From owner-7tms_r@net.bio.net Sun Nov 20 22:00:00 1994 Path: biosci!BIONET.IG.COM!kristoff From: kristoff@BIONET.IG.COM (David Kristofferson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: test of 7tms_r@net.bio.net Date: 21 Nov 1994 14:15:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Please refrain from posting to the list until we post an all clear message. We are working on setting up the parallel USENET newsgroup and the mailing list at our U.K. site. David Kristofferson BIOSCI/bionet Manager From owner-7tms_r@net.bio.net Sun Nov 20 22:00:00 1994 Path: biosci!biosci!not-for-mail From: kristoff@net.bio.net (David Kristofferson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: test of bionet.molbio.proteins.7tms_r Date: 21 Nov 1994 14:24:01 -0800 Organization: BIOSCI International Newsgroups for Biology Lines: 3 Distribution: world Message-ID: <3ar6m1$585@net.bio.net> NNTP-Posting-Host: net.bio.net Test of the bionet.molbio.proteins.7tms_r USENET newsgroup and the news-to-mail gateway. From owner-7tms_r@net.bio.net Sun Nov 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: David Kristofferson Newsgroups: bionet.molbio.proteins.7tms_r Subject: test of 7tms_r@daresbury.ac.uk Date: 22 Nov 1994 01:50:44 -0000 Lines: 4 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3aripk$6h5@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk test of 7tms_r@daresbury.ac.uk Tetsing the UK mail-to-news gateway. From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!ucsvc.ucs.unimelb.edu.au!U5636655 From: U5636655@ucsvc.ucs.unimelb.edu.au Newsgroups: bionet.molbio.proteins.7tms_r Subject: CHO-K1 cells Date: 22 Nov 1994 14:28:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HJTAN34C828WXKLL@ucsvc.ucs.unimelb.edu.au> NNTP-Posting-Host: net.bio.net In response to Dave Port's query on receptors in CHO-K1 cells. One of the receptors present in this cell line is the calcitonin receptor. Whether it stimulates protein kinase C in this cell line has not been tested, however, CTRs are capable of increasing IP3 levels and mobilising intracelluar calcium and so are presumably capable of stimulating PKC. Patrick Sexton From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!U.WASHINGTON.EDU!nathanso From: nathanso@U.WASHINGTON.EDU (Neil Nathanson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: PLC-coupled receptors in CHO-K1 Date: 22 Nov 1994 14:56:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Askenazi et al (Cell, v.56, 487 [1989]) reported that CHO cells have endogenous CCK and thrombin receptors which activate PLC. Neil M. Nathanson Department of Pharmacology, SJ-30 University of Washington Seattle, WA89195 From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!BIMCORE.EMORY.EDU!medtjm From: medtjm@BIMCORE.EMORY.EDU (T. J. Murphy) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: CHOs Date: 22 Nov 1994 17:50:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411230150.AA27057@bimcore> NNTP-Posting-Host: net.bio.net Dave Port writes: >Does anybody out there know off hand what subtypes of G-protein coupled >receptors are expressed endogenously in CHO-K1 cells? I am interested in >particular if anyone knows if they express significant amouts of an a1-AR >subtype, or if they express any Ang II AT-1s? Actually, any PK-C coupled >receptor would do. No AT1's, in fact, cloned AT1's don't seem to couple well to PLC at all in these cells, see Nature 351:233, 1991. Although that paper had population selected cells, a few clonal cells with good expession (>2pmol/mg membrane protein) weren't any better. Try ATP, it work's well in a lot of places. TJ Murphy Emory University From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!ITSA.UCSF.EDU!sredhar From: sredhar@ITSA.UCSF.EDU (Sunil Sreedharan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: CHOs Date: 22 Nov 1994 19:52:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <9411230150.AA27057@bimcore> NNTP-Posting-Host: net.bio.net There are low levels of type I VIP receptors expressed on CHO K1 cells, as determined by binding studies and by PCR. Although the human receptors couple to increases in intracellular calcium when stably transfected in CHO cells, the endogenous receptors show no noticible effect on intracellular calcium (or cAMP) levels when activated. ________________________________________________________________________ Sunil P. Sreedharan, Ph.D. Assistant Professor, UCSF Div. of Allergy & Immunology Dept. of Medicine, UB8B San Francisco, CA 94143-0711 Tel. (415)476-9895 FAX: (415)476-6915 email: sredhar@itsa.ucsf.edu From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!NEURON1.BERKELEY.EDU!stevens From: stevens@NEURON1.BERKELEY.EDU (Raymond C. Stevens) Newsgroups: bionet.molbio.proteins.7tms_r Subject: 7TM's in yeast Date: 22 Nov 1994 08:44:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411221645.AA10502@neuron1.berkeley.edu> NNTP-Posting-Host: net.bio.net What tricks are neccessary to get mammalian 7TM's to express in yeast? For example, how much of the N-termini of STE2 is needed to get other receptors to function/express in yeast? Thanks for any info, Ray Stevens From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!DEFIANCE.HSC.COLORADO.EDU!port_d From: port_d@DEFIANCE.HSC.COLORADO.EDU ("Dave Port") Newsgroups: bionet.molbio.proteins.7tms_r Subject: CHOs Date: 22 Nov 1994 08:15:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <199411221620.JAA26601@essex.hsc.colorado.edu> NNTP-Posting-Host: net.bio.net Subject: Time:9:14 AM OFFICE MEMO CHOs Date:11/22/94 Does anybody out there know off hand what subtypes of G-protein coupled receptors are expressed endogenously in CHO-K1 cells? I am interested in particular if anyone knows if they express significant amouts of an a1-AR subtype, or if they express any Ang II AT-1s? Actually, any PK-C coupled receptor would do. Thanks Dave Port port_d@defiance.hsc.colorado.edu From owner-7tms_r@net.bio.net Mon Nov 21 22:00:00 1994 Path: biosci!biosci!not-for-mail From: biohelp@net.bio.net (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins.7tms_r Subject: G-PROTEIN-COUPLED-RECEPTOR/bionet.molbio.proteins.7tms_r is ready! Date: 22 Nov 1994 16:41:47 -0800 Organization: BIOSCI International Newsgroups for Biology Lines: 198 Distribution: world Message-ID: <3au34b$3md@net.bio.net> NNTP-Posting-Host: net.bio.net The G-PROTEIN-COUPLED-RECEPTOR/bionet.molbio.proteins.7tms_r newsgroup is ready for operation. PLEASE NOTE that many USENET sites do not allow automatic creation of new USENET groups!!! If you do not see bionet.molbio.proteins.7tms_r in your newsreader within another day or two, ask your news system administrator to act on our "newgroup" message to enable the group at your site. We have already done several tests and are certain that the group is currently propagating around the network. 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Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-7tms_r@net.bio.net Tue Nov 22 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: tensen@chem.vu.nl (Kees Tensen) Newsgroups: bionet.molbio.proteins.7tms_r Subject: endogenous receptors CHO-cells Date: 23 Nov 1994 08:44:29 -0000 Lines: 3 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3auvdd$8aj@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk We use ATP as a positive control for Ca++ mobilisation in CHO-cells (at a concentration of 10-5). From owner-7tms_r@net.bio.net Tue Nov 22 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!news.sprintlink.net!pipex!lyra.csx.cam.ac.uk!NewsWatcher!user From: sjm@mole.bio.cam.ac.uk (Steven J. Mcclue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: first post? Date: Wed, 23 Nov 1994 09:16:15 +0000 Organization: Dept of Genetics, University of Cambridge Lines: 6 Message-ID: NNTP-Posting-Host: cok-mac4.gen.cam.ac.uk X-Newsreader: Value-Added NewsWatcher 2.0b19.1+ Greetings all! Am I the first poster here? If so, I'd like to start the ball rolling by asking whether people believe that the "Quaternary complex" model of Lefkowitz et.al. actually describes what happens to a receptor when it binds an agonist and subsequently activates its G-protein. If not, what are the best alternative models? Steve --------------------------------------------------------------------- How America was saved from communism - Elvis shot JFK... From owner-7tms_r@net.bio.net Tue Nov 22 22:00:00 1994 Path: biosci!SIUMED.EDU!clawyer From: clawyer@SIUMED.EDU (Carl Lawyer) Newsgroups: bionet.molbio.proteins.7tms_r Subject: possible mechs GPLR dysfunction Date: 23 Nov 1994 16:38:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411240034.AA04433@siumed.edu> NNTP-Posting-Host: net.bio.net A variety of human diseases appear to involve signal pathways that act through GPLRs. We may not yet appreciate all the potential mechanisms for dysfunction of G-protein linked receptors. Can GPLR enthusiasts add to, modify, or comment on this list? Potential mechanisms for dysfunction of G-protein linked receptors: * mutations-genomic, somatic * autoimmunity to extracellular loops * dysregulation of: linking to G-proteins phosphorylation glycosylation-routing to proper site in cell membrane, rate of catabolism gene expression variable splicing palmitylation ligand catabolism * G-protein malfunction From owner-7tms_r@net.bio.net Tue Nov 22 22:00:00 1994 Path: biosci!RECEPTOR.MGH.HARVARD.EDU!lfk From: lfk@RECEPTOR.MGH.HARVARD.EDU (Lee F. (Frank) Kolakowski) Newsgroups: bionet.molbio.proteins.7tms_r Subject: TOC Excerpts for 7TMS_R from JBC 11-18->12-9 Date: 23 Nov 1994 19:44:46 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 139 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411240339.AA07317@receptor> NNTP-Posting-Host: net.bio.net AU