Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m312671200
ISSN1083-351X
AutoresSrinivasan Madabushi, Alecia K. Gross, Anne Philippi, Elaine C. Meng, Theodore G. Wensel, Olivier Lichtarge,
Tópico(s)Chemokine receptors and signaling
ResumoG protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions. G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions. The profusion and diversity of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; TM, transmembrane; ET, evolutionary tracing. give them a central role in health and disease. In humans, over 1000 genes encode these receptors (1Nestler E.J. Landsman D. Nature. 2001; 409: 834-835Crossref PubMed Scopus (82) Google Scholar), each of which responds to a single or few ligands by activating G proteins, which then modulate enzymes and channels to initiate highly amplified signaling cascades. Such cascades control sight, taste, smell, slow neurotransmission and the responses to most water-soluble hormones and chemokines. In fact, GPCRs are so ubiquitous that, although they are the targets of nearly 50% of current drugs (2Drews J. Nat. Biotechnol. 1996; 14: 1516-1518Crossref PubMed Scopus (203) Google Scholar), this is still a small fraction of their pharmacological potential (3Flower D.R. Biochim. Biophys. Acta. 1999; 1422: 207-234Crossref PubMed Scopus (227) Google Scholar). Some of the major questions relevant to GPCR pharmacology include the following: What residues are critical for ligand binding and G protein activation? What do different receptor families have in common with regard to their activation mechanism? From a structural perspective, it is known that all GPCRs form a seven transmembrane (TM) α-helical bundle, connected by three intracellular and three extracellular loops, with an extracellular N terminus and an intracellular C terminus (4Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (349) Google Scholar, 5Kolakowski L.F.J. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). However, low overall sequence identity of 25% even within class A GPCRs (6Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5095) Google Scholar, 7Horn F. Bywater R. Krause G. Kuipers W. Oliveira L. Paiva A.C. Sander C. Vriend G. Receptors Channels. 1998; 5: 305-314PubMed Google Scholar, 8Donnelly D. FEBS Lett. 1997; 409: 431-436Crossref PubMed Scopus (69) Google Scholar) suggests that significant deviations can occur in ligand binding pockets and in interhelical contacts that stabilize or mediate the transition between active and inactive conformations (9Chothia C. Lesk A.M. EMBO J. 1986; 5: 823-826Crossref PubMed Scopus (2030) Google Scholar). From an experimental perspective, while there is a wealth of data on a handful of GPCRs, most are known only from translated DNA sequences, hence the need for computational methods to extract functional information from those sequences. For example, correlated mutational analysis (10Oliveira L. Paiva A.C. Vriend G. Chembiochem. 2002; 3: 1010-1017Crossref PubMed Scopus (72) Google Scholar) and sequence-based entropy (11Suel G.M. Lockless S.W. Wall M.A. Ranganathan R. Nat. Struct. Biol. 2003; 10: 59-69Crossref PubMed Scopus (667) Google Scholar) have been used to detect networks of functional residues. Other studies focused on receptor topology (12Baldwin J.M. EMBO J. 1993; 12: 1693-1703Crossref PubMed Scopus (892) Google Scholar), functional fingerprints (13Attwood T.K. Findlay J.B. Protein Eng. 1993; 6: 167-176Crossref PubMed Scopus (48) Google Scholar), dimerization interfaces (14Gouldson P.R. Higgs C. Smith R.E. Dean M.K. Gkoutos G.V. Reynolds C.A. Neuropsychopharmacology. 2000; 23: S60-S77Crossref PubMed Scopus (127) Google Scholar), and modeling of receptor-ligand binding (15Vaidehi N. Floriano W.B. Trabanino R. Hall S.E. Freddolino P. Choi E.J. Zamanakos G. Goddard 3rd, W.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12622-12627Crossref PubMed Scopus (255) Google Scholar, 16Bissantz C. Bernard P. Hibert M. Rognan D. Proteins. 