limbird, Ph.D. Department of Pharmacology Vanderbilt University Telephone 615-322-2207 Fax 615-343-1084 email: lee.limbird@mcmail.vanderbilt.edu Stephen M. Lanier, Ph.D. Department of Pharmacology Medical University of South Carolina Telephone 803-792-2574 fax 803-792-2475 email: laniersm@musc.edu The ASPET colloquium is intended to bring together academic and industrial scientists to discuss various aspects of signalling, regulation and trafficking of alpha2-adrenergic receptors and their use as experimental templates of the superfamily of G-protein coupled receptors for understanding the above issues and defining novel therapeutic targets. Participation of students and young investigators is strongly encouraged. Abstract deadline: August 1, 1995 Contact S Lanier for registration form and program. From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!LYNX.DAC.NEU.EDU!rdeth From: rdeth@LYNX.DAC.NEU.EDU (richard deth) Newsgroups: bionet.molbio.proteins.7tms_r Subject: GTP-induced affinity changes Date: 22 Jun 1995 14:44:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506222141.RAA00223@lynx.dac.neu.edu> Although the lines are quiet (July 1 proposal deadline??) I thought I would respond to Rick Neubig's comment on Gq-coupled receptors having a smaller GTP-induced shift for agonist displacement curves: 1. When comparing the GTP shift for alpha-1 vs. alpha-2 receptors in the same membrane preparation (JPET 243:430 and 252:1184) we found that epinephrine showed a similar affinity for the R state (3.2 uM and 4.3 uM respectively) but a five-fold lower affinity for R* at alpha 2 receptors (9.6 and 47 nM respectively). This resulted in a larger GTP shift at alpha-1 receptors if you just considered the Kd values. Notably, the alpha-2 values were obtained in 100mM Na+, since GTP is rather ineffectual in its absence. The actual size of the space between displacement curves is, however, also dependent upon the proportion of receptors involved in the ARG complex. Since Gi-coupled receptors such as the alpha-2 have a higher native affinity for G proteins (i.e. higher tendency to be in the R* state), a higher proportion of them are in the ARG complex than is the case for alpha-1 receptors. 2.In the case of the cloned alpha-2D receptor, we measured the overall size of the GTP shift for a series of full and partial agonists and found a direct proportionality between this value and the size of the stimulated GTP binding produced by each agent. Thus agonist efficacy in the forward direction was directly related to the negative influence of GTP on RG association in the backwards direction. This really emphasizes the role of agonists as allosteric modulators of the receptor surface which contributes to the R/G interface. P.S. On the earlier issue of naming antagonists which reduce spontaneous receptor activity, why not use the term "negative antagonist" as suggested by Tommy Costa? This is associated with the term "null antagonist" for agents which occupy the agonist binding site without favoring the R or R* conformations. To me it is clear that there are three categories of ligands: agonist, null antagonists and negative antagonists. The first and last categories contain a range of partial to full allosteric influences, depending upon the degree to which they prefer the two postulated receptor conformations. It so happens that nature has not utilized negative allosterism as a message. Hmmmm or could it be that we haven't recognized them yet. Comments? Richard Deth Northeastern University rdeth@lynx.neu.edu From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!CODON.NIH.GOV!usdin From: usdin@CODON.NIH.GOV (Ted Usdin) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Filtration manifolds Date: 22 Jun 1995 05:32:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: To those who bind, I am curious about current preferences in binding technology. For small assays we have been using the Millipore bucket-thing and plenty of people around here have Brandel harvesters. I am considering buying a gizmo for moderate sized assays. I would like to hear peoples thoughts regarding ease of use, reliability, consistency, rinsing uniformity, speed, clogging problems (all the important minutiae) of the currently available gadgets. Regards, ****************************** Ted B. Usdin, M.D., Ph.D. Laboratory of Cell Biology, NIMH Bldg 36/Rm3A17 {36 Convent Dr MSC 4090 (U.S. mail)} BETHESDA MD 20892-4090 e-mail usdin@codon.nih.gov