Becker-Andre-M. Wiesenberg-I. Schaeren-Wiemers-N. Andre-E. Missbach-M. Saurat-J-H. Carlberg-C. TI Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 28531. AU Gao-J-L. Murphy-P-M. TI Human cytomegalovirus open reading frame US28 encodes a functional {beta} chemokine receptor SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 28539. AU Kim-J-Y. Devreotes-P-N. TI Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium. SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 28724. AU Migeon-J-C. Thomas-S-L. Nathanson-N-M. TI Regulation of cAMP-mediated gene transcription by wild type and mutated G-protein {alpha} subunits. Inhibition of adenylyl cyclase activity by muscarinic receptor-activated and constitutively activated G0{alpha}. SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 29146. AU Liu-K. Bergson-C. Levenson-R. Schmauss-C. TI On the origin of mRNA encoding the truncated dopamine D3-type receptor D3nf and detection of D3nf-like immunoreactivity in human brain SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 29220. AU Carruthers-C-J-L. Unson-C-G. Kim-H-N. Sakmar-T-P. TI Synthesis and expression of a gene for the rat glucagon receptor. Replacement of an aspartic acid in the extracellular domain prevents glucagon binding. SO J-Biol-Chem. 1994 Nov. 18. 269(46). p 29321. AU Bozic-C-R. Gerard-N-P. Uexkull-Guldenband-C-. Kolakowski-L-F-Jr.. Conklyn-M-J. Breslow-R. Showell-H-J. Gerard-C. TI The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29355. AU Montero-M. Garcia-Sancho-J. Alvarez-J. TI Activation by chemotactic peptide of a receptor-operated Ca2+ entry pathway in differentiated HL60 cells SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29451. AU Lepretre-N. Mironneau-J. Morel-J-L. TI Both {alpha}1A- and {alpha}2A-adrenoreceptor subtypes stimulate voltage-operated L-type calcium channels in rat portal vein myocytes. Evidence for two distinct transduction pathways. SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29546. AU Ceresa-B-P. Limbird-L-E. TI Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of {alpha}2-adrenergic receptor-G-protein interactions. A negative charge at amino acid residue 79 forecasts {alpha}2A-adrenergic receptor sensitivity to allosteric modulation by monovalent cations and fully effective receptor/G-protein coupling. SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29557. AU Hunt-T-W. Carroll-R-C. Peralta-E-G. TI Heterotrimeric G proteins containing G{alpha}i3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase. SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29565. AU Hawes-B-E. Touhara-K. Kurose-H. Lefkowitz-R-J. Inglese-J. TI Determination of the G{beta}{gamma}-binding domain of phosducin. A regulatable modulator of G{beta}{gamma} signaling. SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29825. AU Kinsella-B-T. O'Mahony-D-J. FitzGerald-G-A. TI Phosphorylation and regulated expression of the human thromboxane A2 receptor SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29914. AU Codina-J. Birnbaumer-L. TI Requirement for intramolecular domain interaction in activation of G protein {alpha} subunit by aluminum fluoride and GDP but not by GTP{gamma}S SO J-Biol-Chem. 1994 Nov. 25. 269(47). p 29339. AU Hjorth-S-A. Adelhorst-K. Pedersen-B-B. Kirk-O. Schwartz-T-W. TI Glucagon and glucagon-like peptide 1: selective receptor recognition via distinct peptide epitopes SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30121. AU Quick-M-W. Simon-M-I. Davidson-N. Lester-H-A. Aragay-A-M. TI Differential coupling of G protein {alpha} subunits to seven-helix receptors expressed in Xenopus oocytes SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30164. AU Xue-J-C. Chen-C. Zhu-J. Kunapuli-S. DeRiel-J-K. Yu-L. Liu-Chen-L-Y. TI Differential binding domains of peptide and non-peptide ligands in the cloned rat {kappa} opioid receptor SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30195. AU Kozell-L-B. Machida-C-A. Neve-R-L. Neve-K-A. TI Chimeric D1/D2 dopamine receptors. Distinct determinants of selective efficacy, potency, and signal transduction. SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30299. AU Yarfitz-S-L. Deer-J-L-R. Froelick-G. Colley-N-J. Hurley-J-B. TI In situ assay of light-stimulated G protein activity in Drosophila photoreceptor G protein {beta} mutants SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30340. AU Kawate-N. Menon-K-M-J. TI Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor. SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30651. AU Gadbut-A-P. Toupin-D-K. Kilbourne-E-J. Galper-J-B. TI Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G{alpha}i2, and the acetylcholine-sensitive K+ channel SO J-Biol-Chem. 1994 Dec. 2. 269(48). p 30707. AU Nelson-E-J. Hinkle-P-M. TI Thyrotropin-releasing hormone activates Ca2+ efflux. Evidence suggesting that a plasma membrane Ca2+ pump is an effector for a G-protein-coupled Ca2+-mobilizing receptor. SO J-Biol-Chem. 1994 Dec. 9. 