From owner-7tms_r@net.bio.net Sun Jan 01 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!news.starnet.net!wupost!howland.reston.ans.net!pipex!uunet!newstf01.news.aol.com!not-for-mail From: danielbram@aol.com (Danielbram) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Nice serpent pictures Date: 2 Jan 1995 11:30:13 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 19 Sender: root@newsbf02.news.aol.com Message-ID: <3e99ml$i8a@newsbf02.news.aol.com> References: <3df1a2$5sc@lastactionhero.rs.itd.umich.edu> Reply-To: danielbram@aol.com (Danielbram) Rick The pictures your referred to in your Dec, 23 posting can be produced relatively easily in most graphics software packages. The software I use is MSWorks and Canvas for PC and Mac as well as Showcase for UNIX applications. Any of these packages can be used to produce the pictures you desire in about 1 to 1.5hrs. I have found it most helpful to 1) make one circle then use Edit|Copy|Paste to reproduce the circle, I then repeat the process of Edit|Copy|Paste over so that I now have four circles etc.. When 20 circles are available I, 2) place each amino acid into an individual circle and group the letter and circle. Next 3) use the Edit|Copy|Paste to reproduce each circled amino acid untill there is approximently 30 of each amino acid. When I start to build the protein structure all I have to do is select the proper circled amino acid and place it accordingly. IMHO this is an easy way to produce these types of pictures. Feel free to email me if you have any questions. Daniel Kennesaw State College From owner-7tms_r@net.bio.net Mon Jan 02 22:00:00 1995 Path: biosci!MEMBRAN1.WEIZMANN.AC.IL!bmweis From: bmweis@MEMBRAN1.WEIZMANN.AC.IL (yehudit weisinger) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 3 Jan 1995 01:20:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501030821.AA27931@membran1.weizmann.ac.il> NNTP-Posting-Host: net.bio.net SUBSCRIBE -- Sincerly, Yehudit Weisinger BMWEIS@MEMBRAN1.WEIZMANN.AC.IL From owner-7tms_r@net.bio.net Mon Jan 02 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: Cornelius Krasel Newsgroups: bionet.molbio.proteins.7tms_r Subject: Internalization (was Re: carboxy-tails) Date: 3 Jan 1995 19:45:41 -0000 Lines: 42 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3ec9h5$jgm@mserv1.dl.ac.uk> Original-To: 7tms_r@dl.ac.uk Dan Strahs (strahs@vaxa.crc.mssm.edu) wrote: > The asparagine of > the "NPxxY" motif in helix 7 has been shown to interact with the conserved > aspartate in helix 2 (Zhou et al. (1994) Mol. Pharm. 45(2), 165-170). While > the Lefkowitz's lab interpretation of the conserved tyrosine in this motif as ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > required for sequestration is probably correct, it is not required that it ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > be in the cytoplasm. I am not so sure about this. There are IMHO at least two papers who challenge this interpretation. Keefer et al. (JBC 269, 16425-16433) have shown that the Y in the NPxxY motif is not required for transcytosis of the alpha2-AR in MDCK cells but seems to impair proper folding of the receptor. Slice et al. (JBC 269, 21755-21762) looked at the sequestration of a mutant of the Gastrin-releasing peptide receptor (the tyrosine at the end of the 7th TM helix occurs here in a NPxxxY context) and did not find any requirement for a tyrosine. (There might even be the possibility that there is no universal internalization mechanism for 7TMH receptors since Lefkowitz' group has shown that phosphorylation is not required for sequestration of the b2AR, but it is apparently required for mAChR sequestration.) I think it is quite safe to assume this region to be an alpha helix (see also a modelling study by Vriend and others, published last year in TIPS which specifically discusses the role of this tyrosine residue in signal transduction) but the question is how far this helix extends further into the cytoplasm. Maybe it would possible to crystallize the C-terminal tail since it has been shown that it can be phosphorylated by GRKs when expressed as a GST fusion protein; so it might be that it has a quite stable conformation. BTW, it is just in Germany that news distribution is so far behind email distribution? In our newsgroup, articles appear at least two days late. Just a few random thoughts, --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!VAXA.CRC.MSSM.EDU!STRAHS From: STRAHS@VAXA.CRC.MSSM.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: RE: Internalization (was Re: carboxy-tails) Date: 4 Jan 1995 13:45:38 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 66 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501042145.NAA27453@net.bio.net> Cornelius Krasel (krasel@alf.biochem.mpg.de) wrote: >Dan Strahs (strahs@vaxa.crc.mssm.edu) wrote: > >> The asparagine of >> the "NPxxY" motif in helix 7 has been shown to interact with the conserved >> aspartate in helix 2 (Zhou et al. (1994) Mol. Pharm. 45(2), 165-170). While >> the Lefkowitz's lab interpretation of the conserved tyrosine in this motif as > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >> required for sequestration is probably correct, it is not required that it > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >> be in the cytoplasm. >I am not so sure about this. There are IMHO at least two papers who challenge >this interpretation. Keefer et al. (JBC 269, 16425-16433) have shown that >the Y in the NPxxY motif is not required for transcytosis of the alpha2-AR >in MDCK cells but seems to impair proper folding of the receptor. Slice et >al. (JBC 269, 21755-21762) looked at the sequestration of a mutant of the >Gastrin-releasing peptide receptor (the tyrosine at the end of the 7th TM >helix occurs here in a NPxxxY context) and did not find any requirement for >a tyrosine. (There might even be the possibility that there is no universal >internalization mechanism for 7TMH receptors since Lefkowitz' group has >shown that phosphorylation is not required for sequestration of the b2AR, >but it is apparently required for mAChR sequestration.) To address the last point first, I agree that there is probably no universal mechanism for internalization. However, common mechanisms may well exist among receptors of high homology, such as the catecholaminergic neurotransmitter GPCRs. However, a case for a conserved mechanism for internalization via Y7.53 has yet to be made. I'm not sure that the papers detailed above adequately dispute the Lefkowitz lab contention (and by the implication that I'm arguing it, my opinion/contention). Although internalization may have been measured for the mutant Y7.53(426)A alpha-2A adrenergic receptor (per private conversation with Dr. Limbird), this is not discussed in the paper. Moreover, the alpha-2A receptor possesses unique sequences in the third intracellular loop, which appear to stabilize the expression of the receptor at the basolateral membrane. It's not clear that the internalization measured in the paper corresponds to agonist-stimulated sequestration, which, in any event, may be directly inhibited in this receptor by the third intracellular loop sequences. The GRP receptor is divergent; most GPCR's have the sequence NPxxY while this receptor is NPxxxY. This variation is a conserved variation in two receptor families (the gastrin and endothelin receptors) and does not ever occur in any other family (I'm basing this on a quick analysis of 250+ GPCR sequences). This conserved variation in a highly conserved (very close to 100%) residue argues that this region probably serves a different purpose in these two families. The title of the paper in question should probably have been worded as 'The Conserved NPX(3)Y Motif ... Is Not A General ^^^^^^^ Sequestration Sequence' rather than as 'The Conserved NPX(n)Y Motif ... Is Not ^^^^^^^ A General Sequestration Sequence'. There is certainly more work here to be done... the Lefkowitz lab results may hold only in the beta-2 receptor or in CHO cells. Even more remarkable than their results is that so little attention has been paid to this conserved residue. Other extremely notable exceptions where completely conserved residues have been ignored or practically ignored include Gly (G1.49), Asn (N1.50), Trp (W4.50), Leu (L2.46), Trp (W6.48) and Cys (C6.47). Dan Strahs From owner-7tms_r@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: Helix 7 & C-terminal tails etc. Date: 4 Jan 1995 02:58:32 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <20408D40_000E2D00.00989F49CF598840$11_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net Cornelius Krasel wrote: "I think it is quite safe to assume that this region (i.e. the NPxxY region of helix 7) to be alpha helix (...) but the question is how far this helix extends further into the cytoplasm". If you build a GPCR model based on bacteriorhodopsin using an alignment in which helix 7 of the GPCR is aligned to that of bR by assuming that the retinal attachment points in rhodopsin and bacteriorhodopsin are at equivalent positions, then by definition the NPxxY region is at the cytoplasmic end of the helix. bR NIETLLFMVLDVSAKVGFGLILLRS Rhod PIFMTIPAFFAKSAAIYNPVIY * ^^ ^ However, this is a rather large assumption. Our analysis of GPCR sequence alignments suggests that the retinal attachment poit in rhodopsin is close to the extracellular side of the bilayer (Receptor and Channels 1994, 2: 61- 78). This places the NPxxY sequence middway through the bilayer rather than at the cytoplamic end. Happy New Year, Dan Donnelly bmb6hdd@biovax.leeds.ac.uk University of Leeds, U.K. From owner-7tms_r@net.bio.net Tue Jan 03 22:00:00 1995 Path: biosci!WEIZMANN.WEIZMANN.AC.IL!BMNATOCH From: BMNATOCH@WEIZMANN.WEIZMANN.AC.IL Newsgroups: bionet.molbio.proteins.7tms_r Subject: Subscribe Date: 4 Jan 1995 01:11:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <950104.111146.