tel 301-402-4161 fax 301-402-1748 From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Newsgroups: bionet.molbio.proteins.7tms_r Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!NewsWatcher!user From: usdin@codon.nih.gov (Ted Usdin) Subject: Radioligand Binding Technology Message-ID: Followup-To: bionet.molbio.proteins.7tms_r Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.99.30 Organization: NIMH Date: Fri, 23 Jun 1995 00:20:18 GMT Lines: 14 To those who bind, I am curious about current preferences in binding technology. For small assays we have been using the Millipore bucket-thing and plenty of people around here have Brandel harvesters. I am considering buying a gizmo for moderate sized assays. I am interested, and perhaps others are in thoughts regarding ease of use, reliability, consistency, rinsing uniformity, speed, clogging problems (all the important minutiae) of the currently available gadgets. Regards, Ted Usdin From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!emory!usenet From: medtjm@bimcore.emory.edu (T. J. Murphy) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: AT1R -> JAK/STAT Date: 22 Jun 1995 21:23:57 GMT Organization: Biomolecular Computing Resource, Emory University Lines: 28 Distribution: world Message-ID: <3scn1d$qa2@emory.mathcs.emory.edu> References: Reply-To: medtjm@bimcore.emory.edu NNTP-Posting-Host: bimcore.emory.edu In article 95Jun21102319@receptor.receptor.mgh.harvard.edu, lfk@receptor.mgh.harvard.edu (Lee F. (Frank) Kolakowski) writes: > > >Last month in Nature K. E. Bernstein and colleagues published data >showing that stimulation of the AT1R in RASM cells promoted the >phosphorylation of JAK/STAT family members. > >They then showed that with a polyclonal against the C-term of the AT1R, >they were able to immunoprecipitate STAT. > >They then concluded that the AT1R talked directly to STAT without >a G-protein. I believe that the AT receptors also show poor GTP >shifts. Might this be because thay don't talk well to alphas? > > >-- >Frank Kolakowski First things first, did the antibodies actually immunoprecipitate the AT1 receptor??? Only time will tell. --- TJ Murphy Asst. Professor Dept of Pharmacology Emory University School of Medicine From owner-7tms_r@net.bio.net Wed Jun 21 23:00:00 1995 Path: biosci!bms.com!watson_j From: watson_j@bms.com (John Watson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe Date: 22 Jun 1995 10:51:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net subscribe 7tms_r /--------------------------------------------------------------\ \--------------------------------------------------------------/ || A. John Watson, Ph.D. || || Research Investigator II || || Bristol-Myers Squibb Pharmaceutical Reseach Institute || || Central Nervous System Biology, Department 404 || || 5 Research Parkway || || Wallingford, CT 06492 || || (203) 284-6745 || || (203) 284-7569 fax || || watson_j@bms.com || /--------------------------------------------------------------\ \--------------------------------------------------------------/ From owner-7tms_r@net.bio.net Thu Jun 22 23:00:00 1995 Path: biosci!rnisd0.DNET.roche.com!margolsr From: margolsr@rnisd0.DNET.roche.com Newsgroups: bionet.molbio.proteins.7tms_r Subject: postdoctoral positions Date: 23 Jun 1995 11:07:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506231804.AA24682@mailgate.roche.com> NNTP-Posting-Host: net.bio.net Postdoctoral positions available at Linguagen, a northern New Jersey biotechnology company. Candidates should have experience in molecular biology, pharmacology, protein biochemistry as it relates to G proteins, G protein coupled receptors, or signal transduction. Send CVs to Robert F. Margolskee, Linguagen, PO Box 395, Basking Ridge, NJ 07920-0395. From owner-7tms_r@net.bio.net Thu Jun 22 23:00:00 1995 Path: biosci!rug.ac.be!JeanLuc.Verschelde From: JeanLuc.Verschelde@rug.ac.be (Jean-Luc Verschelde) Newsgroups: bionet.molbio.proteins.7tms_r Subject: unsubscribe Date: 23 Jun 1995 00:51:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net From owner-7tms_r@net.bio.net Thu Jun 22 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!usc!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!soap.news.pipex.net!pipex!edi.