269(49). p 30854. AU Degtyarev-M-Y. Spiegel-A-M. Jones-T-L-Z. TI Palmitoylation of a G protein {alpha}i subunit requires membrane localization not myristoylation SO J-Biol-Chem. 1994 Dec. 9. 269(49). p 30898. AU Hjorth-S-A. Schambye-H-T. Greenlee-W-J. Schwartz-T-W. TI Identification of peptide binding residues in the extracellular domains of the AT1 receptor SO J-Biol-Chem. 1994 Dec. 9. 269(49). p 30953. From owner-7tms_r@net.bio.net Wed Nov 23 22:00:00 1994 Path: biosci!agate!news.ucdavis.edu!dale!ez040939 From: ez040939@dale.ucdavis.edu (Joel Brockman) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Dumb Newby questions about 7TM's Date: 23 Nov 1994 04:54:33 GMT Organization: University of California, Davis Lines: 22 Message-ID: <3auhu9$8e3@mark.ucdavis.edu> NNTP-Posting-Host: dale.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] 1.) Have any 7TM's been localized to intracellular membranes, or are they always in the plasma membrane? 2.) The literature I've read so far suggests that most 7TM's bind ligands in the transmembrane region. So why do some 7TM's have a large soluble amino terminal end? The cannabinoid receptor in nerve cells (CB1) binds very hydrophobic ligands, but it has about 14 kDa of peptide hanging out on the extracellular side of the membrane. It seems unlikely that this portion of the protein would have a functional role in ligand binding or coupling to G-proteins. Is the cell just wasting energy making this part of the protein, or is it more likely that it has some other function? Please post or e-mail answers. Thanks for your time. Joel Brockman Nobody of particular importance UC Davis From owner-7tms_r@net.bio.net Wed Nov 23 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: tensen@chem.vu.nl (Kees Tensen) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Signal peptide sequences in GPCRs Date: 24 Nov 1994 14:57:27 -0000 Lines: 15 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3b29kn$44m@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk Dear Netters, I'm keen on getting information about the presence of signal peptide (hydrophobic leader) sequences in GPCRs. I know that the glycoproteinhormone receptors are predicted to contain a signal peptide. Are there anymore examples? How can receptor proteins be routed to the plasmamembrane, are any of them predicted to be synthesized on free ribozymes? If so, where are the proteins glycosylated? Finally, when a receptor does contain a signal peptide, is this sequence also necessary for proper heterologous expression in e.g. CHO cells? Thanks ! Kees Tensen From owner-7tms_r@net.bio.net Thu Nov 24 22:00:00 1994 Path: biosci!PO.CWRU.EDU!pre From: pre@PO.CWRU.EDU (Paul R. Ernsberger) Newsgroups: bionet.molbio.proteins.7tms_r Subject: The 7TMS_R orphanage Date: 25 Nov 1994 08:25:10 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: <199411251625.LAA07412@owl.INS.CWRU.Edu> Reply-To: pre@po.CWRU.Edu (Paul R. Ernsberger) NNTP-Posting-Host: net.bio.net > >>4) Biological Resources >> a) distribution of receptor types in cell lines >> b) distribution of g-protein types in cell lines >> c) pointers to transgenic and targeted disruptions of relevant genes >> >> >>If any of these topics are available already, please forward them to >>me. I don't want to do this unless you want this also. >> >>Thanks >> >>Frank Kolakowski >> > >A couple of lists/questions that have been discussed would be worth adding >to the Biological Resources section of the FAQ since they will continue to >be asked and a current update would be helpful for all: the uncloned >receptors that are known (and suspected?)to be GPCRs, and the known gene >structures (perhaps grouped by number of exons in coding sequence with >notes as to additional noncoding exons which may not be as well >characterized). > >Tom I. Bonner To that I would add a database of known and suspected orphan GPCR's with data on their cell distribution as well as sequence. Someone posted a message on this topic some time ago. -- Paul Ernsberger, Ph.D., Depts. of Medicine and Neuroscience Case Western Reserve University School of Medicine Cleveland, OH 44106-4982//pre@po.cwru.edu//(216)368-4738 phone (216)368-4752 telefax **Receptor Neuropharmacology** From owner-7tms_r@net.bio.net Fri Nov 25 22:00:00 1994 Path: biosci!MC.DUKE.EDU!PREMO003 From: PREMO003@MC.DUKE.