+0200.BMNATOCH@WEIZMANN.WEIZMANN.AC.IL> Subcribe From owner-7tms_r@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!merck.de!rippmann From: rippmann@merck.de (Friedrich Rippmann) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Helix 7 & C-terminal tails etc. Date: 5 Jan 1995 00:33:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 230 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501050832.AAA27818@net.bio.net> References: <20408D40_000E2D00.00989F49CF598840$11_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net >If you build a GPCR model based on bacteriorhodopsin using an alignment >in which helix 7 of the GPCR is aligned to that of bR by assuming that >the retinal attachment points in rhodopsin and bacteriorhodopsin are at >equivalent positions, then by definition the NPxxY region is at the cytoplasmic >end of the helix. > >bR NIETLLFMVLDVSAKVGFGLILLRS >Rhod PIFMTIPAFFAKSAAIYNPVIY > * ^^ ^ > >However, this is a rather large assumption. Our analysis of GPCR sequence >alignments suggests that the retinal attachment poit in rhodopsin is close >to the extracellular side of the bilayer (Receptor and Channels 1994, 2: 61- >78). This places the NPxxY sequence middway through the bilayer rather than >at the cytoplamic end. Dan raises a very interesting point: Should we rely on the equivalence of Lys positions (the retinal attachment point) in bacteriorhodopsin and rhodopsin? His sequence alignment analysis, and also mine, included below, indicate that the idea of a positional equivalence is probably not a good one, and a few authors have in fact shifted the alignment in helix 7 so that there is nolonger a Lys in corresponding positions (e.g Kuipers94 = Kuipers, W., Van Wijngarden, I., IJzerman, A.P., A model of the 5-HT1A receptor: Agonist and antagonist binding sites, Drug Design and Discovery, 11(3), 231-249, 1994; Cronet_93 = Cronet, P., Sander, C., Vriend, G., Modeling of transmembrane seven helix bundles, Prot. Eng., 6, 59-64, 1993). Especially Cronet's paper is interesting, as it uses a semi-automatic method ('environmental preference parameters') to fit the sequence onto the bacteriorhodopsin fold. The underlying problem here is the question of an evolutionary relationship of bacteriorhodopsin and rhodopsin. If there is, I would expect the Lys in equivalent positions, and could use the bacteriorhodopsin fold for modelling GPCRs. If there is not, as the alignments indicate for helix 7, I should forget about bacteriorhodopsin as a useful template entirely. Are there any good arguments out there for an evolutionary relationship, or is there just some distant topological relationship between bacteriorhodopsin and rhodopsin? Are there membrane proteins with six helices, or eight? Are they as 'homologous' to GPCRs as bacteriorhodopsin is to rhodopsin? - Friedrich - ------------------------------------------------------------------------------- Dr. Friedrich Rippmann work: home: c/o E. MERCK Schroederstrasse 72 Med. Chemistry/Drug Design 69120 Heidelberg 64271 Darmstadt, Germany +49-6221-413366 +49-6151-726290 Email: rippmann@merck.de Fax ... 782149 -------------------------------------------------------------------------------- What follows is a sequence alignment for helix 7 of almost all human GPCRs, underlined with 'xxxxxxx' where the method of Jones et al. (Jones, D.T.,Taylor, W.R.,Thornton, J.M.,A model recognition approach to the prediction of all-helical membrane protein structure and topology, Biochemistry,33,3038-3049,1994)[program MEMSAT] predicts the transmembrane stretch for an individual sequence. On top of this, I have added the alignment relative to bacteriorhodopsin, as published by various authors. Most authors rely on the equivalence in Lys positions in bacteriorhodopsin and rhodopsin. %Kuipers94 --------NIETLLFMVLDVSAKVGFGLILLRE %Rippmann_94 ---NIETLLFMVLDVSAKVGFGLILLRE~~~~~ %Vriend ---NIETLLFMVLDVSAKVGFGLILLR-~~~~~ %Cronet_93 -------NIETLLFMVLDVSAKVGFGLILLRE~ %Hibert_91 ---NIETLLFMVLDVSAKVGFGLILLR-~~~~~ %Trumpp_92 ---NIETLLFMVLDVSAKVGFGLILLR-~~~~~ %Westkae93 ---NIETLLFMVLDVSAKVGFGLILLR-~~~~~ %Mc4r_Human -ycvcfmshfnlylilimcnsiidpliyalrsq %Mc4r_Pred -----------xxxxxxxxxxxxxxxxxxx %Mc5r_Human -ycsrfmshfnmylilimcnsvmdpliyafrsq %Mc5r_Pred -----------xxxxxxxxxxxxxxxxxxx %Actr_Human -ycacymslfqvngmlimcnavidpfiyafrsp %Actr_Pred -------------xxxxxxxxxxxxxxxxx %Mshr_Human -tcgcifknfnlflaliicnaiidpliyafhsq %Mshr_Pred -----------xxxxxxxxxxxxxxxxxxx %Canr_Human ----liktvfafcsmlcllnstvnpiiyalrsk~ %Canr_Pred --------xxxxxxxxxxxxxxxxxxxxxx %Edg1_Human -cdilfraeyfl--vlavlnsgtnpiiytltnk~ %Edg1_Pred ---------xxx--xxxxxxxxxxxxxxxx %Aa2a_Human --shaplwlmylaivlshtnsvvnpfiyayrir~ %Aa2a_Pred ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ %Aa2b_Human --knkpkwamnmaillshansvvnpivyayrnr~ %Aa2b_Pred -----------xxxxxxxxxxxxxxxxxxx %Aa1r_Human --chkpsiltyiaiflthgnsamnpivyafriq %Aa1r_Pred -------xxxxxxxxxxxxxxxxxxxxxxx %5h2a_Human ---dvigallnvfvwigylssavnplvytlfnk~ %5h2a_Pred ----xxxxxxxxxxxxxxxxxxxxxxxx %5h2c_Human ---klmekllnvfvwigyvcsginplvytlfnk~ %5h2c_Pred -----------xxxxxxxxxxxxxxxxxxxx %Dadr_Human --fcidsntfdvfvwfgwansslnpiiy-afna~ %Dadr_Pred -----------xxxxxxxxxxxxxxxxx-xx %Dbdr_Human gfpcvsettfdvfvwfgwansslnpviy-afna~ %Dbdr_Pred ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ %B1ar_Human ----vpdrlfvffnwlgyansafnpiiy-crsp~ %B1ar_Pred --------xxxxxxxxxxxxxxxxxxxx-x %B2ar_Human ----irkevyillnwigyvnsgfnpliy-crsp~ %B2ar_Pred ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ %B3ar_Human ----vpgpaflalnwlgyansafnpliy-crsp~ %B3ar_Pred --------xxxxxxxxxxxxxxxxxxxx-x %A1aa_Human ----psegvfkvifwlgyfnscvnpliypcssr~ %A1aa_Pred -----------xxxxxxxxxxxxxxxxx %Hh2r_Human ----inevleaivlwlgyansalnpilyaalnr~ %Hh2r_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Acm2_Human ----ipntvwtigywlcyinstinpacyalcna %Acm2_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Acm4_Human ----ipdtvwsigywlcyvnstinpacyalcna %Acm4_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Acm1_Human ----vpetlwelgywlcyvnstinpmcyalcnk %Acm1_Pred -----------xxxxxxxxxxxxxxxxxxxx %Acm3_Human ----ipktfwnlgywlcyinstvnpvcyalcnk %Acm3_Pred -----------xxxxxxxxxxxxxxxxxxxx %Acm5_Human ----vpvtlwhlgywlcyvnstvnpicyalcnr %Acm5_Pred -----------xxxxxxxxxxxxxxxxxxxx %D2dr_Human --cnippvlysaftwlgyvnsavnpiiyttfni %D2dr_Pred -------xxxxxxxxxxxxxxxxxxxxx %D4dr_Human --csvpprlvsavtwlgyvnsalnpviytvfna %D4dr_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %A2aa_Human --csvprtlfkfffwfgycnsslnpviytifnh %A2aa_Pred -----------xxxxxxxxxxxxxxxxxxxx %A2ac_Human --cqvpgplfkfffwigycnsslnpviytvfnq %A2ac_Pred -----------xxxxxxxxxxxxxxxxxxxx %A2ab_Human --ckvphglfqfffwigycnsslnpviytifnq %A2ab_Pred -----------xxxxxxxxxxxxxxxxxxxx %5h1b_Human --cwfhlaifdfftwlgylnslinpiiytmsne %5h1b_Pred ---xxxxxxxxxxxxxxxxxxxxxxxxx %5h1d_Human --cwihpalfdfftwlgylnslinpiiytvfne %5h1d_Pred -----------xxxxxxxxxxxxxxxxxxxx %5h1e_Human --ytvssevadfltwlgyvnslinpllytsfne %5h1e_Pred -----------xxxxxxxxxxxxxxxxx %5h1f_Human --ckiseemsnflawlgylnslinpliytifne %5h1f_Pred -----------xxxxxxxxxxxxxxxxxxxx %5h1a_Human --chmptllgaiinwlgysnsllnpviyayfnk %5h1a_Pred -------xxxxxxxxxxxxxxxxxxxxxxxx %Oxyr_Human --apkeasafiivmllaslnsccnpwiymlftg~ %Oxyr_Pred ------xxxxxxxxxxxxxxxxxxxxxxxxx %V2r_Human --aplegapfvllmllaslnsctnpwiyasfss %V2r_Pred ---------xxxxxxxxxxxxxxxxxxxx %Grhr_Human mlnrlsdpvnhffflfaflnpcfdpliygyfsl %Grhr_Pred ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ %Opsg_Human --ypfhplmaalpaffaksatiynpviyvfmnr %Opsg_Pred -------xxxxxxxxxxxxxxxxxxxxxxxx %Opsr_Human --yafhplmaalpayfaksatiynpviyvfmnr %Opsr_Pred -------xxxxxxxxxxxxxxxxxxxxxxxx %Opsb_Human --hgldlrlvtipsffsksaciynpiiycfmnk %Opsb_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Opsd_Human --snfgpifmtipaffaksaaiynpviyimmnk %Opsd_Pred -------xxxxxxxxxxxxxxxxxxxxxx %Nk3r_Human nrwkyiqqvylasfwlamsstmynpiiycclnk~ %Nk3r_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Opr1_Human nrwkyiqqvylasfwlamsstmynpiiycclnk~ %Opr1_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Nk1r_Human ylkkfiqqvylaimwlamsstmynpiiycclnd~ %Nk1r_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Nk2r_Human ychkfiqqvylalfwlamsstmynpiiycclnh~ %Nk2r_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Cckr_Human aerrlsgtpisfilllsytsscvnpiiycfmnk~ %Cckr_Pred ---------xxxxxxxxxxxxxxxxxxxxxx %Gasr_Human ahralsgapisfihllsyasacvnplvycfmhr~ %Gasr_Pred ---------xxxxxxxxxxxxxxxxxxxxxx %Ny1r_Human iatcnhnllfllchltamistcvnpifygflnk~ %Ny1r_Pred -------xxxxxxxxxxxxxxxxxxxxxxxx %Et1r_Human ellsflllmdyiginlatmnscinpialyfvsk~ %Et1r_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Etbr_Human ellsfllvldyiginmaslnscinpialylvsk~ %Etbr_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Grpr_Human dtsmlhfvtsicarllaftnscvnpfalyllsk~ %Grpr_Pred ------xxxxxxxxxxxxxxxxxxxxxxxxx %Nmbr_Human dpslghmivtlvarvlsfgnscvnpfalyllse~ %Nmbr_Pred ------xxxxxxxxxxxxxxxxxxxxxxxxx %Brs3_Human dpsamhfiftifsrvlafsnscvnpfalywlsk~ %Brs3_Pred ------xxxxxxxxxxxxxxxxxxxxxxxxx %Ebi2_Human sqrhsfqislhftvclmnfnccmdpfiyffack~ %Ebi2_Pred -------xxxxxxxxxxxxxxxxxxxxxxxxx %Thrr_Human stteaayfayllcvcvssisscidpliyyyass~ %Thrr_Pred ------xxxxxxxxxxxxxxxxxxxxxxxx %Il8a_Human errnnigraldateilgflhsclnpiiyafigq~ %Il8a_Pred --------------xxxxxxxxxxxxxxxxx %Il8b_Human errnhidraldateilgilhsclnpliyafigq~ %Il8b_Pred --------------xxxxxxxxxxxxxxxxx %Blr1_Human klngslpvaitmceflglahcclnpmlytfagv~ %Blr1_Pred --------------xxxxxxxxxxxxxxxxxxx %Ebi1_Human elskqlniaydvtyslacvrccvnpflyafigv~ %Ebi1_Pred -----------xxxxxxxxxxxxxxxxxxxxxx %Ny3r_Human efentvhkwisitealaffhcclnpilyaflga~ %Ny3r_Pred --------xxxxxxxxxxxxxxxxxxxxxxxxx %Ag2r_Human riadivdtampiticiayfnnclnplfygflgk~ %Ag2r_Pred --------xxxxxxxxxxxxxxxxxxxxxxx %Ckr1_Human eqsrhldlavqvteviaythccvnpviyafvge~ %Ckr1_Pred --------------xxxxxxxxxxxxxxxxx %Rdc1_Human rlehalftalhvtqclslvhccvnpvlysfinr~ %Rdc1_Pred ----xxxxxxxxxxxxxxxxxxxxxxxx %Ssr1_Human -da----tvsqlsvilgyanscanpilygflsd~ %Ssr1_Pred -----------xxxxxxxxxxxxxxxxxxxx %Ssr4_Human -da----tvnhvslilsyanscanpilygflsd~ %Ssr4_Pred -----------xxxxxxxxxxxxxxxxxxxx %Ssr2_Human -ptpalkgmfdfvvvltyanscanpilyaflsd~ %Ssr2_Pred -----------xxxxxxxxxxxxxxxxxxxx %Ssr3_Human -eepaffglyflvvalpyanscanpilygflsy~ %Ssr3_Pred ------xxxxxxxxxxxxxxxxxxxxxxxxx %Fml1_Human -kykiilvlinptsslaffnsclnpilyvfmgr %Fml1_Pred ----xxxxxxxxxxxxxxxxxxxxxxxxx %Fml2_Human -kykiidilvnptsslaffnsclnpmlyvfvgq %Fml2_Pred ---------------xxxxxxxxxxxxxxxxx %Fmlr_Human -mykeigiavdvtsalaffnsclnpmlyvfmgq %Fmlr_Pred -----xxxxxxxxxxxxxxxxxxxxxxxx %C5ar_Human -tflllnkldslcvsfayinccinpiiyvvagq %C5ar_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Brb2_Human qderiidvitqiasfmaysnsclnplvyvivgk~ %Brb2_Pred -------xxxxxxxxxxxxxxxxxxxxxxxx %Pafr_Human fhqainda-hqvtlcllstncvldpviycfltk~ %Pafr_Pred -----------xxxxxxxxxxxxxxxxxxxx %Ntr_Human flydfyhyfymvtnalfyvsstinpilynlvsa~ %Ntr_Pred -------------------------xxxxxxxx %Mas_Human eywstfgnlhhisllfstinssanpfiyffvgs %Mas_Pred -----------xxxxxxxxxxxxxxxxxxxx %Lshr_Human ----------vllvlfypinscanpflyaiftk~ %Lshr_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Tshr_Human ----------illvlfyplnscanpflyaiftk~ %Tshr_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Fshr_Human ----------illvlfhpinscanpflyaiftk~ %Fshr_Pred ----------xxxxxxxxxxxxxxxxxxxxx %Olfi_Human ----------vmammytvvtpmlnpfiyslrnr %Olfi_Pred ----------xxxxxxxxxxxxxxxxxxxx %Olfj_Human ----------lisvtytvitpllnpvvytlrnk %Olfj_Pred ----------xxxxxxxxxxxxxxxxxxxx %Ta2r_Human qlsrttekelliylrvatwnqildpwvyilfrr %Ta2r_Pred ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-7tms_r@net.bio.net Wed Jan 04 22:00:00 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: Postdoc positions available Date: 5 Jan 1995 08:22:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net The following positions relevant to those with in interest in GPCRs are presently available. POSTDOCTORAL POSITION Available immediately to study the binding site structure of serotonin 5-HT2 receptors using chemical and molecular approaches. Unique environment in a research group encompassing molecular biology, pharmacology, biophysics and computational molecular modeling. An independent, academically oriented candidate is desired with experience in physical chemistry of macromolecules, and/or molecular engineering. Interest and experience in cell biology and/or molecular pharmacology are preferred. Send curriculum vitae and telephone numbers of 3 references to: S.C.Sealfon, Neurobiology, Box 1065, (212) 241-7075; Fax (212) 996-9785; sealfon@msvax.mssm.edu or H. Weinstein, Department of Physiology and Biophysics, Box 1218, (212) 241-6530; Fax (212) 860-3369, Mount Sinai School of Medicine, New York, NY 10029-6574. POSTDOCTORAL POSITIONS Available immediately to investigate the activation of dopamine receptors in tissue culture and transgenic animals. Unique collaborative research environment encompassing molecular biology, pharmacology, computational molecular modeling and transgenic mouse research. An independent, academically oriented candidate is desired with facility in molecular engineering. Experience in molecular pharmacology and tissue culture is preferred. Send curriculum vitae and telephone numbers of 3 references to: S.C.Sealfon, Neurobiology, Box 1065, (212) 241-7075; Fax (212) 996-9785; sealfon@msvax.mssm.edu; Mount Sinai School of Medicine, New York, NY 10029-6574. From owner-7tms_r@net.bio.net Wed Jan 04 22:00:00 1995 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: helix 7 Date: 5 Jan 1995 10:18:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 84 Sender: daemon@net.bio.net Distribution: world Message-ID: <204081BC_000DF7E0.0098A049854EA400$15_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net Dear All, I would be interested to hear what people think about various data concerning helix 7 of the GPCRs which, at least to me, seem to be rather difficult to use together. (Interested readers might like to grab hold of a pen and paper and begin drawing helical wheels). Helix 7 has three very conserved residues, usually referred to as NPxxY, which we have heard about in several recent messages. Since conserved residues are usually in the core of the protein structure, these residues can be used to define the "inner face" of helix 7. On an ideal helical wheel (i.e. 100 degrees between each consectutive residue), these three residues are contained within an arc of 100 degrees. Just outside of this arc lies the position of the retinal attachment point in rhodopsin (Lys-296). This is 20 degrees from the Pro, 80 degrees from the Tyr and 120 degrees from the Asn. Assuming that this residue is also on the "inner face" of the helix, an arc of 120 degrees is now defined with Lys-296 at one end and the conserved Asn at the other. Rao et al (Nature 367: 639-642) have shown that the Lys-296 (h7) and Glu-113 (h3) positions are probably close to Gly-90 (in helix 2). I was happy to see the paper since our model fitted with this result (i.e. helices 2, 3 and 7 were in close proximity at the Gly-90, Glu-113 and Lys-296 positions respectively). In this model, the Lys-296 position on the helical wheel contacts helix 2 while the conserved Asn contacts helix 6. It all seemed perfectly logical until Harel Weinstein sent me a reprint of Zhou et al. (1994) Molecular Phamacology 45: 165-170. In this paper, it is suggested that the conserved Asn in helix 7 (actually an Asp in this case) interacts with helix 2. If this is also the case in rhodopsin, it would seem to result in the Lys-296 being orientated towards the lipid in our model. Hence my first question is: Can the Rao et al. and the Zhou et al. results be incorporated together into a consistent GPCR model? Given the above, it also seems that Asn-312 (Mol Pharm 44:111-114) and possibly Ser-319 (J Biol Chem 264:13573-13578) in the human beta2-AR are involved in ligand binding - or at least are likely in be on the "inner face" of helix 7 in the vicinity of the binding site. These residues are both inside the 120 degree arc described above. But what about the Trp identified by Wong et al. (1988) J. Biol. Chem. 263:7925-7928 ????? This is well conserved, especially in the amine binding GPCRs. It is at the 313 position in the beta-2 adrenergic receptor and is 180 degrees from the conserved Asn (i.e. they're completely opposite). In most models, this region (Asn-312, Trp312) is part of the seventh transmembrane helix. Hence questions 2 and 3 are: How can Trp-313 be involved in ligand binding if the above data is correct? and Why is the Wong et al. data very rarely (ever?) mentioned in modelling papers? In our model, the transmembrane part of helix 7 begins at about Ile-314 (human beta2-AR: or at Phe-294 in human rhodopsin) i.e. Asn-312 and Trp-313 are not part of the seventh transmembrane helix. The following 20 residues show very strong alpha-helical periodicity - consitent with a transmembrane helix. However, there is also a region of helical periodicity prior to this transmembane helix. Interestingly, the more conserved face of this helical region (i.e. the "inner face") is also the most hydrophobic face. This suggests that the exterior of the helix faces a more polar environment and is consistent with a helix on the extracellular side of the bilayer. It does not seem to be a simple extention of helix 7 that protrudes from the bilayer since the "inner faces" of the two helical regions are not in phase with each other. Moreover, the region between the two helical regions consists of residues Asn-312 and Trp-313 - both mentioned above as possible ligand binding residues. However, I don't know exactly how to build this in 3D. I'd be very interested to hear other views on the subject. Best Regards, Dan Donnelly Dept. Biochemistry and Molecular Biology University of Leeds Leeds LS2 9JT U.K. Tel. +44 532 333117 E-Mail: bmb6hdd@biovax.leeds.ac.uk From owner-7tms_r@net.bio.net Thu Jan 05 22:00:00 1995 Path: biosci!GSBS.GS.UTH.TMC.EDU!dclark From: dclark@GSBS.GS.UTH.TMC.EDU (Dick Clark) Newsgroups: bionet.molbio.proteins.7tms_r Subject: NPxxY and coupling Date: 6 Jan 1995 15:54:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501062354.AA13191@gsbs> NNTP-Posting-Host: net.bio.net With regard to the proposal by Barak et al (JBC 269, 2790-2795) that their Y326A substitution specifically blocks internalization, we have calculated from their data that the coupling efficiency of this mutant is approximately 5-10 % of the WT =DFAR (see Whaley et al Mol. Pharm.= 45:481-489 for the calculation of coupling efficiency). This calculation indicates that the substitution causes delocalized perturbations of the receptor, and would support the contention of Keefer et al that this substitution may impair proper folding of the receptor. Certainly more mutations and studies of this region are needed to test the proposal that the NPxxY is a recognition domain for internalization.=20 An additional point. Barak et al also imply that internalization is not a mechanism of desensitization. Any event that separates the receptor from Gs/adenylyl cyclase, as is the case with internalization, will desensitize. It is my opinion that not only is internalization a means of desensitizing the receptor, but that it may be intimately intertwined with the function of =DFARK, perhaps along the lines suggested by the recent work of Tsuga et al (JBC 269:32522) on the m2 AChR. From owner-7tms_r@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!GIBBS.OIT.UNC.EDU!tropsha From: tropsha@GIBBS.OIT.UNC.EDU (Alex Tropsha) Newsgroups: bionet.molbio.proteins.7tms_r Subject: 3D models of rhodopsin Date: 9 Jan 1995 12:30:30 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi folks: I am interested in 3D modeling of mammalian rhodopsin. I would appreciate any references that describe such efforts. I will summarize the answers for the net. With best regards, Alexander Tropsha, Ph.D. Assistant Professor, Director the Laboratory for Molecular Modeling CB # 7360, Beard Hall School of Pharmacy University of North Carolina Chapel Hill, NC 27599-7360 Tel. (919) 966-2955 Fax (919) 966-6919 From owner-7tms_r@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!JULIET.CALTECH.EDU!vslepak From: vslepak@JULIET.CALTECH.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Pertussis toxin sensitivity of "PTX-insensitive receptors". Date: 9 Jan 1995 10:38:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <950109103734.2aa35e3a@Juliet.Caltech.Edu> NNTP-Posting-Host: net.bio.net Dear netters, I am collecting the information on the receptors that are found to be pertussis toxin-insensitive in vitro (like Gq-family coupled M2-MAchR, for example) but behave as PTX-sensitive in vivo. I will appriciate any references to published papers as well as personal experience and comments on this subject. Vlad Slepak, PhD. Biology 147-75 Caltech, Pasadena CA 91125 From owner-7tms_r@net.bio.net Sun Jan 08 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!news.starnet.net!wupost!howland.reston.ans.net!cs.utexas.edu!atlantis.utmb.edu!news From: rwildin@beach.utmb.edu (Bob Wildin, M.D.) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Endothelin-B receptor and Hirshprung's Date: 9 Jan 1995 15:10:26 GMT Organization: University of Texas Medical Branch, Galveston Lines: 13 Sender: rwildin@beach.utmb.edu@beach.utmb.edu Message-ID: <3erjl2$nmb@atlantis.utmb.edu> NNTP-Posting-Host: wildin.utmb.edu X-Posted-From: InterNews 1.0.4@129.109.6.79 X-Authenticated: rwildin on POP host beach.utmb.edu FYI: Another human disease associated with a 7tms receptor: Puffenberger, E.K, et al., A missense mutation of the endothelin-B receptor gene in multigenic Hirshprung's disease. Cell 79:1257-1266, Dec. 30, 1994. Authors show W276C mutation (conserved residue of tm helix 5) in large Mennonite pedigree. Two following articles from same group show that mouse piebald-lethal (natural) mutation is a mutation in this gene, and that an intentional knockout of this gene give the same phenotype as seen in humans. From owner-7tms_r@net.bio.net Mon Jan 09 22:00:00 1995 Path: biosci!nencki.gov.pl!baclofen From: baclofen@nencki.gov.pl ("Marcin Gierdalski") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Cannabinoid receptor(s) Ab Date: 10 Jan 1995 02:34:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <1893.baclofen@ameba.nencki.gov.pl_POPMail.PC_3.2.2> NNTP-Posting-Host: net.bio.net Dear netters, I am intersting in localization of cannabinoid receptor protein in central nervous system. Is a specific antibody (poly- or monoclonal) available anywhere? I suppose it could be as the receptor protein was highly purified by one group (I don't remember which one, unfortunately). Best regards Marcin Gierdalski ---------------------------------------------------------------------- Marcin Gierdalski internet: baclofen@nencki.gov.pl Nencki Institute of Exp. Biol. baclofen@plearn.edu.pl Dept. of Neurophysiology bitnet: baclofen@plearn.bitnet 3 Pasteur St Warsaw POLAND Fax: +(48-22)-22-53-42 ---------------------------------------------------------------------- From owner-7tms_r@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!MEMBRAN1.WEIZMANN.AC.IL!bmweis From: bmweis@MEMBRAN1.WEIZMANN.AC.IL (yehudit weisinger) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 11 Jan 1995 02:25:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501110926.AA13642@membran1.weizmann.ac.il> NNTP-Posting-Host: net.bio.net SUBSCRIBE -- Sincerly, Yehudit Weisinger BMWEIS@MEMBRAN1.WEIZMANN.AC.IL From owner-7tms_r@net.bio.net Tue Jan 10 22:00:00 1995 Path: biosci!MRC.COM!buggy From: buggy@MRC.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Glp-IR anti-body any-body? Date: 11 Jan 1995 05:26:50 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501111324.AA25372@mrcs1> Has anyone out there ever heard of an antibody raised against the Glp-I Receptor? ...I would be most grateful for any leads! THanks, Joe Buggy BUGGY@mrc.com From owner-7tms_r@net.bio.net Thu Jan 12 22:00:00 1995 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cs.utexas.edu!news.sprintlink.net!howland.reston.ans.net!news.cac.psu.edu!news.pop.psu.edu!psuvax1!news.cc.swarthmore.edu!image-slave.swarthmore.edu!user From: ehom1@cc.swarthmore.edu (Erik Forbes Y. Hom) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Quenstion: protein concentration in a cell Date: 13 Jan 1995 20:12:24 GMT Organization: Swarthmore College Lines: 13 Message-ID: NNTP-Posting-Host: image-slave.swarthmore.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit Hi netters. Can anyone be of help by telling me where/how I might be able to find/calculate a "typical" concentration (mg/ml or something of the like) of a housekeeping protein like tubulin in a "typical" mammalian cell. I need a ball park figure. As far as I know, about 16-20% of a cell's weight is protein (but what's the typical weight of a "typical" mammalian cell?), volume is readily estimated from cell diameter, and a highly expressed protein could be about 1% of the total protein in a cell (so for, say tubulin, about 0.1-0.2% of the cell is protein by weight (?)). Any suggestions, info???? Please email. Cheers, erik From owner-7tms_r@net.bio.net Fri Jan 13 22:00:00 1995 Path: biosci!Merck.Com!keith_elliston From: keith_elliston@Merck.Com (Keith Elliston) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: "Simplesse" by NutraSweet (fwd) Date: 14 Jan 1995 11:03:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501141909.AA18029@igw.merck.com> NNTP-Posting-Host: net.bio.net Forwarded message: > From: leigh@nbi.com (Leigh Melton) > Subject: Re: "Simplesse" by NutraSweet > > NutraSweet is a brand name of an artificial sweetener. The company name > you are looking for is Archer Daniels Midland. Why not give them a > ring? I'm sure they would be more than happy to supply you with press > releases. > > Simple Pleasures is no longer on the market in the US as far as I know. > I believe ADM pulled their fat substitute from production. One of the > problems with it is that it breaks down rapidly unless kept down to a > certain temperature. That is why it was only marketed in ice cream. > > This is all from memory. I suggest you contact ADM to get the real... > scoop. :) The Nutrasweet company distributes aspartame as nutrasweet. The nutrasweet company is (or was as of last year) a division of the Monsanto Corporation, in St. Louis MO. I suggest you go to them for your information. Keith -- Keith O. Elliston | Phone: (908)594-6099 Dept. of Bioinformatics | Fax: (908)594-2929 Merck Research Laboratories | Email: Elliston@merck.com Box 2000 / Mail Stop RY80A-1 | Elliston@aol.com Rahway, NJ 07065 U.S.A. | Elliston@mbcl.rutgers.edu From owner-7tms_r@net.bio.net Sun Jan 15 22:00:00 1995 Path: biosci!rnisd0.DNET.roche.com!margolsr From: margolsr@rnisd0.DNET.roche.com Newsgroups: bionet.molbio.proteins.7tms_r Subject: reply to Simplesse by NutraSweet Date: 16 Jan 1995 07:14:38 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501161507.AA16925@mailgate.roche.com> NNTP-Posting-Host: net.bio.net If my memory serves me right, Simplesse was created and manufactured by the NutraSweet Company. I believe that it is just whipped eggwhite protein and that the trapped air/protein mix gives the "mouthfeel" of real fat. It is supposed to be very unstable and ice cream was apparently one application where it worked reasonably well. However, Consumer Reports sensory evaluation claimed that it was somewhat grainy in texture and not a very good substitute for the silky feel of high fat ice cream. It will be interesting to determine if "mouthfeel" is transduced by 7TM r type receptors and G proteins. Present information suggests that bitter is transduced by the gustducin G protein and that sweet is transduced by Gs. Salt and sour are apparently transduced by apical sodium and potassium channels. RFM From owner-7tms_r@net.bio.net Mon Jan 16 22:00:00 1995 Path: biosci!ALCOR.BMS.COM!krystek From: krystek@ALCOR.BMS.COM (Stanley Krystek) Newsgroups: bionet.molbio.proteins.7tms_r Subject: is it quiet or is it me? Date: 17 Jan 1995 08:52:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501171652.AA04701@alcor> NNTP-Posting-Host: net.bio.net i have not got any messages this week, is the list that quiet or is it me? stan Stanley R. Krystek, Jr., Ph.D. Bristol-Myers Squibb Pharmaceutical Research Institute Department of Macromolecular Modeling P.O. Box 4000, Room H.3812, Princeton, N.J. 08543-4000 (609) 252-4100 FAX: (609) 252-6030 INTERNET: krystek@bms.com From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Path: biosci!MSMAIL.BMS.COM!Monshizadegan_H.BioChemistry From: Monshizadegan_H.BioChemistry@MSMAIL.BMS.COM (Monshizadegan H) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Subscribe me too Date: 18 Jan 1995 07:40:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Interested, subscribe please. From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Path: biosci!MSMAIL.BMS.COM!Green_D.BioChemistry From: Green_D.BioChemistry@MSMAIL.BMS.COM (Green D) Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe Date: 18 Jan 1995 07:37:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net subscribe From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Path: biosci!MSMAIL.BMS.COM!Green_D.BioChemistry From: Green_D.BioChemistry@MSMAIL.BMS.COM (Green D) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 18 Jan 1995 07:34:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Greetings...looking forward to info exchange From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Path: biosci!greencross.co.jp!inoue From: inoue@greencross.co.jp (Yoshihisa Inoue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Helix 7 & C-terminal tails Date: 18 Jan 1995 05:34:34 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 54 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501181129.UAA27424@gccss2.greencross.co.jp> NNTP-Posting-Host: net.bio.net Dear Netters, I want to discuss about "Helix 7 and C-terminal tails" again. (1)J.Marie et al. showed that Tyr292(TM7) seems to bind with Asp74(TM2) in AT1 and is essential for its coupling to phospholipase C.(JBC, 269,20815-20818). But this Tyr is not the one of NPxxY. cf. C.Bihoreau et al.,ProNAS,90,5133-5137 (1993). As for beta2 receptor, see F.Z.Chang et al.,JBC,266,14953-14957 (1991). (2)H.T.Schambye et al. showed that the antagonists bind differently with Asn295Ser mutant of AT1 receptor(Br.J.Pharmacol.,113,331-333 (1994)). In all 4 pairs of insurmountable and competitive antagonists, the latter were affected more by these substitutions than the former. Their results might suggests the difference between the competitive and insurmountable antagonists. Some researchers say that insurmountable antagonists bind with the receptor too much long time, but these results might not support this idea. Their another paper is ProNAS,91,7046-7050 (1994). (3)H.Ji et al. reported that Leu300 and Phe301 are a little bit important for DuP753 binding in AT1 receptor. They are xx amino acids in NPxxY (JBC,269,16533-16536(1994)). (4)S.A.Hjorth et al. reported that the AII binding sites are a litte bit different from the non-peptide antagonist L-158809 binding sites. (JBC,269,30953-30959 (1994)) (5)J.-P. Berlose et al. reported the NMR data of S/T-X-X-N-P-X-X-Y peptide. I want to wait their future report (Eur.J.Biochem.,225, 827-843 (1994)). But M.J.Miller et al. reported "The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H+ translocating function retained". This is not G-protein coupled receptor, but membrane-consisting 2 helices(ProNAS,87,4900-4904(1990)). They showed that Gly in helix1 is near Asp in helix2. Do you think it corresponds to TM1 and TM2? (6)E.A.Dratz et al. reported "NMR structure of a receptor-bound G-protein peptide(Nature,363,276-281 (1993)). And M.Mahmoudian reported "the complex of human Gs protein with the beta3 adrenergic receptor" (J.Mol.Graphics,12,22-28 & 34 (1994). This report will be interesting to the researchers who study inner loops or C-tails. (7) As for Dr.Donnelly's question, I think trp is very much important for ligand binding, and/or for formation of the salt bridge at the surface of the receptor. They provide a 'floor' to the site (D.J.Underwood et al., Chemistry & Biology,1(4),211-221 (1994)). I'd be very interested to hear other views on the subject, too. Best Regards, ____/ ___/ ___/ Yoshihisa INOUE (^_^) the Green Cross Corp. / / / 2-25-1 Shodai-Ohtani,Hirakata,Osaka 573 JAPAN / _ / / / tel: +81-720-56-9328 / / / / fax: +81-720-68-9597 _____/ _____/_____/ E-mail: inoue@greencross.co.jp From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Newsgroups: bionet.molbio.proteins.7tms_r Path: biosci!rutgers!umdnj!soma!butler From: butler@soma.UMDNJ.EDU (Gary Butler) Subject: SDS gel artifact 50-50k bands Message-ID: Summary: SDS gel artifact 50-60k bands Keywords: SDS gel artifact 50-60k bands Sender: news@umdnj.edu () Organization: Univ. of Medicine and Dentistry of NJ Date: Wed, 18 Jan 1995 17:39:16 GMT Lines: 12 -- Gary H. Butler, Ph.D. Coriell Institute for Medical Research 401 Haddon Ave. Camden, NJ 08103 Tel: (609) 757-9716 Fax: (609) 964 0254 Attn: Butler Email: butler@UMDNJ.edu From owner-7tms_r@net.bio.net Tue Jan 17 22:00:00 1995 Path: biosci!greencross.co.jp!inoue From: inoue@greencross.co.jp (Yoshihisa Inoue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: 7TMS_R model list. Part.3 Date: 18 Jan 1995 05:16:36 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501181128.UAA27415@gccss2.greencross.co.jp> NNTP-Posting-Host: net.bio.net Dear netters, I found several 3D models after I posted in November. AU T.Naka TI Angiotensin II receptor antagonist activities and mode of action of benzimidazole-7-carboxylic acid derivatives (Japanese) SO Nihon Rinsho,51(6),167-171 (1993) COM Angiotensin II receptor AU M.Mahmoudian TI The complex of human Gs protein with the beta3 adrenergic receptor: A computer-aided molecular modeling study SO J.Mol.Graphics,12(1),22-28,34 (1994) COM beta3 receptor and Gs protein AU V.P.Gerskowitch, D.Girdlestone, and D.H.Jenkinson TI Receptors on the agenda: a symposium to honour Sir James Black SO TiPS, 15 (10),355-357 (1994) COM beta2 receptor and NK1 receptor, models were provided by C.D.Strader and D.Underwood. AU D.J.Underwood, C.D.Strader, R.Rivero, A.A.Patchett, W.Greenlee, and K.Prendergast TI Structural model of antagonist and agonist binding to the Angiotensin II, AT1 subtype, G protein coupled receptor SO Chemistry and Biology, 1(4), 211-221 (1994) COM beta2 model was initially built, then AT1 receptor. Docking model (beta2-isoproterenol),(AT1-L159282),(AT1-L162313) are shown. If you know more, please let me know. Thank you in advance. Best regards, ____/ ___/ ___/ Yoshihisa INOUE (^_^) the Green Cross Corp. / / / 2-25-1 Shodai-Ohtani,Hirakata,Osaka 573 JAPAN / _ / / / tel: +81-720-56-9328 / / / / fax: +81-720-68-9597 _____/ _____/_____/ E-mail: inoue@greencross.co.jp From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 1995 Path: biosci!EMBL-HEIDELBERG.DE!Gert.Vriend From: Gert.Vriend@EMBL-HEIDELBERG.DE Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 19 Jan 1995 13:54:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HM1PRJX7S2MC3DZO@EMBL-Heidelberg.DE> NNTP-Posting-Host: net.bio.net I am perfectly willing to read mails from this group, and I frequently answer questions (by sending mail directly to the asker if it is not of general interest, and by bouncing it to the group if I think more people could use the answer). I get 40-60 mail messages per day, a serious fraction of those come from this group. Deleteing messages about cheese and nonfat fat is taking so much time that I consider unsubscribing. Please stop this junk mail. Gert. From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 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: "Simplesse" by NutraSweet Date: 19 Jan 1995 14:02:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501192155.AA01695@receptor> References: <1995Jan19.181541.1953@adobe.com> NNTP-Posting-Host: net.bio.net Would people please stop posting this discussion to bionet.molbio.proteins.7tms_r. The pharmacology of these materials are perhaps worth discussion, but we have diverged from that topic. Thanks Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-735-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 1995 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: tasty GPCRs? Date: 19 Jan 1995 01:48:09 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <2040BC1D_000E52E8.0098AB0956584320$11_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net Have I accidently subscribed to a "cookery-net" or is there a connection between "Simplesse" and GPCRs that I've missed? Turkey-cheese pockets!?*! Help!!!!!!!! Dan From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 1995 Path: biosci!sb.com!Anthony_M_Brown%notes From: Anthony_M_Brown%notes@sb.com (Anthony M Brown) Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe Date: 19 Jan 1995 03:28:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501191426.AA6923@pho018.sb.com> NNTP-Posting-Host: net.bio.net subscribe From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 1995 Path: biosci!JULIET.CALTECH.EDU!vslepak From: vslepak@JULIET.CALTECH.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Stupid ""Simplesse" by NutraSweet" discussion Date: 19 Jan 1995 21:47:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: <950119214736.2ba05585@Juliet.Caltech.Edu> NNTP-Posting-Host: net.bio.net I am sorry, why don't you guys discuss all this "Simplesse" by NutraSweet stuff over the phone? I am sick of deleting this junk. Don't they have a weight-loss newsgroup? Vlad. From owner-7tms_r@net.bio.net Wed Jan 18 22:00:00 1995 Path: biosci!RISC.IDG.FI.CNR.IT!giolitti From: giolitti@RISC.IDG.FI.CNR.IT (Alessandro Giolitti) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Constitutive receptors & antagonists Date: 19 Jan 1995 22:58:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Does anyone know if antagonists suppress the activity of constitutively active receptors? I would expect they do, but is there experimental evidence? Alessandro Giolitti _______________________________________ Alessandro Giolitti Drug Design Dept. Menarini s.r.l. Via Sette Santi, 3 50131 Firenze Italy Ph. : +39-55-5680240 FAX : +39-55-5680419 e-mail: giolitti@risc.idg.fi.cnr.it _______________________________________ From owner-7tms_r@net.bio.net Thu Jan 19 22:00:00 1995 Path: biosci!RISC.IDG.FI.CNR.IT!giolitti From: giolitti@RISC.IDG.FI.CNR.IT (Alessandro Giolitti) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Constitutive receptors & antagonists Date: 20 Jan 1995 14:30:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >This is a list of constitutively-active receptor references. The two starred >papers in particular show that some antagonists can at least partially reverse >the constitutive activation of the receptor. Very few systems have been >tested >so far, as you can see, and it is not yet clear if these are only partially >efficacious inverse agonists, or whether these activated receptors cannot be >totally repressed. > >Richard Premont >Duke University Medical Center >Durham, NC 27710 > Thanks for the many references: I will try to check all those I will find. I should, however, make the question more definite: I know (better: our pharmacologists know) the Lefkowitz' work about constitutive receptors, but it should be in transfected cells expressing receptors. Does the same apply to receptors in the tissue, that is: in a different biological environment than the aforementioned? All the endogenous agonists should be depleted, for instance. What could be a reliable test? Alessandro _______________________________________ Alessandro Giolitti Drug Design Dept. Menarini s.r.l. Via Sette Santi, 3 50131 Firenze Italy Ph. : +39-55-5680240 FAX : +39-55-5680419 e-mail: giolitti@risc.idg.fi.cnr.it _______________________________________ From owner-7tms_r@net.bio.net Thu Jan 19 22:00:00 1995 Path: biosci!MC.DUKE.EDU!PREMO003 From: PREMO003@MC.DUKE.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Constitutive receptors & antagonists Date: 20 Jan 1995 09:39:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 114 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HM2IST7TZ6000040@mc.duke.edu> NNTP-Posting-Host: net.bio.net Date: Fri, 20 Jan 1995 13:18 -0500 (EST) From: premo003%Hades@ccmail.duke.edu Subject: Re: Constitutive receptors & antagonists To: giolitti@RISC.IDG.FI.CNR.IT, 7tms_r.net.bio.net@mc.duke.edu MIME-version: 1.0 MIME-version: 1.0 Content-type: TEXT/PLAIN Alessandro: This is a list of constitutively-active receptor references. The two starred papers in particular show that some antagonists can at least partially reverse the constitutive activation of the receptor. Very few systems have been tested so far, as you can see, and it is not yet clear if these are only partially efficacious inverse agonists, or whether these activated receptors cannot be totally repressed. Richard Premont Duke University Medical Center Durham, NC 27710 CAM receptor references: Allen, L. F., R. J. Lefkowitz, M. G. Caron and S. Cotecchia (1991). "G-protein-coupled receptor genes as protooncogenes: Constitutively activating mutation of the alpha1B-adrenergic receptor enhances mitogenesis and tumorigenicity." Proc Natl Acad Sci Usa 88(24): 11354-11358. * Barker, E. L., R. S. Westphal, D. Schmidt and E. Sanders-Bush (1994). "Constitutively active 5-hydroxytryptamine2C receptors reveal novel inverse agonist activity of receptor ligands." J Biol Chem 269(16): 11687-11690. Boone, C., N. G. Davis and G. F. Sprague (1993). "Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype." Proc. Natl. Acad. Sci. USA 90: 9921-9925. Cotecchia, S., S. Exum, M. G. Caron and R. J. Lefkowitz (1990). "Regions of the a1-adrenergic receptor involved in coupling to phosphatidlylinostiol hydrolysis and enhanced sensitivity of biological function." Proc. Natl. Acad. Sci. USA 87: 2896-2900. Kjelsberg, M. A., S. Cotecchia, J. Ostrowski, M. G. Caron and R. J. Lefkowitz (1992). "Constitutive activation of the a1B-adrenergic receptor by all amino acid substitution at a single site." J. Biol. Chem. 267: 1430-1433. Kosugi, S., Okajima, F., Ban, T., Hidaka, A., Shenker, A., and Kohn, L. D. (1993) Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinosotide and 3',5'-cyclic adenosine monophosphate signal generation. Mol. Endocrinol. 7: 1009-1020. Lefkowitz, R. J. (1993). "Turned on to ill effect." Nature` 365: 603-604. Lefkowitz, R. J., Cotecchia, S., Samama, P., and Costa, T. (1993) Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol. Sci. 14: 303-307. Parker, E. M. and E. M. Ross (1991). "Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian b-adrenergic receptor." J. Biol. Chem. : 9987-9995. Parma, J., L. Duprez, J. Van Sande, P. Cochaux, C. Gervy, J. Mockel, J. Dumont and G. Vassart (1993). "Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas." Nature 365(6447): 649-651. Pei, G., Samama, P., Lohse, M., Wang, M., Codina, J., and Lefkowitz, R. J. (1994) A constitutively active mutant beta2-adrenergic receptor is constitutively desensitized and phosphorylated. Proc. Natl. Acad. Sci. (USA) 91: 2699-2702. Rao, V. R., G. B. Cohen and D. D. Oprian (1994). "Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness." Nature 367: 639-642. Ren, Q., Kurose, H., Lefkowitz, R. J., and Cotecchia, S. (1993) Constitutively active mutants of the alpha2-adrenergic receptor. J. Biol. Chem. 268: 16483-16487. Robbins, L. S., Nadeau, J. H., Johnson, K. R., Kelly, M. A., Roselli- Rehfuss, L., Baack, E., Mountjoy, K. G., and Cone, R. D. (1993) Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72: 827-834. Robinson, P. R., G. B. Cohen, E. A. Zhukovsky and D. D. Oprian (1992). "Constitutively active mutants of rhodopsin." Neuron 9: 719-725. Robinson, P. R., Buczylko, J., Ohguro, H., and Palczewski, K. (1994) Opsins with mutations at the site of chromophore attachment constitutively activate transducin but are not phosphorylated by rhodopsin kinase. Proc. Natl. Acad. Sci. (USA) 91: 5411-5415. Samama, P., Cotecchia. S., T. Costa and R. J. Lefkowitz (1993). "A mutation- induced activated state of the b2-adrenergic receptor: Extending the ternary complex model." J. Biol. Chem. 268: 4625-4636. * Samama, P., Pei, G., Costa, T., Cotecchia, S., and Lefkowitz, R. J. (1994) Negative antagonists promote an inactive conformation of the beta2-adrenergic receptor. Mol. Pharmacol. 45: 390-394. Shenker, A., L. Laue, S. Kosugi, J. J. Morendino, T. Minegishik and G. B. Cutler (1993). "A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty." Nature 365: 652-654. _______________________________________________________________________________ Subject: Constitutive receptors & antagonists From: giolitti@RISC.IDG.FI.CNR.IT at Internet Date: 1/20/95 1:58 AM Does anyone know if antagonists suppress the activity of constitutively active receptors? I would expect they do, but is there experimental evidence? Alessandro Giolitti From owner-7tms_r@net.bio.net Thu Jan 19 22:00:00 1995 Path: biosci!PT.CYANAMID.COM!hadcockj From: hadcockj@PT.CYANAMID.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Constitutive receptors & antagonists Date: 20 Jan 1995 09:38:09 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 37 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HM2E98J6N68WW1Q2@ptag2.pt.Cyanamid.COM> NNTP-Posting-Host: net.bio.net There are several examples of reverse agonists/negative antagonists that suppress the activities of constitutively active beta-adrenergic receptors. I am sure there are other papers on alpha adrenergic receptors out there. Chidiac et al (1994) Mol Pharm 45:490-499 Samama et al (1994) Mol Pharm 45:390-394 John Hadcock Hadcockj@pt.cyanamid.com ______________________________ Reply Separator _________________________________ Subject: Constitutive receptors & antagonists Author: giolitti@RISC.IDG.FI.CNR.IT at GATEWAY Date: 1/20/95 1:58 AM Does anyone know if antagonists suppress the activity of constitutively active receptors? I would expect they do, but is there experimental evidence? Alessandro Giolitti _______________________________________ Alessandro Giolitti Drug Design Dept. Menarini s.r.l. Via Sette Santi, 3 50131 Firenze Italy Ph. : +39-55-5680240 FAX : +39-55-5680419 e-mail: giolitti@risc.idg.fi.cnr.it _______________________________________ From owner-7tms_r@net.bio.net Fri Jan 20 22:00:00 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: off the topic Date: 21 Jan 1995 08:43:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501211649.JAA25694@essex.hsc.colorado.edu> NNTP-Posting-Host: net.bio.net Subject: Time:9:36 AM OFFICE MEMO off the topic Date:1/21/95 I concur with Gert Vriend and others. What happened, did Martha Stewart start running this news group? Lets stick to receptors and G-proteins. From owner-7tms_r@net.bio.net Fri Jan 20 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news.sprintlink.net!pipex!warwick!news.dcs.warwick.ac.uk!str-ccsun!strath-cs!st-and!Aberdeen!ort020 From: ort020@nof.abdn.ac.uk (a.vijayan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: JOBSTREAM Date: 21 Jan 1995 14:28:51 GMT Organization: University of Aberdeen, Scotland Lines: 3 Message-ID: <3fr5n3$btq@nof.abdn.ac.uk> NNTP-Posting-Host: med.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] Sorry, I could not read anything what you wrote. Could you please repeat your problem. From owner-7tms_r@net.bio.net Fri Jan 20 22:00:00 1995 Path: biosci!greencross.co.jp!inoue From: inoue@greencross.co.jp (Yoshihisa Inoue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: NP**Y and C-terminal tail Date: 21 Jan 1995 01:16:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 87 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501210838.RAA03142@gccss2.greencross.co.jp> NNTP-Posting-Host: net.bio.net Dear Netters, I want to discuss about the "NP**Y"-related topics again. On Jan 6, Dr.Dick Clark wrote: > With regard to the proposal by Barak et al (JBC 269, 2790-2795) that >their Y326A substitution specifically blocks internalization, we have >calculated from their data that the coupling efficiency of this mutant is >approximately 5-10 % of the WT =DFAR (see Whaley et al Mol. Pharm.45:481-489 >for the calculation of coupling efficiency). >.... > An additional point. Barak et al also imply that internalization is >not a mechanism of desensitization. Any event that separates the receptor >from Gs/adenylyl cyclase, as is the case with internalization, will >desensitize. It is my opinion that not only is internalization a means of >desensitizing the receptor, but that it may be intimately intertwined with >the function of =DFARK, perhaps along the lines suggested by the recent work >of Tsuga et al (JBC 269:32522) on the m2 AChR. Internalization and desensitization is different. The desensitization mechanism of G-protein coupled receptors has been extensively studied with regard to beta2 receptor. Two different kinases, protein kinase A and beta- ARK were proven to be responsible for the desensitization through phosphory- lating Ser/Thr residues in C-terminal tail. But the desentitization mechamism of phospholipase C-coupled receptors is little known(TiPS,14,279-285 (1994)). M3, formylpeptide, C5a, alpha1, cholecystokinin receptors are phosphorylated in an agonist-dependent manner. Takano et al reported that the C-terminal tail, especially distal Ser/Thr residues, has a critical role in desensitization of the signaling pathway that leads to phospholipase-C/calcium response axis in PAF receptor (JBC,269,22453-22458 (1994)). And Prossnitz et al reported that the phosphorylation of N-terminal Ser/Thr of C-terminal tail is required for the subsequent phosphorylation of C-terminal residues by GRK2 (JBC,270, 1130-1137 (1995)). And Hyunady et al reported (JBC 269, 24798-24804 (1994)) that they mutated Asp74(TM2) in AT1a into Asn, His, or Tyr and found that each of these mutants showed markedly different internalization kinetics and IP3 stimulation. Asp74 was shown in my last post as > J.Marie et al. showed that Tyr292(TM7) seems to bind with Asp74(TM2) > in AT1 and is essential for its coupling to phospholipase C.(JBC, > 269,20815-20818). But this Tyr is not the one of NPxxY. and they deleted 215-220(TM6 to inner-loop 3), 221-226(inner-loop 3), 227- 231(inner-loop 3) and found that D(215-220) exhibited markedly impaired and slower internalization kinetics. I think D(215-220) contains TM6 parts. Pei et al mutated L266S,K267R,H269K, and L272A (inner-loop 3) of beta2 receptor (ProNAS,91,2699-2702 (1994)) and found that not only is the mutant beta2 receptor constitutively active, it is also constitutively desensitized and down-regulated, and is phosphorylated in the absence of agonist, in a fashion comparable to the agonist-occupied wild-type receptor. So, the confomation of the mutant receptor is equivalent to the active conformation. And as for inner-loop 3, Blueml et al inserted 1 to 5 Ala just after R252, which is thought to be the begining of inner-loop 3(ProNAS,91,7980-7984), and found that the N-terminal portion of inner-loop 3 forms an amphiphilic alpha helix and that the hydrophobic side of this helix represents the G-protein recognition surface. Signaling via growth factors involves phosphorylation of tyrosin and a series of protein-protein interactions, mediated via SH2 and SH3 domains, leading to serial activation of p21ras, Raf-1 kinase and MAP kinase. I am a molecular modeller and a stranger in this field. So I do not know whether the report by C.T.Sigal et al (ProNAS,91,12253-12257 (1994)) is related or not. They say that Myr-GSSKSKPKDPSQRRR peptide is the membrane-targeting motif. Does it relate to the results of the Nishimoto group that the basic amino acid in inner loop is important. By the way, M.Hirata et al reported Prostaglandin D receptor sequence and I found it has "DP**F" instead of "NP**Y", and mouse PGI2 receptor, too (ProNAS,91,11192-11196 (1994)). Do these receptors internalize? If so, can we explain the report by Barak et al(JBC 269, 2790-2795)? And I found interesting report, but it might be wrong idea. K.S.Campbell et al reported (ProNAS,91,6344-6348 91994)) that NPTY sequence is required for interaction between Middle Tumor Antigen(MT) and SHC protein, a Src homology 2 (SH2)-containing protooncogene product implicated in activating Ras via association with GRB2 protein. SHC is phosphorylated on tyrosine and associateds with GRB2 in MT-transformed cells. Maybe I am wrong, but any comments? I'd be very interested to hear other views on the subject, too. Best Regards, ____/ ___/ ___/ Yoshihisa INOUE (^_^) the Green Cross Corp. / / / 2-25-1 Shodai-Ohtani,Hirakata,Osaka 573 JAPAN / _ / / / tel: +81-720-56-9328 / / / / fax: +81-720-68-9597 _____/ _____/_____/ E-mail: inoue@greencross.co.jp From owner-7tms_r@net.bio.net Fri Jan 20 22:00:00 1995 Path: biosci!lmcp.jussieu.fr!callebau From: callebau@lmcp.jussieu.fr (Isabelle Callebaut) Newsgroups: bionet.molbio.proteins.7tms_r Subject: unssucribe Date: 21 Jan 1995 05:49:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501211355.AA11300@lmcp.jussieu.fr> NNTP-Posting-Host: net.bio.net Please, skip me out from the 7YTMS list. thank you From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!bmc.uu.se!DANL From: DANL@bmc.uu.se Newsgroups: bionet.molbio.proteins.7tms_r Subject: Unsubscribe Date: 22 Jan 1995 10:16:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <950122191547.202a32d8@bmc.uu.se> NNTP-Posting-Host: net.bio.net unsubscribe 7tms_r From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!medgen.uu.se!dan.larhammar From: dan.larhammar@medgen.uu.se (Dan Larhammar) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Subscribe Date: 22 Jan 1995 10:16:33 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501221815.AA11066@alpha.medgen.uu.se> NNTP-Posting-Host: net.bio.net Subscribe 7tms_r Dan L. ---------------------------------------------- Dan Larhammar, Professor Dept of Medical Genetics Uppsala University Box 589 S-751 23 Uppsala, SWEDEN Tel 46-18 17 43 13 Fax 46-18 52 68 49 From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!rutgers!mcrcr6!cmcl2!yale.edu!yale!yale!hsdndev!nmr-z.mgh.harvard.edu!nmr-z.mgh.harvard.edu!lfk Newsgroups: bionet.molbio.proteins.7tms_r Subject: Inappropriate Messages Message-ID: From: lfk@receptor.mgh.harvard.edu (Lee F. (Frank) Kolakowski) Date: 22 Jan 1995 17:02:42 GMT Distribution: world Organization: Mass. General Hospital, Renal Unit NNTP-Posting-Host: receptor.mgh.harvard.edu Lines: 34 Dear 7TMS_R Readers, As we are no longer a mailing list, but a Newsgroup, sometimes messages which are initially only a little of target, get further off target.This happens because in newsreaders followups to this messages carry the list of news groups to "cross-post" the message to. In the case recently the news groups were: Newsgroups: misc.health.diabetes, bionet.molbio.proteins.7tms_r, ba.food, alt.food.professionals, alt.food.ice-cream, alt.food.fat-free The best way to stop this is to reply to the messages suggesting that follows be directed away from 7tms_r. It is impossible to stop this kind of traffic. In many systems there is also something called a kill-file which allows you to automatically delete messages with particular subject lines or addresses of particular authors. I have posted follow-ups to all the messgages removing 7tms_r from the list. I hope this lessens the flow. -- Frank Kolakowski Email: lfk@receptor.mgh.harvard.edu 617-735-7515 (LAB) GCRDb-WWW From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!chem.gla.ac.uk!chrisw From: chrisw@chem.gla.ac.uk (Chris Wood) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Modeling Post-Doc? Date: 22 Jan 1995 22:10:14 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <12942.199501230610@nernst.chem.gla.ac.uk> NNTP-Posting-Host: net.bio.net Hi, If anyone is looking for a molecular modeler (re: GPCRs) let me know .... I need a post-doc! The group to which i am attached is very strong in protein crystallography - but my area was protein modeling, programming massively parallel machines - i am quite fluent in 3L Parallel FORTRAN. I am just finishing writing up of Ph.D. (GPCR Protein Modeling). As i had a commercial sponsor - my work was subject to a publication embargo. If anyone wants my CV ... you only have to email me your request - thanks Chris Wood From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!VAX.GRC.NIA.NIH.GOV!chahrzad From: chahrzad@VAX.GRC.NIA.NIH.GOV Newsgroups: bionet.molbio.proteins.7tms_r Subject: GLP receptor antibody Date: 22 Jan 1995 11:15:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <0098AD8A.FD3AC900.3@vax.grc.nia.nih.gov> NNTP-Posting-Host: net.bio.net Has anybody answered to the question about existance of a GLP receptor antibody ? I would appreciate if you would relay the answer to me as well. I am interested in finding an antibody against the GLP receptor. Thank you in advance. Chahrzad Montrose Tel number 410-5588199 Fax number 410-5588381 Email address Chahrzad@vax.grc.nia.nih.gov From owner-7tms_r@net.bio.net Sat Jan 21 22:00:00 1995 Path: biosci!LYNX.DAC.NEU.EDU!rdeth From: rdeth@LYNX.DAC.NEU.EDU (richard deth) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Post-doc Opportunities? Date: 22 Jan 1995 12:37:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501222036.PAA20723@lynx.dac.neu.edu> NNTP-Posting-Host: net.bio.net Dear Fellow Heptahelical Groupies: I am trying to help a graduate student who is finishing up her thesis in my lab to find a post-doc in the northern New Jersey/NYC region. Her name is Arti Jinsi, and her thesis dealt with the coupling of alpha 2D adrenergic receptors to phospholipase D, in both cultured cells containing the cloned receptor and in vascular tissues. In particular she focused on the regulation of this coupling by tyrosine kinase activity and low molecular weight G proteins. She knows a wide range of relevant lab techniques including GTP binding and radioligand binding assays, SDS-PAGE and immunoblotting, molecular modeling and some melcular biology skills, as well as phospholipase D and vascular contraction assays. Arti also has a good working knowledge about many of the issues current to this discussion group including the structural basis for agonist and antagonist effects, constituitive activity etc. If you have an opening in the next few months or know of someone who does please drop a line to me (rdeth@lynx.neu.edu) and I will have her contact you. Both academic and industrial opportunities are of interest. Thanks for the help!! Richard Deth Northeastern University Tel. (617) 373-4064 FAX (617) 266-6756 E-mail rdeth@lynx.neu.edu From owner-7tms_r@net.bio.net Sun Jan 22 22:00:00 1995 Path: biosci!MZDMZA.ZDV.UNI-MAINZ.DE!SCHROEDER From: SCHROEDER@MZDMZA.ZDV.UNI-MAINZ.DE Newsgroups: bionet.molbio.proteins.7tms_r Subject: Unsubscribe Date: 23 Jan 1995 02:10:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HM6MKJ6X02HWA42V@MZDMZA.ZDV.UNI-MAINZ.DE> NNTP-Posting-Host: net.bio.net Unsubscribe 7tms_r From owner-7tms_r@net.bio.net Sun Jan 22 22:00:00 1995 Path: biosci!greencross.co.jp!inoue From: inoue@greencross.co.jp (Yoshihisa Inoue) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Why our messages were not appeared in the Bionet Newsgroup Date: 23 Jan 1995 03:10:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501230930.SAA14678@gccss2.greencross.co.jp> NNTP-Posting-Host: net.bio.net Dear Netters, Do you think it strange that in the Newsgroup bionet.molbio.proteins.7tms_r only "NutraSweet" were appeared, but the messages sent used in the "mailing list" have been not appeared. So if we post that "please stop NutraSweet" using "mailing list", it seems to be in vain. Any comments? ____/ ___/ ___/ Yoshihisa INOUE (^_^) the Green Cross Corp. / / / 2-25-1 Shodai-Ohtani,Hirakata,Osaka 573 JAPAN / _ / / / tel: +81-720-56-9328 / / / / fax: +81-720-68-9597 _____/ _____/_____/ E-mail: inoue@greencross.co.jp From owner-7tms_r@net.bio.net Sun Jan 22 22:00:00 1995 Path: biosci!MIRIS.MED.YALE.EDU!mike From: mike@MIRIS.MED.YALE.EDU ("Michael Singer") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Mac Software? Date: 23 Jan 1995 13:46:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501231649.ZM1933@miris.med.yale.edu> NNTP-Posting-Host: net.bio.net I model GPCRs on a workstation, but I'd like to show 3-D pictures of the receptor (or at least ligand) models on the MacIntosh. Does anyone know any 3-D molecule rendering programs? Mike Singer From owner-7tms_r@net.bio.net Mon Jan 23 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!msunews!caen!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Mac Software? Date: 24 Jan 1995 18:16:07 GMT Organization: University of Michigan Lines: 32 Message-ID: <3g3g57$5vo@lastactionhero.rs.itd.umich.edu> References: <9501231649.ZM1933@miris.med.yale.edu> NNTP-Posting-Host: warbler.med.umich.edu mike@MIRIS.MED.YALE.EDU ("Michael Singer") wrote: > > I model GPCRs on a workstation, but I'd like to show 3-D pictures of the > receptor (or at least ligand) models on the MacIntosh. Does anyone know any > 3-D molecule rendering programs? > > Mike Singer > I just ran across a Public domain program called RasMol which is available on Windows, Mac (including powermac versions) and Unix. It is an incredibly powerful program for displaying protein and macromolecular structures. You can do a simple display just by using menu choices but there is also a command line option to permit you to customize your image to almost any degree you want. For instance, I created a display of the transducin structure with the backbone as a stick (line with 3-D effect), the tryptophans marked in red, the cysteines in yellow, the GTP as a space-filled model with CPK colors and the Mg in green space-fill. It is really very impressive. It was developed by a guy at Glaxo. Here's a clip from the blurb describing it. The source code is public domain and freely distributable provided that the original author is suitably acknowledged. The complete source code and user documentation may be obtained by anonymous FTP from ftp.dcs.ed.ac.uk [129.215.160.5] in the directory /pub/rasmol. The source code, documentation and Microsoft Windows executables are stored in several files appropriate for the receiving operating system. Please read the "README" file in the distribution directory. UNIX and VAX systems should retreive either RasMol2.tar.Z, RasMol2.tar.gz. Apple Mac users should retrieve rasmac.sit.hqx. 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 Mon Jan 23 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!msunews!caen!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Constitutive receptors & antagonists Date: 24 Jan 1995 18:08:34 GMT Organization: University of Michigan Lines: 78 Message-ID: <3g3fn2$5vo@lastactionhero.rs.itd.umich.edu> References: NNTP-Posting-Host: warbler.med.umich.edu giolitti@RISC.IDG.FI.CNR.IT (Alessandro Giolitti) wrote: > > Thanks for the many references: I will try to check all those I will find. > I should, however, make the question more definite: I know (better: our > pharmacologists know) the Lefkowitz' work about constitutive receptors, but > it should be in transfected cells expressing receptors. Does the same apply > to receptors in the tissue, that is: in a different biological environment > than the aforementioned? All the endogenous agonists should be depleted, > for instance. What could be a reliable test? > > > Alessandro Alessandro, There was substantial evidence for "precoupling" of GPCR's to G proteins in native tissues which predated the recent interest in "constituitive" activation of receptors. The methods used for some of these studies (especially the kinetics of agonist binding to alpha2 and FMLP receptors) are not influenced by the presence of contaminating agonist. These studies measure a different aspect of receptor-G protein coupling (high affinity binding) but do indicate that you don't need an overexpressed system to observe relatively tight RG interactions. 1. Wreggett, K. A. and A. De Lean. 1984. The ternary complex model. Its properties and application to ligand interactions with the D2-dopamine receptor of the anterior pituitary gland. Mol. Pharmacol 26:214-227. 2. Nerme, V., Y. Severne, T. Abrahamsson, and G. Vauquelin. 1986. Spontaneous coupling of the beta-adrenergic receptor to Ns in mammalian cardiac membranes. Mol. Pharmacol 30:1-5. 3. Neubig, R. R., R. D. Gantzos, and W. J. Thomsen. 1988. Mechanism of agonist and antagonist binding to a2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex. Biochemistry 27:2374-2384. 4. Costa, T., J. Lang, C. Gless, and A. Herz. 1990. Spontaneous association between opioid receptors and GTP-binding regulatory proteins in native membranes: specific regulation by antagonists and sodium ions. Mol. Pharmacol 37:383-394. 5. Jagadeesh, G., E. J. Cragoe,Jr., and R. C. Deth. 1990. Modulation of bovine aortic alpha-2 receptors by Na+, 5'-guanylylimidodiphosphate, amiloride and ethylisopropylamiloride: evidence for receptor G-protein precoupling. J Pharmacol Exp Ther 252:1184-1196. 6. Leung, E., K. A. Jacobson, and R. D. Green. 1990. Analysis of agonist-antagonist interactions at A1 adenosine receptors. Mol. Pharmacol 38:72-83. 7. Fay, S. P., R. G. Posner, W. N. Swann, and L. A. Sklar. 1991. Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. Biochemistry 30:5066-5075. 8. Costa, T., Y. Ogino, P. J. Munson, H. O. Onaran, and D. Rodbard. 1992. Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol. Pharmacol 41:549-560. You may also be interested in a recent review that I wrote reviewing evidence that the "precoupling" may be a bit more complicated than just a "spontaneous" interaction of the R and G. I think the idea that RG interactions are regulated by more than just the affinity of R for G is going to be very important. Neubig, R. R. 1994. Membrane organization in G protein mechanisms. FASEB J 8:939-946. Rick Neubig http://www.umich.edu/~rneubig From owner-7tms_r@net.bio.net Mon Jan 23 22:00:00 1995 Path: biosci!cict.fr!bourbon From: bourbon@cict.fr (Henri-Marc Bourbon) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Unsubscribe Date: 24 Jan 1995 01:28:51 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501240932.AA21587@cix.cict.fr> NNTP-Posting-Host: net.bio.net Unsubscribe 7tms_s From owner-7tms_r@net.bio.net Tue Jan 24 22:00:00 1995 Path: biosci!chem.gla.ac.uk!chrisw From: chrisw@chem.gla.ac.uk (Chris Wood) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Postdoc please! - email address Date: 25 Jan 1995 18:56:50 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 46 Sender: daemon@net.bio.net Distribution: world Message-ID: <6400.199501260256@nernst.chem.gla.ac.uk> NNTP-Posting-Host: net.bio.net Hi, If anyone is looking for a molecular modeler (re: GPCRs) let me know .... I need a post-doc! The group to which i am attached is very strong in protein crystallography - but my area was protein modeling, programming massively parallel machines - i am quite fluent in 3L Parallel FORTRAN. I am just finishing writing up of Ph.D. (GPCR Protein Modeling). As i had a commercial sponsor - my work was subject to a publication embargo. If anyone wants my CV ... you only have to email me your request Forgive this double posting, but i failed to clearly give my email address; it is: gacu57@udcf.gla.ac.uk or: chrisw@nernst.chem.gla.ac.uk Also, please feel free to email my supervisor, who will furnish you with a very positive vibes about me! My supervisor is: Prof. Neil Isaacs. His details are: ______________________________________________________________________ | Neil Isaacs Tel: (041) 339 8855 ext 5954 | | Department of Chemistry FAX: (041) 330 4888 | | University of Glasgow email: neil@islay.chem.gla.ac.uk | | Glasgow G12 8QQ | | Scotland | |______________________________________________________________________| PS Please use -v option to ensure your email is forwarded properly: mail -v neil@islay.chem.gla.ac.uk From owner-7tms_r@net.bio.net Tue Jan 24 22:00:00 1995 Path: biosci!GEMINI.OSCS.MONTANA.EDU!uched From: uched@GEMINI.OSCS.MONTANA.EDU (Ed Dratz) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Chris Wood's CV Date: 25 Jan 1995 18:28:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501260228.AA15622@terra.oscs.montana.edu> NNTP-Posting-Host: net.bio.net Chris Wood, you did not include your e-mail address in your postdoc request. From owner-7tms_r@net.bio.net Tue Jan 24 22:00:00 1995 Path: biosci!RISC.IDG.FI.CNR.IT!giolitti From: giolitti@RISC.IDG.FI.CNR.IT (Alessandro Giolitti) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Constitutive activity Date: 25 Jan 1995 13:14:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >We recently published on constitutive activity of the mu opioid >receptor (Wang et al., Life Sci.54, PL339-350 (1994) in neuroblastoma cells. >Constitutive activity appeared to be regulated by phosphorylation >and could represent a key element in narcotic addiction. Kinase inhibitor >H& reversed tolerance/dependence in mice, and also blocked mu >receptor phosphorylation (to be published). Naloxone is a negative >antagonist, and CTAP a neutral antagonist. >Let me know if you have any comments. >Wolfgang Sadee > I will check the paper you indicated. We are presently involved in neurokinin receptors, and in some cases a decrease in tone of tissue preparation have been observed while treating them with antagonists but without any agonist added. I would expect, considering the active conformation of the receptor as one already accessible to it, and therefore present (with a probability varying with the receptor and environment), that a negative antagonist suppresses this "constitutive" activity. The problem is: how to demonstrate this with receptors in a physiological condition, not in transfected ("artificial system") cells? Alessandro Giolitti _______________________________________ Alessandro Giolitti Drug Design Dept. Menarini s.r.l. Via Sette Santi, 3 50131 Firenze Italy Ph. : +39-55-5680240 FAX : +39-55-5680419 e-mail: giolitti@risc.idg.fi.cnr.it _______________________________________ From owner-7tms_r@net.bio.net Tue Jan 24 22:00:00 1995 Path: biosci!CGL.UCSF.EDU!sadee From: sadee@CGL.UCSF.EDU (Wolfgang Sadee) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Constitutive receptor activity Date: 25 Jan 1995 09:58:15 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501251758.JAA10526@socrates.ucsf.EDU> NNTP-Posting-Host: net.bio.net Recent reference posted did not contain our paper on this topic: Constitutive mu opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence. Wang,Bilsky, Porreca, Sadee, Life Sci. 54 PL 339-350 (94). This work was done with the native mu receptor in neuroblastoma cells. Regulation of constitutive receptor activity, possibly by phosphorylation (now supported by data in mu receptor transfected cells), was porposed as a novel mechanism of receptor regulation, which may play a key role in narcotic tolerance and dependnece. Does anybody have any comments on this topic? q From owner-7tms_r@net.bio.net Tue Jan 24 22:00:00 1995 Path: biosci!agate!news.duke.edu!usenet From: thomasr@acpub.duke.edu (Ronald Thomas) Newsgroups: bionet.molbio.proteins.7tms_r Subject: EGFR-western blot problem Date: 25 Jan 1995 10:55:06 -0500 Organization: Duke University; Durham, NC; USA Lines: 2 Message-ID: <3g5s8q$qbq@news.duke.edu> NNTP-Posting-Host: raphael.acpub.duke.edu In an attempt to localize the 170kD EGF receptor (from fetal rat lung tissue cx) on PAGE/Western blot using primary antibody, biotinylated protein From owner-7tms_r@net.bio.net Wed Jan 25 22:00:00 1995 Path: biosci!MV3B.CRYST.BBK.AC.UK!UBCG17C From: UBCG17C@MV3B.CRYST.BBK.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 26 Jan 1995 06:01:10 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <199501261400.GAA05728@net.bio.net> NNTP-Posting-Host: net.bio.net I am interested in expressing the human ETA receptor using alternative leader sequences. It appears however, that there is confusion as to what the N-terminal sequence of the mature ETA receptor protein actually is. Can anyone point me to good experimental evidence identifying the authentic mature N-terminal sequence of human ETA receptor? Phil Nugent, Birkbeck College, University of London. From owner-7tms_r@net.bio.net Thu Jan 26 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!cs.utexas.edu!swrinde!pipex!warwick!news.dcs.warwick.ac.uk!str-ccsun!strath-cs!st-and!Aberdeen!ort020 From: ort020@nof.abdn.ac.uk (a.vijayan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Summer job Date: 27 Jan 1995 18:14:22 GMT Organization: University of Aberdeen, Scotland Lines: 8 Message-ID: <3gbd5u$2et@nof.abdn.ac.uk> NNTP-Posting-Host: med.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] I am a biochemist working in the area of protein chemistry. Presently I am working with extracellular matrix proteins of skin dermis. Before this I was working with cartilage proteoglycans. My Masters degree was on complement receptors. Durring summer I am planning to have a working holiday in Toronto and New Jersey areas. Could any body in this group help me to find a suitable laboratory to work for a month or so in view of gaining more experience. You could respond to my e-mail address above. From owner-7tms_r@net.bio.net Fri Jan 27 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!spool.mu.edu!torn!nott!dgbt!netfs.dnd.ca!ub!csn!yuma!purdue!news.bu.edu!MED-ORALBIO7.BU.EDU!klennon From: klennon@acs.bu.edu Newsgroups: bionet.molbio.proteins.7tms_r Subject: silver staining Date: Fri, 27 Jan 1995 21:49:47 GMT Organization: Boston University Lines: 3 Message-ID: NNTP-Posting-Host: med-oralbio7.bu.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Help!! I am trying to use a silver staining technique for proteins immobilized on an SDS-PAGE gel that is not working at all. If anyone has any advice, please forward it to kalen@conncoll.edu From owner-7tms_r@net.bio.net Tue Jan 31 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!pipex!sunsite.doc.ic.ac.uk!nntp0.brunel.ac.uk!strath-cs!st-and!Aberdeen!ort020 From: ort020@nof.abdn.ac.uk (a.vijayan) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Summer job wanted Date: 1 Feb 1995 14:53:00 GMT Organization: University of Aberdeen, Scotland Lines: 12 Message-ID: <3go78c$8tp@nof.abdn.ac.uk> NNTP-Posting-Host: med.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] I have an M.Sc degree by research in biochemistry. I am working as a Research Biochemist in the Dept of Surgery, University of Aberdeen, Scotland, U.K. I am working on a project related to wound healing. My particular interest is in skin dermis proteoglycans. I am familiar with most of the techniques related to protein purification and characterisation. My masters thesis was on receptors for complement component C3a. I will be grateful if somebody could help me to find a suitable place either in States or Canada during summer holidays to broaden my expertise. Please respond to my e-mail address above. Thank you in advance. From owner-7tms_r@net.bio.net Tue Jan 31 22:00:00 1995 Path: biosci!NWU.EDU!j-takahashi From: j-takahashi@NWU.EDU (Joseph S. Takahashi) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 1 Feb 1995 15:28:57 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <199502012328.AA261431320@casbah.acns.nwu.edu> NNTP-Posting-Host: net.bio.net unsubscribe Joseph S. Takahashi Phone: (708) 491-4598 j-takahashi@nwu.edu New FAX: (708) 467-4065 or jtakahas@casbah.acns.nwu.edu From owner-7tms_r@net.bio.net Tue Jan 31 22:00:00 1995 Path: biosci!NWU.EDU!j-takahashi From: j-takahashi@NWU.EDU (Joseph S. Takahashi) Newsgroups: bionet.molbio.proteins.7tms_r Subject: (none) Date: 1 Feb 1995 15:45:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <199502012345.AA289262327@casbah.acns.nwu.edu> NNTP-Posting-Host: net.bio.net Unsubscribe 7tms_s Joseph S. Takahashi Phone: (708) 491-4598 j-takahashi@nwu.edu New FAX: (708) 467-4065 or jtakahas@casbah.acns.nwu.edu