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!serra.unipi.it!usenet From: buccione@cmns.mnegri.it (Roberto Buccione) Newsgroups: bionet.molbio.proteins.7tms_r Subject: intracellular 7TM receptors? Date: 23 Jun 1995 07:09:54 GMT Organization: Consorzio Mario Negri Sud Lines: 14 Sender: -Not-Authenticated-[2686] Message-ID: <3sdpc2$vbc@serra.unipi.it> NNTP-Posting-Host: neurobio03.cmns.mnegri.it X-Posted-From: InterNews 1.0.7@neurobio03.cmns.mnegri.it Xdisclaimer: No attempt was made to authenticate the sender's name. Is there strong evidence showing an intracellular localization of 7tm receptors other than on the PM, (besides when they are overexpressed)? And anyway, what would be the meaning of this? In any case, heterotrimeric G proteins happen to be all over the cell, certainly not only on the PM... We would like to discuss this, especially in the light of possible homeostatic functions they might have in intracellular regulation (metabolism, cytoskeleton, etc.). Roberto Buccione Molecular Neurobiology Laboratory Consorzio Mario Negri Sud S. Maria Imbaro (Ch) From owner-7tms_r@net.bio.net Sat Jun 24 23:00:00 1995 Path: biosci!rutgers!oitnews.harvard.edu!das-news2.harvard.edu!fas-news.harvard.edu!newspump.wustl.edu!news.ecn.bgu.edu!news.moneng.mei.com!howland.reston.ans.net!news.sprintlink.net!demon!plug.news.pipex.net!pipex!edi.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!ghost.dsi.unimi.it!news.graphics.cornell.edu!newsstand.cit.cornell.edu!cornell.edu!pf13 From: pf13@cornell.edu (Peter Fraissinet) Newsgroups: bionet.molbio.proteins.7tms_r Subject: AD: FS: Advances in Electrophoresis vol. 7 Date: Thu, 22 Jun 1995 09:55:43 UNDEFINED Organization: Cornell University Lines: 9 Sender: pf13@cornell.edu (Verified) Message-ID: NNTP-Posting-Host: 128 X-Newsreader: Trumpet for Windows [Version 1.0 Rev B] Advances in Electrophoresis, vol. 7. 1994. Edited by A. Chrambach, M.J. Dunn, B.J. Radola. Weinheim: VCH. xii, 487 pp. Cloth. As new. $40.00 postpaid in USA. Email me to reserve it. pf13@cornell.edu From owner-7tms_r@net.bio.net Sun Jun 25 23:00:00 1995 Path: biosci!PT.CYANAMID.COM!hadcockj From: hadcockj@PT.CYANAMID.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re[2]: AT1R -> JAK/STAT Date: 26 Jun 1995 06:16:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HS5N9NCBMEBOT69W@ptag2.pt.Cyanamid.COM> NNTP-Posting-Host: net.bio.net For Fergus McKenzie and others proposing a new class of G protein-coupled receptors where agonist binding is relatively insensitive to GTP analogs some caution should be used. He points out that agonist binding to the somatostatin receptor subtype I (SSTR1) is insensitive to GTP analogs. This was originally observed in one of the earliest publications on SSTR1 from Terry Reisine's lab. However, following this publication at least three labs have shown that SSTR1 behaves as a "typical" GPCR in that agonist binding is sensitive to GTP analogs. All of the following publications were from analysis performed in cell lines different from the one used by Terry Reisine's lab. Thus, SSTR1 does not show this atypical behavior in many cell lines but does in others. The references are listed below. A receptor that does show this "atypical" behavior, however, is the turkey erthrocyte beta-adrenergic receptor. John Hadcock References for GTP sensitivity of agonist binding to SSTR1: 1. Hou et al (1994) JBC 269:10357-10362 2. Hadcock et al (1994) Mol Pharm 45:410-416 3. Patel Et al (1994) BBRC 198: 605-612 From owner-7tms_r@net.bio.net Sun Jun 25 23:00:00 1995 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: GTP shifts Date: 26 Jun 1995 08:27:01 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <37A0F0C8_001EE6A0.009927645EBA34C9$13_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net While the subject of "GTP shifts" continues, can I bring up the point I made in reply to Rick Neubig. Is the size of the GTP shift important? Does the affinity of an agonist for the R form of the receptor have any physiological relevance? The role of an agonist is presumably to promote the formation of the active (R*) form of the receptor and a high affinity for this form of the receptor is advantagous. Is there any reason why the inactive (R) form of the recpetor should have a high or low affinity for the agonist - or is this affinity a reflection of how the binding site of the R form has altered during the conformational change from R* ? My feeling is that so long as the agonist promotes formation of R*, its affinity for thr R form is not important for the function of the receptor. Of course, my feeling was that England would win the Rugby Union World Cup last week and in the end that was horribly wrong! Any