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Dumb Newby questions Date: 26 Nov 1994 12:35:10 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 47 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HJXUWMLQFM00125K@mc.duke.edu> NNTP-Posting-Host: net.bio.net >Joel Brockman asks: >1.) Have any 7TM's been localized to intracellular membranes, or are >they always in the plasma membrane? Rhodopsin and the color iodopsins are the premiere examples of non-plasma membrane GPCRs. These are localized to intradiscal membranes in the retinal rod (or cone) outer segments. In addition, many laboratories have found that putative olfactory receptors expressed in many cell lines are localized to intracellular compartments. This is why they remain "putatives". One possibility is that they are consistently misfolded in non-olfactory cells and never mature to a plasma membrane form. In the olfactory neuroepithelium, these receptors are thought to be found only in the apical cilia, and perhaps they need a targeting or folding companion not found in COS cells, etc. An alternative is that they are supposed to be found within the cell - after all, odorants are often membrane permeant. G protein subunits have been reported to be associated with Golgi membranes (particular Gi-alpha), so perhaps there are some receptor-like proteins there as well. >2.) The literature I've read so far suggests that most 7TM's bind >ligands in the transmembrane region. So why do some 7TM's have a large >soluble amino terminal end? The cannabinoid receptor in nerve cells (CB1) >binds very hydrophobic ligands, but it has about 14 kDa of peptide >hanging out on the extracellular side of the membrane. It seems unlikely >that this portion of the protein would have a functional role in ligand >binding or coupling to G-proteins. Is the cell just wasting energy >making this part of the protein, or is it more likely that it has some >other function? Several classes of receptors have a large extracellular domain, and it certainly plays a functional role. Mutations in extracellular conserved residues in the GHRH receptor and in the parathyroid Ca2+-sensing receptor render them nonfunctional and lead to disease states. I'm sure there are also examples of in vitro mutations, but I don't recall which receptors offhand. I think that the general emphasis on binding within the membrane spans is not always correct. It is an artefact of the identification of receptors for small ligands first: rhodopsin, b-adrenergic, muscarinic. The intramembrane span pocket simply is not large enough for a large polypeptide or glycoprotein hormone to bind there. I think that the extracellular domains allow binding and orientation of the ligand so that only a small portion of it (or even of a part of the receptor, ala thrombin) is presented to the membrane regions to achieve an activated conformation. Richard Premont Dept Medicine, Box 3821 Duke University Medical Center From owner-7tms_r@net.bio.net Fri Nov 25 22:00:00 1994 Path: biosci!MC.DUKE.EDU!PREMO003 From: PREMO003@MC.DUKE.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Signal peptide sequences in GPCRs Date: 26 Nov 1994 12:55:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 37 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HJXVM2EFIA00125K@mc.duke.edu> NNTP-Posting-Host: net.bio.net >Kees Tensen asked: >>I'm keen on getting information about the presence of signal peptide >(hydrophobic leader) sequences in GPCRs. I know that the >glycoprotein hormone receptors are predicted to contain a signal peptide. >Are there any more examples? How can receptor proteins be routed to the >plasma membrane, are any of them predicted to be synthesized on free >ribozymes? If so, where are the proteins glycosylated? Finally, when a >receptor does contain a signal peptide, is this sequence also necessary for >proper heterologous expression in e.g. CHO cells? Many receptors contain signal sequences. Essentially all receptors with large extracellular domains do. In particular, all members of the metabotropic glutamate receptor family have signal peptides, as do all members of the GI hormone family (secretin, VIP, glucagon, PACAP, PTH, calcitonin, etc). To my knowledge, no one has removed a signal sequence from a receptor which naturally has one. I suspect it would kill expression. On the other hand, Brian Kobilka did the opposite: he put a signal sequence on the b2-adrenergic receptor, which naturally lacks one. The expression level is significantly increased with the signal sequence. As I understand it, transmembrane proteins without signal leader sequences insert into the ER post-translationally rather than co-translationally. I don't know if this translation is a free ribosome or rough ER event. Post- translationally is most probably the less efficient way to insert, hence the increased expression by adding a leader sequence. Also co-insertion of membrane span pairs is more efficient than single span insertions (one reason for the preponderance of channels, etc with an even number of spans). The span orientation is decided by the charge distribution at the ends of the hydrophobic stretches which will be membrane spans. Nevertheless, once inserted into the ER, these receptor proteins will be glycosylated just as would any other ER resident protein. Hope this helps clarify things. It is still a murky area. Richard Premont Dept Medicine, Box 3821 Duke University Medical Center From owner-7tms_r@net.bio.net Sun Nov 27 22:00:00 1994 Path: biosci!PT.CYANAMID.COM!hadcockj From: hadcockj@PT.CYANAMID.