2003; 50: 5-25Crossref PubMed Scopus (311) Google Scholar, 17Attwood T.K. Croning M.D. Gaulton A. Protein Eng. 2002; 15: 7-12Crossref PubMed Scopus (32) Google Scholar). This study aims to test two specific and complementary hypotheses. First, that all GPCRs share a common mechanism of activation and G protein coupling. This hypothesis is consistent with common and often promiscuous activation of G proteins by GPCRs, even after whole TMs are swapped to form chimeric receptors (18Kudo M. Osuga Y. Kobilka B.K. Hsueh A.J. J. Biol. Chem. 1996; 271: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It also implies the presence of a ubiquitous GPCR activation switch made up of residues that are functionally important in all receptors. Our second hypothesis predicts that residues involved in ligand specificity are different in different receptors. This would be consistent with the enormous diversity of ligand sizes and types, ranging from ions and small molecules to peptides and large glycoproteins, and suggests that some residues will be important in some but not all GPCRs. The search for these functionally important residues is based on the evolutionary trace method (hereafter ET, or tracing), which identifies sequence positions where variations among related proteins always correlate with evolutionary divergences (19Lichtarge O. Bourne H.R. Cohen F.E. J. Mol. Biol. 1996; 257: 342-358Crossref PubMed Scopus (1028) Google Scholar). Control studies (20Lichtarge O. Bourne H.R. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7507-7511Crossref PubMed Scopus (169) Google Scholar, 21Lichtarge O. Yamamoto K.R. Cohen F.E. J. Mol. Biol. 1997; 274: 325-337Crossref PubMed Scopus (98) Google Scholar, 22Sowa M.E. He W. Wensel T.G. Lichtarge O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1483-1488Crossref PubMed Scopus (72) Google Scholar), genuine predictions followed by mutagenesis studies (23Sowa M.E. He W. Slep K.C. Kercher M.A. Lichtarge O. Wensel T.G. Nat. Struct. Biol. 2001; 8: 234-237Crossref PubMed Scopus (116) Google Scholar), and large scale testing by us (24Madabushi S. Yao H. Marsh M. Kristensen D.M. Philippi A. Sowa M.E. Lichtarge O. J. Mol. Biol. 2002; 316: 139-154Crossref PubMed Scopus (176) Google Scholar, 25Yao H. Kristensen D.M. Mihalek I. Sowa M.E. Shaw C. Kimmel M. Kavraki L. Lichtarge O. J. Mol. Biol. 2002; 326: 255-261Crossref Scopus (161) Google Scholar) or others (26Pritchard L. Dufton M.J. J. Mol. Biol. 1999; 285: 1589-1607Crossref PubMed Scopus (59) Google Scholar, 27Landgraf R. Fischer D. Eisenberg D. Protein Eng. 1999; 12: 943-951Crossref PubMed Scopus (69) Google Scholar, 28Innis C.A. Shi J. Blundell T.L. Protein Eng. 2000; 13: 839-847Crossref PubMed Scopus (126) Google Scholar) show that trace residues cluster in the three-dimensional structure of proteins and that these clusters predict binding or catalytic sites. Moreover, differential ET analysis, which subtracts residues traced on many evolutionary branches of a protein family from those traced on a subset of these branches, can also identify positions that are important to that subset but not to the entire protein family (21Lichtarge O. Yamamoto K.R. Cohen F.E. J. Mol. Biol. 1997; 274: 325-337Crossref PubMed Scopus (98) Google Scholar). In keeping with the hypotheses, we find that a trace of diverse receptors identifies a common functional site shared by all GPCRs and that differential ET identifies ligand-specific functional sites. The published mutational record confirms the former is a generic activation switch. Moreover, novel mutations at two trace residues reveal that they are linked to constitutive activity and protein stability demonstrating the predictive value of the evolutionary trace model to guide rational mutagenesis of GPCRs. GPCR Family Alignments—BLAST (29Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (73559) Google Scholar) retrieved 129 visual opsins, 69 bioamine, 58 olfactory, and 82 chemokine class A receptor sequences from the NCBI data base (see additional information available at imgen.bcm.tmc.edu/molgen/labs/lichtarge/gpcr/). Each family was aligned with ClustalW (30Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56668) Google Scholar) separately, and their TM segments were excised based on exclusion of charged residues and poorly conserved residues near the TM boundaries. Using the bovine rhodopsin numbering, segment 36–64 is TM1, 72–97 is TM2, 108–141 is TM3, 153–175 is TM4, 202–230 is TM5, 250–277 is TM6, and 286–311 is TM7. The segments were concatenated, and alignment was pruned to produce a gapless alignment of 330 GPCRs containing 195 TM residues. Global and Specific Determinants—Generically important residues were obtained by tracing this alignment (20Lichtarge O. Bourne H.R. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7507-7511Crossref PubMed Scopus (169) Google Scholar, 24Madabushi S. Yao H. Marsh M. Kristensen D.M. Philippi A. Sowa M.E. Lichtarge O. J. Mol. Biol. 2002; 316: 139-154Crossref PubMed Scopus (176) Google Scholar, 25Yao H. Kristensen D.M. Mihalek I. Sowa M.E. Shaw C. Kimmel M. Kavraki L. Lichtarge O. J. Mol. Biol. 2002; 326: 255-261Crossref Scopus (161) Google Scholar), ranking the relative importance of each position, and selecting those in the top 5th, 10th, 15th, and 20th percentile ranks. The rank of a given residue is the number of branches of the phylogenetic tree at which it becomes invariant in each branch, starting from the root branch as 1. To identify trace residues uniquely important to each family, we subtracted the generic ones from those that were important in ET analysis of that family, at the same percentile rank (see Supplementary Material). Mutational Data—Functional data on trace residues were gathered from the tGRAP mutant data base (32Kristiansen K. Dahl S.G. Edvardsen O. Proteins. 1996; 26: 81-94Crossref PubMed Scopus (72) Google Scholar), the protein mutant database (33Nishikawa K. Ishino S. Takenaka H. Norioka N. Hirai T. Yao T. Seto Y. Protein Eng. 1994; 7: 773Crossref Scopus (25) Google Scholar), and the literature. For global trace residues we considered mutations at cognate residues across all class A receptors, but for opsin-specific trace residues we used opsin mutations only. A ligand binding effect means that binding assays showed altered affinity or specificity. A G protein-coupling effect signifies altered GTP binding or in inositol phosphate production under appropriate controls. Constitutive activity means signaling activity (inositol phosphate production/cAMP accumulation/G protein activation) in a non-activated (ligand-free) receptor. Folding or expression effects mean considerably less mutant receptor expression under identical conditions as wild-type. Subtle changes were not taken into account, and given multiple functional effects a residue was classified by the one most frequently observed (Fig. 1 and Table II). Two-tailed χ2 tests were performed as described in a previous study (34Brown B.W. Hollander M. Statistics: A Biomedical Introduction, Wiley Series in Probability and Mathematical Statistics. John Wiley & Sons, New York1977: 196-201Google Scholar).Table IIMutational effects of globally important (cognate) residues in different receptor families Mutations of cognate trace residues (bovine rhodopsin numbering) in various receptors obtained from t-GRAP mutant database/alignments and protein mutant database and in some cases from literature search. 211 out of 239 (88%) mutations in 37 residues had functional effects. Ile-75 and Leu-79 had no mutational data. + refers to mutation causing a functional change; - indicates mutation that had no recorded effect. The numbers of receptor families in which trace residue position was mutated are listed. Refer to Supplementary Material for a detailed version of this table.Residue (bovine rhodopsin)No. of mutantsNo. of receptor familiesDominant mutational effectaCA, constitutive activity; LB, ligand binding effects; ME, mechanistic effects observed in structural studies (does not count as functional effect); Exp, decreased expression; G protein, G-protein coupling decreased/completely uncoupled/decreased G-protein stimulation+-5533Mixed (CA, LB, Exp.)6222Mixed/ G protein7211G protein effects7332Expression757685Mixed (CA, [↑ cAMP])78212Mixed (G-protein, CA)798533Ligand binding86614Ligand binding87526Ligand binding9122Ligand binding1101515Ligand binding118729Ligand binding120119Ligand binding121548Ligand binding12811CA131112Mixed (CA, LB)132325Ligand binding134199CA135128Mixed (CA, ME, G uncoupling)136614G protein effects13833G protein effects15322Mixed (LB, expression)16110210Ligand binding204738Ligand binding21533Ligand binding22222CA22333G protein effects254104G protein effects25733CA265927Ligand binding26764Mixed (CA, folding)3028210Mixed (folding/expression, LB)303116Folding/expression30553Ligand binding30610410G protein effects307213Mixed (LB, ME)31021G protein effectsa CA, constitutive activity; LB, ligand binding effects; ME, mechanistic effects observed in structural studies (does not count as functional effect); Exp, decreased expression; G protein, G-protein coupling decreased/completely uncoupled/decreased G-protein stimulation Open table in a new tab DNA Mutagenesis and Expression of Opsins—All opsin variants were expressed using modified forms of a plasmid containing a synthetic opsin gene cDNA in a pMT3-based vector (35Ferretti L. Karnik S.S. Khorana H.G. Nassal M. Oprian D.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 599-603Crossref PubMed Scopus (171) Google Scholar, 36Franke R.R. Sakmar T.P. Oprian D.D. Khorana H.G. J. Biol. Chem. 1988; 263: 2119-2122Abstract Full Text PDF PubMed Google Scholar). Mutations were introduced using the QuickChange® mutagenesis kit (Stratagene). Expression in COS-1 cells, membrane isolation, reconstitution with 11-cis-retinal, and protein purification using 1D4 antibody affinity chromatography were carried out as described previously (36Franke R.R. Sakmar T.P. Oprian D.D. Khorana H.G. J. Biol. Chem. 1988; 263: 2119-2122Abstract Full Text PDF PubMed Google Scholar, 37Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (407) Google Scholar, 38Robinson P.R. Buczylko J. Ohguro H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5411-5415Crossref PubMed Scopus (51) Google Scholar, 39Gross A.K. Rao V.R. Oprian D.D. Biochemistry. 2003; 42: 2009-2015Crossref PubMed Scopus (63) Google Scholar, 40Zhukovsky E.A. Robinson P.R. Oprian D.D. Science. 1991; 251: 558-560Crossref PubMed Scopus (133) Google Scholar). Absorbance spectrophotometry was carried out using an Olis/SLM-Aminco DW-2000 dual-beam instrument adapted for darkroom use. Photolysis of rhodopsin was carried out using a continuous wave argon ion laser directed into the sample compartment from above by a concave mirror that dispersed the beam evenly over the cuvette, with beam gating carried out with a manual shutter. Complete photoisomerization of rhodopsin and all mutants was achieved without significant heating in less than 1 s. Transducin activation assays were carried out as described previously (39Gross A.K. Rao V.R. Oprian D.D. Biochemistry. 2003; 42: 2009-2015Crossref PubMed Scopus (63) Google Scholar). Global Determinants—To find residues mediating a generic signal transduction mechanism, we traced jointly four evolutionarily distant families: the visual rhodopsin, bioamine, olfactory, and chemokine receptors. The 39 residues ranked in the top 20th percentile (Fig. 1 and Tables I and II) are predicted to be generically important. When they are mapped onto the rhodopsin structure, they form a three-dimensional structural cluster that is internal, mostly located in the cytoplasmic half of the membrane, and statistically significant (p = 0.0002, which is the probability that the same number of randomly selected residues would cluster as tightly in three dimensions (see Ref. 24Madabushi S. Yao H. Marsh M. Kristensen D.M. Philippi A. Sowa M.E. Lichtarge O. J. Mol. Biol. 2002; 316: 139-154Crossref PubMed Scopus (176) Google Scholar)). The cluster includes critical GPCR motifs and residues, such as DRY motif in TM3, NPXXY motif in TM7, Cys-110 in TM3 (disulfide-bonded), and Asn-55 in TM1. Most of its trace residues belong to TM2, TM3, TM7, and to a smaller extent TM6, consistent with mutagenesis, spectroscopy, and cross-linking evidence that the latter three helices undergo rigid-body motions upon activation (41Sheikh S.P. Zvyaga T.A. Lichtarge O. Sakmar T.P. Bourne H.R. Nature. 1996; 383: 347-350Crossref PubMed Scopus (399) Google Scholar, 42Sheikh S.P. Vilardarga J.P. Baranski T.J. Lichtarge O. Iiri T. Meng E.C. Nissenson R.A. Bourne H.R. J. Biol. Chem. 1999; 274: 17033-17041Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 43Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1124) Google Scholar, 44Gether U. Ballesteros J.A. Seifert R. Sanders-Bush E. Weinstein H. Kobilka B.K. J. Biol. Chem. 1997; 272: 2587-2590Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 45Fanelli F. Barbier P. Zanchetta D. De Benedetti P.G. Chini C. Mol. Pharm. 1999; 56: 214-225Crossref PubMed Scopus (93) Google Scholar).Table IThe top 20% of trace residues in global and opsin family analyses Residues specific to the opsin family are underlined. Refer to the Supplementary Material for percentile ranks and functional classification of global trace residues. The top 20% of trace residues from global (class A) and opsin family analyses are shown.TMGlobalOpsin1Asn-55, Thr-62Asn-55, Gly-51, Thr-582Leu-72, Asn-73, Ile-75, Leu-76, Asn-78, Leu-79, Phe-85, Met-86, Val-87, Phe-91Leu-72, Asn-73, Leu-76, Asn-78, Gly-90, Phe-913Cys-110, Thr-118, Gly-120, Gly-121, Leu-128, Leu-131, Ala-132, Glu-134, Arg-135, Tyr-136, Val-138Cys-110, Glu-113, Gly-121, Leu-125, Leu-128, Leu-131, Glu-134, Arg-1354Ala-153, Trp-161Ala-153, Gly-156, Trp-161, Cys-167, Pro-171, Trp-1755Val-204, Pro-215, Cys-222, Tyr-223Met-207, Pro-215, Cys-222, Tyr-223, Val-2306Val-254, Met-257, Trp-265, Pro-267Val-250, Met-253, Val-254, Pro-2677Asn-302, Pro-303, Ile-305, Tyr-306, Ile-307, Asn-310Met-288, Phe-294, Ala-295, Lys-296, Asn-302, Pro-303, Tyr-306, Ile-307, Asn-310 Open table in a new tab To assess the functional importance of these 39 trace residues, we reviewed the published record of their mutations (Table II) and found 239 mutations (excluding structural studies) distributed among 37 trace residues. 211 (88%) of these mutations gave rise to functional differences, including at least one at each of the 37 trace positions. Residues mutated in 2 receptors affected both in 6 of 7 (86%) cases, those mutated in 3 receptors affected all three 7 out of 8 (87%) times and at least 2 in all 8 (100%) instances. Nineteen positions were mutated in 4 or more receptors. In all 19 cases function was disrupted in at least 3 different receptor types, and in 18 cases (95%) function was affected in 4 or more receptor types. This confirms the generic role of these residues in GPCR signaling. To correlate the structural location of these trace residues with their function, we further sub-classified them according to their most frequently observed mutational effect and mapped this information onto the rhodopsin structure (see Fig. 1 and Supplementary Material). Fourteen residues, colored cyan in Fig. 1B, predominantly affect ligand binding (Table II) and segregate in the extracellular half of the cluster. Sixteen residues, colored blue in Fig. 1C, cause constitutive activity, folding, or expression defects, and these segregate roughly in the middle of the cluster (Table II). Seven residues, colored magenta in Fig. 1D, primarily affect G protein coupling and signaling, and those fill the cytoplasmic base of the transmembrane domain. Thus specific functional effects localize strikingly in three regions: a trigger region, composed of residues most immediately involved in ligand-induced conformational changes, a coupling region adjacent to the cytoplasmic loops where G proteins bind, and between them a linking core, composed of residues that stabilize TM interactions and folding. Two trace residues in this linking core, Ile-75 and Leu-79, have not been previously mutated, to our knowledge, and we mutated Leu-79 to test the functional importance of our prediction. As a negative control, we also analyzed the mutational data on the bottom 20% of evolutionarily important positions (39 residues). 3 positions had no mutational data, 14 had mutations that had no effect in any receptors, and only 3 residues had mutations that affected a generic GPCR function, namely expression (1 case) and G protein interaction (2 cases). Mutations of the remaining 19 residues affected ligand interaction, which is a ligand-specific function. In terms of mutations, 60 out of 112 (54%) reported mutations that have no apparent effect on function as opposed to only 12% at trace residues (Table III and Supplementary Material). This greater than 4-fold difference is significant with a p value of less than 0.0005 on a χ2 test. Of the 52 mutations at low ranked positions that have functional effects, 27 mutations (52%) are in the top 20% of family-specific residues identified by differential trace analysis (data not shown). Finally, although 27 of 37 (73%) top ranked trace residues affect function in at least 3 receptors, only 7 of 36 (19%) low ranked positions do so (see Supplementary Material). Thus top ranked trace residues affect generic functions, and they do so in multiple receptor families, whereas bottom ranked ones often have no effect, and when they do this is mostly limited to ligand interactions in one or two receptors.Table IIIDistribution of mutations for the top and bottom 20% of ET residues Mutational effectiveness at a trace versus non-trace residue is significantly different with a χ2 test p value of 0.0005 or better. Refer to Supplementary Material for related tables. Number of mutations for top and bottom 20% of ET residues are shown.Fraction of ET residuesNo. of mutations with recorded effectNo. of mutations with no recorded effectTop 20%21128Bottom 20%5260 Open table in a new tab Retinal Binding Site in Opsin Family—We asked next whether differential trace analysis (21Lichtarge O. Yamamoto K.R. Cohen F.E. J. Mol. Biol. 1997; 274: 325-337Crossref PubMed Scopus (98) Google Scholar) could highlight functional determinants unique to the visual rhodopsin family. We traced 129 rhodopsin sequences and subtracted from the resulting trace residues the global ET residues (Fig. 1) at the same percentile rank (Fig. 2). At the top 20th percentile rank, 17 opsin-specific trace residues were found, of which 11 cluster near the retinal binding pocket (p value < 0.001) (Table I and Supplementary Material). The other opsin-specific residues appear at the cytoplasmic edge or near it. A retrospective analysis of the literature, summarized in Table IV, shows 32 known opsin family mutations at 12 opsin-specific trace residues. Of these, 28 mutations (87%) in 12 trace residues are linked to spectral shifts, constitutive activity, night blindness, or retinitis pigmentosa. Interestingly, Trp-175 is the single apparent false positive. This is surprising given that it is conserved across all opsin sequences, yet past mutations to other aromatic residues caused no spectral shifts and no loss of G protein activation (46Lin S.W. Sakmar T.P. Biochemistry. 1996; 35: 11149-11159Crossref PubMed Scopus (225) Google Scholar). We therefore focused on this amino acid, Trp-175, for further study.Table IVMutational effects of opsin family specific trace residues Recorded functional effects of mutations in opsins are listed. Gly-156, Met-207, Val-230, Met-288, and Phe-294 had no available mutational data. Overall, 28 of 32 (87%) mutations had functional effects. Refer to Supplementary Material for references of mutations listed. Mutational effects of opsin family specific trace residues are shown.TMResidue (bovine rhodopsin no.)Mutational effect151ADRPaADRP, autosomal dominant retinitis pigmentosa58ADRP, ↓ transducin activity, expression290Congenital stationary night blindness, blue shift, CA3113Blue shift, CA, spectral shift125ADRP, ↓ retinal binding, misfolding, expression, no spectral activity ↓ transducin activation, ↓ time in M-II4156167ADRP, low expression levels, misfolding171ADRP, low expression levels, misfolding, localization175Normal spectrum and G protein activation520752306250↓ G-protein coupling, switched G-protein specificity253Spin labeling: rhodopsin7288294295Spectral shift296CA, ↓ expression levels, arrestin and rhodopsin kinase mediated effects affecteda ADRP, autosomal dominant retinitis pigmentosa Open table in a new tab Trace Residue Mutations in Rhodopsin—As a bona fide test of the ability of ET to guide function-altering mutations in GPCRs, we prepared mutants at one globally important residue Leu-79 (L79A and L79S) and at one opsin-specific residue Trp-175 (W175A, W175C, and W175H). All expressed at sufficient levels to allow isolation by immunoaffinity chromatography in detergent following reconstitution with 11-cis-retinal. The absorbance spectra in the rhodopsin form (with 11-cis-retinal bound) were essentially identical to wild-type (data not shown). All displayed wild-type ability to activate transducin upon exposure to light (Fig. 3). There were, however, functional differences between the mutant proteins and wild-type opsin. As shown in Fig. 4, substitutions at both positions (W175A, W175C, W175H, and L79A) led to constitutive activity, i.e. G protein activation in the absence of ligand. Unlike wild-type opsin (47Gross A.K. Xie G. Oprian D.D. Biochemistry. 2003; 42: 2002-2008Crossref PubMed Scopus (32) Google Scholar), none of the mutant opsins was sufficiently stable for isolation in detergent without added 11-cis-retinal, except L79S, which was obtained in very low yield (data not shown). To determine the ability of the mutants to regenerate the dark photoreceptive state following photoactivation, we used a laser flash to effect nearly instantaneous quantitative photoisomerization of bound 11-cis-retinal to the all-trans form in the presence of excess 11-cis-retinal and then measured the maximum
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