comments will be gratefully received (on the science, not the rugby). Best Regards, Dan From owner-7tms_r@net.bio.net Sun Jun 25 23:00:00 1995 Path: biosci!RECEPTOR.MGH.HARVARD.EDU!lfk From: lfk@RECEPTOR.MGH.HARVARD.EDU (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: subscribe Date: 26 Jun 1995 11:13:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <9506261146.AA04335@receptor> References: NNTP-Posting-Host: net.bio.net Dear John, please send e-mail to 'biosci-server@net.bio.net' and say in the body of the email 'subscribe 7tms_r' Best wishes, Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-355-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Sun Jun 25 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!gaia.ucs.orst.edu!ava.bcc.orst.edu!vogelw From: vogelw@ava.bcc.orst.edu (Walter Vogel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: What's in a name? Negative antagonism or something else. Date: 27 Jun 1995 01:56:20 GMT Organization: Oregon State University, Corvallis, Oregon Lines: 51 Message-ID: <3snog4$54k@gaia.ucs.orst.edu> NNTP-Posting-Host: ava.bcc.orst.edu Subject: What's in a name? Negative antagonism or something else. There should be a distinction between terms that describe an observation and those that attempt to explain the observation. When a ligand binds a receptor and results in activation of an effector (a down stream effect) we call this "agonism" and the ligand an "agonist"; when referring to a ligand that competes with an agonist but does not activate the system we call this ligand an "antagonist" and the phenomenon "antagonism". What remains to be named are terms to describe the phenomenon of ligand induced reducution of basally activated effector systems and a term for those ligands. First, a comparison to a similar situation involving the definition of partial agonism and partial agonists. The phenomenon of partial agonism where a ligand at saturating concentrations results only a fraction of the effect produced by an agonist has long been observed. The ligand that produced this fractional effect is often referred to as partial agonist. But a problem occurs in this definition when considering that the identical receptor and ligand can exhibit partial agonism in one system but full agonism in another. Furthermore, in an overexpressed cell culture system we (1) see partial agonism at low receptor density but at high receptor site density the system displays full agonism. We are left with the uncomfortable antitautology that partial agonists don't always produce partial agonism. Without going into the possible mechanisms capable of explaining this I feel that the term "partial agonist" is a misnomer because it gives the impression that such a ligand is interacting with a given receptor-G protein complex in an essentially different way from a full agonist. This may be (according to one model) or not at all (another model (1) ) but it shouldn't be labeled as such. Better to say that a ligand "displays partial agonism" in a particular system than label it a "partial agonist" because it does. We need a name for the phenomenon of ligand induced reduction of basal effector activation. I side with Richard Deth that "negative antagonism" has precedent (2), even if I don't agree that the mechanism presented in that paper is generally applicable. According to this logic ligands should be referred to as "agonists" or "antagonists" and the phenomena that result from their action would be referred to as "agonism", "partial agonism", "antagonism", "partial negative antagonism", and "negative antagonism". 1. Vogel, W. K. et al. J. Biol. Chem. (1995) 270 (26) in press (expected June 30 issue). 2. Costa, T. et al. Mol. Pharm. (1992) 41, 549. Walter K. Vogel Dept Biochemistry and Biophysics Oregon State University From owner-7tms_r@net.bio.net Mon Jun 26 23:00:00 1995 Path: biosci!DEFIANCE.HSC.COLORADO.EDU!port_d From: port_d@DEFIANCE.HSC.COLORADO.EDU ("Dave Port") Newsgroups: bionet.molbio.proteins.7tms_r Subject: phosphorylation Date: 27 Jun 1995 19:50:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <199506280251.UAA14992@essex.hsc.colorado.edu> NNTP-Posting-Host: net.bio.net phosphorylation Dear fellow 7-spanners: Is anyone out there aware of any information on hierarchical phosphorylation of any G-protein coupled receptor? I am aware of Bouvier's late 80's JBC on long/short truncations of the b2-AR (i.e., mutation of sites close to TM7 versus more distal S/T bARK sites) in terms of function. I don't think there is any info on this subject, but I would like to know if I'm missing something. Thanks J.D. Port UCHSC MED/PHARM port_d@defiance.hsc.colorado.edu From owner-7tms_r@net.bio.net Tue Jun 27 23:00:00 1995 Path: biosci!galaxy.ucr.edu!ratatosk.yggdrasil.com!nntp-sc.barrnet.net!nntp-hub2.barrnet.net!news.Stanford.EDU!bloom-beacon.mit.edu!news.starnet.net!wupost!howland.reston.ans.net!cs.utexas.edu!convex!news.duke.edu!news-feed-1.peachnet.edu!usenet.eel.ufl.edu!news.uoregon.edu!gaia.ucs.orst.edu!ava.bcc.orst.edu!vogelw From: vogelw@ava.bcc.orst.edu (Walter Vogel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: On the meaning of R and R* Date: 28 Jun 1995 18:38:16 GMT Organization: Oregon State University, Corvallis, Oregon Lines: 47 Message-ID: <3ss7io$l6b@gaia.ucs.orst.edu> NNTP-Posting-Host: ava.bcc.orst.edu Perhaps it is only I who is confused but the meaning or rather the identity of the R* state confuses me. Some appear to be using R* to mean the G protein coupled 'RG' state while others are clearly not. In the model proposed by Rodbell (1) and as applied to the coupling of the beta-adrenergic receptor and called the ternary complex model (2), there are two states R and RG of receptor. The R state is free receptor capable of binding agonist (A) with low affinity. The RG state is complexed with G protein and capable of binding A with high affinity, the resulting ARG ternary complex being responsible for signal transduction. The first convincing evidence that this two-state model needed to be extended was reported in a communication from Lefkowitz's group (3). They found that each of 19 site-directed mutations at a particular site in the alpha 1B-adrenergic receptor conferred constitutive phosphoinositide hydrolysis activity (the basal rate was significant, whereas in wild type it was insignificant), increased agonist affinity, but unchanged antagonist affinity when expressed in COS-7 cells. Agonist binding was also observed to be relatively unaffected by guanine nucleotides. Results from the same group published a year later proposed an 'extended ternary complex model' to explain constitutively active beta-adrenergic receptor mutants (4). This model is excessively complex and its implementation is presented as the only one capable of explaining the results when it is not. The essential advance of this model (also found in ref. 3) is that there is an isomerization of the receptor from R to R* and that only R* is capable of interacting with G proteins to form R*G. The R form bind agonist with low affinity while both the R* and the R*G forms bind agonist with high affinity. The model allowed the R* and the R*G states to have different affinities for agonist even though there is no evidence for this and I think no requirement. Note well that I am not addressing the subject of antagonist affinities at all. So in the absence of ligands we have: +G R <==> R* <==> R*G ___________________ 1. Rodbell, M. (1980) Nature 284, 481-489. 2. De Lean, A. et al. (1980) J. Biol. Chem. 255, 71008-7117. 3. Kjelsberg, M. A. et al. (1992) J. Biol. Chem. 267, 1430-1433. 4. Samama, P. et al. (1993) J. Biol. Chem. 268, 4625-4636. Walter K. Vogel Dept Biochemistry and Biophysics Oregon State University From owner-7tms_r@net.bio.net Wed Jun 28 23:00:00 1995 Path: biosci!VIR.COM!biosignal From: biosignal@VIR.COM (BioSignal Inc.) Newsgroups: bionet.molbio.proteins.7tms_r Subject: re: Better name for inverse agonists? Date: 29 Jun 1995 11:34:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net It seems that we're having trouble agreeing on a term for specific ligands which produce direct effects on receptors opposite to those of agonists. Such effects were first reported, I believe, at the benzodiazepine/GABAa receptor complex, where "inverse agonists" were found to allosterically decrease GABA affinity, in contrast to the increases in GABA affinity caused by benzodiazepine "agonists" such as diazepam. A few years later, certain ligands were found to produce effects opposite to those of endogenous transmitters at receptors coupled to G proteins, and the term "negative antagonist" was proposed. Notwithstanding the uncertainty concerning a possible endogenous ligand for the benzodiazepine binding site, it appears that the same general phenomenon is occuring in both cases, and therefore one name should suffice for all "opposite" ligands. Whereas "inverse agonist" suggests an action opposite to that of an agonist, the term "negative antagonist" is less easy to interpret. First of all, it's a double negative. Since the prefix "ant-" means "against" or "opposed to", wouldn't negative antagonists then be "in favor of agonists"? Alternativeley, it's been suggested that the term "antagonist" be reserved for truly "neutral" ligands, in accord with the classical interpretation that hormone-blocking agents lack intrinsic activity. If antagonists have no effect on receptor activity per se, then wouldn't negative antagonists also lack activity (i.e., -0 = 0)? Thus, I think that the term "negative antagonist" is confusing and should be dropped. "Inverse agonist", although not totally satisfactory, seems less objectionable. Webster's defines agonist as, among other things, "one that is engaged in a struggle" (to promote a certain protein conformation?). In that case, an antagonist would be opposed to the struggler; such opposition could be either active or passive (but never negative). It follows that an "antagonist" may or may not posess intrinsic activity. How then do we distinguish between the two types? For the sake of historical consistency, it might be best to retain "antagonist" as a general term for both. Something that struggles in the opposite direction could be referred to as an "inverse agonist" (or maybe a "negative agonist"), while something with no activity at all would be a "passive antagonist". Peter Chidiac BioSignal Inc. From owner-7tms_r@net.bio.net Wed Jun 28 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!news.sprintlink.net!psgrain!nntp.teleport.com!usenet From: Kim Neve Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: On the meaning of R and R* Date: 29 Jun 1995 21:50:52 GMT Organization: Oregon Health Sciences University Lines: 17 Message-ID: <3sv77s$8j0@maureen.teleport.com> References: <3ss7io$l6b@gaia.ucs.orst.edu> NNTP-Posting-Host: ip-pdx1-50.teleport.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) vogelw@ava.bcc.orst.edu (Walter Vogel) wrote: > The essential advance of this >model (also found in ref. 3) is that there is an isomerization of the >receptor from R to R* and that only R* is capable of interacting with G >proteins to form R*G. > This point was also made explicitly by Contreras and Molinoff in two papers published in 1986 (JPET 237:165-172, and JPET 239:136-143). Interactions of Beta-2 receptors with G proteins were prevented by GTP in one paper and by solubilization in the second paper, yet both still provided evidence for an agonist-dependent conformational change from R to R*. Kim Neve Oregon Health Sciences University From owner-7tms_r@net.bio.net Wed Jun 28 23:00:00 1995 Path: biosci!iibfc.uba.ar!ortizm From: ortizm@iibfc.uba.ar ("Marcela Ortiz ") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Information Date: 29 Jun 1995 20:29:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <2ff35c67.iibfc@iibfc.uba.ar> NNTP-Posting-Host: net.bio.net I would like to contact Dr Patrick J.Casey, PhD. Asst. Prof., who have been working at Duke University Medical Centre by last year. I have no other way to know his current address, fax number or E-mail. Can anybody help me on this matter? Thank you very much since now. --- Marcela Ortiz ortizm@iibfc.uba.ar From owner-7tms_r@net.bio.net Thu Jun 29 23:00:00 1995 Path: biosci!IUNHAW1.IUN.INDIANA.EDU!WANDERS From: WANDERS@IUNHAW1.IUN.INDIANA.EDU ("W. Marshall Anderson") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Substance P antagonist Date: 30 Jun 1995 13:18:00 -0700 Organization: Indiana University Northwest Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <38F75B71A5@iunhaw1.iun.indiana.edu> Reply-To: wanders@iunhaw1.iun.indiana.edu NNTP-Posting-Host: net.bio.net What is currently considered to be the best substance P antagonist (with special reference to studies of the eye if possible?) Reply to wanders@iunhaw1.iun.indiana.edu Thanks, W. Marshall Anderson I.U. Sch. Med. NWCME 3400 Broadway Gary, IN 46408 USA phone 219 n980-6534 FAX 219 980-6566 e-mail wanders@iunhaw1.iun.indiana.edu W. Marshall Anderson, Ph.D. Phone: 219 980-6534 FAX: 219 980-6566 E-mail: wanders@iunhaw1.iun.indiana.edu From owner-7tms_r@net.bio.net Thu Jun 29 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!news.sprintlink.net!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!winx03!wrzx12!phak004 From: phak004@wrzx12.rz.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: phosphorylation Date: 30 Jun 1995 08:28:34 GMT Organization: University of Wuerzburg, Germany Lines: 27 Distribution: world Message-ID: <3t0cji$3q9@winx03.informatik.uni-wuerzburg.de> References: <199506280251.UAA14992@essex.hsc.colorado.edu> NNTP-Posting-Host: wrzx12.rz.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Dave Port (port_d@DEFIANCE.HSC.COLORADO.EDU) wrote: > Is anyone out there aware of any information on hierarchical > phosphorylation of any G-protein coupled receptor? I think the group of Kris Palczewski has investigated the phosphorylation of rhodopsin and come up with some information about sequential phosphorylation. Specifically, what you are looking for *might* be this paper: @article{ohguro:94a, author = {Hiroshi Ohguro and Richard S. Johnson and Lowell H. Ericsson and Kenneth A. Walsh and Krzysztof Palczewski}, title = {Control of Rhodopsin Multiple Phosphorylation.}, journal = {Biochemistry}, volume = 33, pages = {1023--1028}, year = 1994 } I am doing this from memory, so I don't guarantee for anything :-) --Cornelius. -- /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */ /* Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game you play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Thu Jun 29 23:00:00 1995 Path: biosci!novo.dk!CES From: CES@novo.dk (CES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Human GTP-binding proteins, gamma subunits Date: 30 Jun 1995 09:31:15 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear netters. Looking through GenBank for sequences for human gamma subunits of g proteins revealed nothing! Do any of you have references of the cloning of any of these? Carsten Stidsen ces@novo.dk From owner-7tms_r@net.bio.net Thu Jun 29 23:00:00 1995 Path: biosci!daresbury!trane.uninett.no!nac.no!Norway.EU.net!EU.net!news.sprintlink.net!europa.chnt.gtegsc.com!library.ucla.edu!info.ucla.edu!nnrp.info.ucla.edu!usenet From: jnakamot@pediatrics.medsch.ucla.edu (Jon Nakamoto) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Human GTP-binding proteins, gamma subunits Date: 1 Jul 1995 02:08:11 GMT Organization: UCLA Medical Center Lines: 16 Sender: -Not-Authenticated-[3482] Distribution: world Message-ID: <3t2amb$i34@saba.info.ucla.edu> References: NNTP-Posting-Host: 149.142.47.12 X-Posted-From: InterNews 1.0.1@149.142.47.12 Xdisclaimer: No attempt was made to authenticate the sender's name. Carsten, With the exception of gamma-1 (Genbank S62026 and S62027), not many human gamma subunit nucleotide sequences are out there (most are bovine and rat). Undoubtedly many groups have some fragments of the human sequence, but it's not worth most people's time to get the entire sequence and do all of the work to confirm it before publishing as a boring one-page communication. However, I bet you'll see a few articles with the human sequences of some of the more recently-identified gamma subunits in the near future. Jon Nakamoto, MD Clinical Instructor of Pediatrics/Endocrinology, UCLA jnakamot@pediatrics.medsch.ucla.edu From owner-7tms_r@net.bio.net Thu Jun 29 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!fu-berlin.de!zrz.TU-Berlin.DE!cs.tu-berlin.de!fauern!winx03!wrzx12!phak004 From: phak004@wrzx12.rz.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: phosphorylation Date: 30 Jun 1995 12:26:31 GMT Organization: University of Wuerzburg, Germany Lines: 28 Distribution: world Message-ID: <3t0qhn$9lp@winx03.informatik.uni-wuerzburg.de> References: <199506280251.UAA14992@essex.hsc.colorado.edu> NNTP-Posting-Host: wrzx12.rz.uni-wuerzburg.de X-Newsreader: TIN [version 1.2 PL2] Dave Port (port_d@DEFIANCE.HSC.COLORADO.EDU) wrote: > Is anyone out there aware of any information on hierarchical phosphorylation > of any G-protein coupled receptor? In addition to the reference by Palczewski which I gave in a previous posting I have come across the following paper: @article{pullen:94, author = {Nicholas Pullen and Muhammad Akhtar}, title = {Rhodopsin Kinase: Studies on the Sequence of and the Recognition Motif for Multiphosphorylation.}, journal = {Biochemistry}, volume = 33, pages = {14536--14542}, year = 1994 } These authors phosphorylated peptides corresponding to the C-terminus of rhodopsin and arrived at similar results as Ohguro et al. Hope that helps, --Cornelius. -- /* Cornelius Krasel, Institut f. Pharmakologie, Versbacher Str. 9, D-97078 */ /* Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de */ /* "Science is the game you play with God to find out what His rules are." */