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: possible mechs GPLR dysfunction Date: 28 Nov 1994 12:40:30 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 37 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HK0NPFR23U8WZ1ZP@ptag2.pt.Cyanamid.COM> NNTP-Posting-Host: net.bio.net I believe that down-regulation of GPCRs can also play an integral role in human disease states. For example, in chronic heart failure, a pronounced decrease in beta1-adrenergic receptors is observed. This is probably due to increased norepinephrine levels which down regulate beta1-adrenergic receptor mRNA levels as well as desensitizing the system. This can also be a big problem in therapies involiving GPCRs. In addition antagonist treament can lead to supersensitization (e.g. propranolol hypersensitivity). John Hadcock Hadcockj@pt.cyanamid.com ______________________________ Reply Separator _________________________________ Subject: possible mechs GPLR dysfunction Author: clawyer@siumed.edu at GATEWAY Date: 11/23/94 7:38 PM A variety of human diseases appear to involve signal pathways that act through GPLRs. We may not yet appreciate all the potential mechanisms for dysfunction of G-protein linked receptors. Can GPLR enthusiasts add to, modify, or comment on this list? Potential mechanisms for dysfunction of G-protein linked receptors: * mutations-genomic, somatic * autoimmunity to extracellular loops * dysregulation of: linking to G-proteins phosphorylation glycosylation-routing to proper site in cell membrane, rate of catabolism gene expression variable splicing palmitylation ligand catabolism * G-protein malfunction From owner-7tms_r@net.bio.net Sun Nov 27 22:00:00 1994 Path: biosci!ROMEO.CALTECH.EDU!ajwatson From: ajwatson@ROMEO.CALTECH.EDU (John Watson) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Newsgroup created yet? Date: 28 Nov 1994 09:36:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HK0AYH35W2B7WNJN@HAMLET.CALTECH.EDU> NNTP-Posting-Host: net.bio.net The G-protein coupled receptor newsgroup bionet.molbio.proteins.7tms_r has not yet shown up at my site. Has it appeared anywhere yet? John /--------------------------------\ \--------------------------------/ || A. John Watson, Ph.D. || || Senior Research Fellow || || Caltech Biology, 147-75 || || Pasadena, CA 91125 || || (818) 395-3781 || || (818) 796-7066 fax || || ajwatson@romeo.caltech.edu || /--------------------------------\ \--------------------------------/ From owner-7tms_r@net.bio.net Sun Nov 27 22:00:00 1994 Path: biosci!agate!news.ucdavis.edu!dale!ez042951 From: ez042951@dale.ucdavis.edu (Chris Haskell) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: possible mechs GPLR dysfunction Date: 28 Nov 1994 16:33:40 GMT Organization: University of California, Davis Lines: 15 Distribution: world Message-ID: <3bd0p4$81r@mark.ucdavis.edu> References: <9411240034.AA04433@siumed.edu> NNTP-Posting-Host: dale.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] Carl Lawyer (clawyer@SIUMED.EDU) wrote: : A variety of human diseases appear to involve signal pathways that act : through GPLRs. We may not yet appreciate all the potential mechanisms for : dysfunction of G-protein linked receptors. : Can GPLR enthusiasts add to, modify, or comment on this list? ..... Desensitization. While this could be a malfunction in phosphorylation, it could also involve other interactions. Chris cahaskell@ucdavis.edu From owner-7tms_r@net.bio.net Mon Nov 28 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.cc.swarthmore.edu!netnews.upenn.edu!msuinfo!uwm.edu!news.doit.wisc.edu!sweetprotein.ahabs.wisc.edu!user From: ming@ahabs.wisc.edu (Ding Ming) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Expression systems for G protein coupled receptors Date: Tue, 29 Nov 1994 10:54:23 -0600 Organization: University of Wisconsin-Madison Lines: 11 Message-ID: NNTP-Posting-Host: sweetprotein.ahabs.wisc.edu Hello, netters, I was discussing with a professor about the expression system for G protein coupled receptors designed to identify 7tms_r by expression cloning. He was arguing that traditional expression systems, e.g., COS cells, used for FACS or panning might not sensitive enough to detect the 7tms_r in a cDNA library, and some amplification are definitely needed to hunt for the receptors. Except Xenopus oocytes and renal epithelial cells, are there any other systems suitable for this purpose? Is the amplification process necessary for this kind of work? I would appreciate your experience and ideas. From owner-7tms_r@net.bio.net Mon Nov 28 22:00:00 1994 Path: biosci!aa.wl.com!levineh From: levineh@aa.wl.com (LEVINEH) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Streaking in SDS-PAGE Date: 29 Nov 1994 11:06:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <0213301329111994.A05763.DOMLAB.118BEB5E0B00*@MHS> References: NNTP-Posting-Host: net.bio.net To reduce streaking and laddering of GPR's don't boil the sample in SDS, just leave at room temperature in sample buffer for 10-15 min. This aggregation phenomenon is very prominent for rhodopsin in ROS preps. From owner-7tms_r@net.bio.net Mon Nov 28 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!uunet!newstf01.news.aol.com!newsbf01.news.aol.com!not-for-mail From: biobone@aol.com (Bio Bone) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Monoclonal Abs to 7TM Receptors Date: 29 Nov 1994 13:40:19 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 9 Sender: news@newsbf01.news.aol.com Message-ID: <3bfsij$jlg@newsbf01.news.aol.com> NNTP-Posting-Host: newsbf01.news.aol.com I would be interested in knowing if anyone has experience in making monoclonal antibodies to any of the 7TM receptors. I am currently involved in making Mabs to a number of these receptors and would appreciate any advise that could be given. In particular, I would especially like to know if anyone has made antibodies recognizing the native extracellular loops of these receptors. Bradley Bone COR Therapeutics, Inc. From owner-7tms_r@net.bio.net Mon Nov 28 22:00:00 1994 Path: biosci!WELCHLINK.WELCH.JHU.EDU!bjmarg From: bjmarg@WELCHLINK.WELCH.JHU.EDU (BARRY J MARGULIES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Streaking in SDS-PAGE Date: 29 Nov 1994 09:33:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Is there a way to prevent/reduce the tendency of GCR proteins to streak during SDS-PAGE? I have heard that diluting the sample before adding solubilizing buffer can help, but this has not totally eliminated the problem. Thanks in advance! -Barry (bjmarg@welchlink.welch.jhu.edu) xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx GEORGIA CONFEDERATE SOLDIERS from a monument at Antietam National Battlefield, just south We sleep here in obedience to law. of The Cornfield. When duty called, we came. And no, I'm not from Georgia, When country called, we died. I just thought it was cool. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx From owner-7tms_r@net.bio.net Tue Nov 29 22:00:00 1994 Path: biosci!MRC.COM!dave From: dave@MRC.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 30 Nov 1994 07:20:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411301518.AA15447@mrcs1> NNTP-Posting-Host: net.bio.net I am starting to construct models of GPCR's and am looking for some software that may prove helpful. Specifically, I am interested in something which will calculate hydrophobic moment vectors for helices, given the primary sequence for that stretch of residues. Something which uses any of the commonly accepted constants for the amino acids (i.e., Janin) would be acceptable. I realize that this is a relatively minor programming task, but why re-invent the wheel. In a similar vein, is anyone aware of code which will calculate a "conservation" moment vector in a similar fashion. That is, it will generate a vector based on degree of residue conservation in a given group of helices. Thanks in advance for any assistance..... Dave ---------------------------------------------------------------- David Hartsough dave@mrc.com Structural Chemistry Miles Research Center 400 Morgan Lane Phone: (203)937-2982 West Haven CT 06516 Fax: (203)937-2650 ---------------------------------------------------------------- The opinions expressed herein are solely those of the author and are not necessarily those of Miles Inc. ---------------------------------------------------------------- From owner-7tms_r@net.bio.net Tue Nov 29 22:00:00 1994 Path: biosci!CS.MONTANA.EDU!jones From: jones@CS.MONTANA.EDU (Chris Jones) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Looking for a good database Date: 30 Nov 1994 08:14:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <9411301614.AA20995@cs.montana.edu> NNTP-Posting-Host: net.bio.net Hi all-- I'm looking for a good database of GCRs aligned by transmembrane segments and sorted by sub-family. Alternatively, I could correlate data from different sources to build such a database, but there's not sense in re-inventing the wheel; if anybody knows of such a database out there somewhere, could you please let me know? Thanks in advance. Chris -- ************************jones@fubar.cs.montana.edu************************ * Chris | "Just say 'NO!' to entropy!" * * Jones | * ************************************************************************** From owner-7tms_r@net.bio.net Tue Nov 29 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!swiss.ans.net!newstf01.news.aol.com!newsbf01.news.aol.com!not-for-mail From: biobone@aol.com (Bio Bone) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Streaking in SDS-PAGE Date: 30 Nov 1994 13:18:08 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 5 Sender: news@newsbf01.news.aol.com Message-ID: <3bifl0$8te@newsbf01.news.aol.com> References: <0213301329111994.A05763.DOMLAB.118BEB5E0B00*@MHS> NNTP-Posting-Host: newsbf01.news.aol.com In article <0213301329111994.A05763.DOMLAB.118BEB5E0B00*@MHS>, levineh@aa.wl.com (LEVINEH) writes: In addition to not boiling the samples, using a Urea sample buffer seems to help. From owner-7tms_r@net.bio.net Tue Nov 29 22:00:00 1994 Path: biosci!SHRSYS.HSLC.ORG!wthomas From: wthomas@SHRSYS.HSLC.ORG Newsgroups: bionet.molbio.proteins.7tms_r Subject: GPCR databases Date: 30 Nov 1994 11:18:26 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <009883E5.B26D7ED7.7@shrsys.hslc.org> NNTP-Posting-Host: net.bio.net Chris Jones asked about GPCR alignment databases. The TM7 database in Heidelberg is very useful. For information, check out TM7@EMBL-Heidelberg.de or ftp.EMBL-Heidelberg.de Wally Thomas Weis Center for Research Danville, PA Wthomas@shrsys.hslc.org From owner-7tms_r@net.bio.net Wed Nov 30 22:00:00 1994 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: GPCR models available Date: 1 Dec 1994 06:19:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <20401DB7_000E5C60.009884AEC9817460$17_2@UK.AC.LEEDS.BIO.VAX> If anybody would like a copy of my GPCR models (Receptor and Channels (1994) 2: 61-78) then please send me an E_mail. Best Regards, Dan Donnelly University of Leeds, U.K. From owner-7tms_r@net.bio.net Wed Nov 30 22:00:00 1994 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: PERSCAN programs Date: 1 Dec 1994 06:19:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <20401DB7_000E6FD8.009884AEB4FEE7C0$17_1@UK.AC.LEEDS.BIO.VAX> David Hartsough asked about programs for calculating conserved faces for predicted transmembrane helices etc. I have written a group of programs called PERSCAN which will calculate these properties. The programs are completely free and will run on sgi/sun workstations and also on VAX/VMS machines. The output is PostScript. If anyone wants a copy then please E_mail me and I will return a 'uuencoded' file containing all the code, manual and example input files. The relevant references are Donnelly et al. FEBS Lett (1989) 251: 109-116 Prot. Sci. (1993) 2: 55-70 Prot. Engng. (1993) 6: 629-635 Receptor & Channels (1994) 2: 61-78 Prot. Engng. (1994) 7: 645-653 Curr, Opinion in Struct. Biol. (1994) 4: 582-589 Dan Donnelly University of Leeds, UK From owner-7tms_r@net.bio.net Wed Nov 30 22:00:00 1994 Path: biosci!WELCHLINK.WELCH.JHU.EDU!bjmarg From: bjmarg@WELCHLINK.WELCH.JHU.EDU (BARRY J MARGULIES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Using PNGase F with GCRs Date: 1 Dec 1994 12:48:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I have another question, still dealing with streaking in SDS-PAGE (same question I asked a few days ago); I'm interested in using PNGase F to see if some of the streaking is due to N-linked glycosylation. However, the protocol I have for PNGase treatment requires boiling in SDS to denature the glycoprotein before addition of NP-40 (to modulate the denaturing effect of the SDS on the PNGase) and the PNGase F. Now, I know that boiling GCRs in SDS will cause them to aggregate, and I obviously want to avoid this if I'm trying to resolve a difference with the PNGase treatment using SDS-PAGE. The question: Does anyone have experience treating GCRs with PNGase and subsequently resolving the products in SDS-PAGE, and NOT using boiling techniques? More simply, does anyone have a protocol for PNGase F treatment of GCRs? Thanks in advance, again. -Barry (bjmarg@welchlink.welch.jhu.edu) From owner-7tms_r@net.bio.net Wed Nov 30 22:00:00 1994 Path: biosci!PT.CYANAMID.COM!hadcockj From: hadcockj@PT.CYANAMID.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Streaking in SDS-PAGE Date: 1 Dec 1994 10:03:43 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HK4P4R2C7Q8X03LL@ptag2.pt.Cyanamid.COM> We do not boil our samples. Instead, we reduce with 20 mM DTT (30 min)at 37oC in sample buffer and then alkylate (45 mM Iodoacetamide) for 15 min at room temp. This seems to help with the streaking problems. John Hadcock Hadcockj@pt.cyanamid.com ______________________________ Reply Separator _________________________________ Subject: Streaking in SDS-PAGE Author: bjmarg@welchlink.welch.jhu.edu at GATEWAY Date: 11/29/94 12:33 PM Is there a way to prevent/reduce the tendency of GCR proteins to streak during SDS-PAGE? I have heard that diluting the sample before adding solubilizing buffer can help, but this has not totally eliminated the problem. Thanks in advance! -Barry (bjmarg@welchlink.welch.jhu.edu) xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx GEORGIA CONFEDERATE SOLDIERS from a monument at Antietam National Battlefield, just south We sleep here in obedience to law. of The Cornfield. When duty called, we came. And no, I'm not from Georgia, When country called, we died. I just thought it was cool. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx