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• W2116764040 abstract "G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (α-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize α-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs. G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (α-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize α-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs. G protein-coupled receptor cyan fluorescent protein fluorescence resonance energy transfer guanine nucleotide binding regulatory protein transmembrane wild type yellow fluorescent protein γ-aminobutyric acid, type B endoplasmic reticulum green fluorescent protein G protein-coupled receptors (GPCRs)1 constitute the largest class of transmembrane receptors in multicellular organisms. GPCRs mediate the actions of an enormous array of peptides, hormones, bioactive lipids, neurotransmitters, and sensory stimuli, and they are the targets of many drugs used in clinical medicine. Members of this class of receptor have a similar structural architecture consisting of an N-terminal extracellular domain, followed by a bundle of seven transmembrane α-helices connected by intracellular and extracellular loops, and terminated with a cytoplasmic tail. Therefore, many aspects of GPCR structure, function, signaling, and regulation are conserved. GPCRs recently have been shown to form homo- and/or heterodimeric-oligomeric complexes in living cells (1Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (577) Google Scholar, 2Rios C.D. Jordan B.A. Gomes I. Devi L.A. Pharmacol. Ther. 2001; 92: 71-87Crossref PubMed Scopus (282) Google Scholar, 3Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (500) Google Scholar, 4Levac B.A. O'Dowd B.F. George S.R. Curr. Opin. Pharmacol. 2002; 2: 76-81Crossref PubMed Scopus (121) Google Scholar). There is evidence that oligomerization is important for receptor biogenesis (5Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 6Grosse R. Schoneberg T. Schultz G. Gudermann T. Mol. 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Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar), and down-regulation (14Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 15Horvat R.D. Barisas B.G. Roess D.A. Mol. Endocrinol. 2001; 15: 534-542Crossref PubMed Scopus (26) Google Scholar). GPCRs appear to have the ability to form specific types of homo- and/or heterodimeric-oligomeric complexes (2Rios C.D. Jordan B.A. Gomes I. Devi L.A. Pharmacol. Ther. 2001; 92: 71-87Crossref PubMed Scopus (282) Google Scholar, 3Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (500) Google Scholar, 8Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar, 10Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (958) Google Scholar, 13AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (425) Google Scholar, 16Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (734) Google Scholar, 17Ayoub M.A. Couturier C. Lucas-Meunier E. Angers S. Fossier P. Bouvier M. Jockers R. J. Biol. Chem. 2002; 277: 21522-21528Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 18Patel R.C. Kumar U. Lamb D.C. Eid J.S. Rocheville M. Grant M. Rani A. Hazlett T. Patel S.C. Gratton E. Patel Y.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3294-3299Crossref PubMed Scopus (172) Google Scholar), potentially increasing the functional diversity of this large family of receptors. Whether GPCRs generally form dimers or higher order oligomers is not clear, although a recent study (17Ayoub M.A. Couturier C. Lucas-Meunier E. Angers S. Fossier P. Bouvier M. Jockers R. J. Biol. Chem. 2002; 277: 21522-21528Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) suggests that melatonin MT1 and MT2 receptors primarily form a heterodimeric complex. Defining the mechanisms that direct the formation of GPCR dimers/oligomers is required to understand how GPCRs activate G proteins and to suggest which GPCRs preferentially form homodimers/oligomers versus those that also interact as heterodimers/oligomers with other GPCRs. Studies of chimeric receptors and computational methods (evolutionary trace and correlated mutations) have suggested that dimerization/oligomerization may occur by exchange of one or more complementary TM domains between receptor subunits (e.g. TM1 and -2 of one receptor are reciprocally exchanged for their counterparts in the second receptor (19Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (295) Google Scholar, 20Gouldson P.R. Reynolds C.A. Biochem. Soc. 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Intermolecular disulfide bonds occur within the N-terminal extracellular domains of calcium-sensing (25Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 26Goldsmith P.K. Fan G.F. Ray K. Shiloach J. McPhie P. Rogers K.V. Spiegel A.M. J. Biol. Chem. 1999; 274: 11303-11309Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 27Pace A.J. Gama L. Breitwieser G.E. J. Biol. Chem. 1999; 274: 11629-11634Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and metabotropic glutamate (28Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 29Ray K. Hauschild B.C. J. Biol. Chem. 2000; 275: 34245-34251Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) receptors and within the second extracellular loop of m3 muscarinic receptors (30Zeng F.Y. Wess J. J. Biol. Chem. 1999; 274: 19487-19497Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). However, these disulfide bonds are dispensable for dimer/oligomerization of these receptors, indicating that non-covalent interactions are sufficient (31Tsuji Y. Shimada Y. Takeshita T. Kajimura N. Nomura S. Sekiyama N. Otomo J. Usukura J. Nakanishi S. Jingami H. J. Biol. Chem. 2000; 275: 28144-28151Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 32Romano C. Miller J.K. Hyrc K. Dikranian S. Mennerick S. Takeuchi Y. Goldberg M.P. O'Malley K.L. Mol. Pharmacol. 2001; 59: 46-53Crossref PubMed Scopus (121) Google Scholar, 33Zhang Z. Sun S. Quinn S.J. Brown E.M. Bai M. J. Biol. Chem. 2001; 276: 5316-5322Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Indeed, the N-terminal extracellular ligand-binding domain of mGluR1 forms a high affinity homodimeric complex (34Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1084) Google Scholar, 35Tsuchiya D. Kunishima N. Kamiya N. Jingami H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2660-2665Crossref PubMed Scopus (320) Google Scholar), which is likely to promote dimer formation by this receptor. Likewise, a C-terminal cytoplasmic coiled-coil motif contributes to the formation of heterodimers between GABAB-R1 and -R2 in the endoplasmic reticulum (36White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (992) Google Scholar, 37Kammerer R.A. Frank S. Schulthess T. Landwehr R. Lustig A. Engel J. Biochemistry. 1999; 38: 13263-13269Crossref PubMed Scopus (83) Google Scholar, 38Kuner R. Kohr G. Grunewald S. Eisenhardt G. Bach A. Kornau H.C. Science. 1999; 283: 74-77Crossref PubMed Scopus (491) Google Scholar, 39Calver A.R. Robbins M.J. Cosio C. Rice S.Q. Babbs A.J. Hirst W.D. Boyfield I. Wood M.D. Russell R.B. Price G.W. Couve A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar, 40Pagano A. Rovelli G. Mosbacher J. Lohmann T. Duthey B. Stauffer D. Ristig D. Schuler V. Meigel I. Lampert C. Stein T. Prezeau L. Blahos J. Pin J. Froestl W. Kuhn R. Heid J. Kaupmann K. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar). However, this coiled-coil motif is not the sole determinant of dimerization because C-terminally truncated GABABreceptors lacking this motif still interact (39Calver A.R. Robbins M.J. Cosio C. Rice S.Q. Babbs A.J. Hirst W.D. Boyfield I. Wood M.D. Russell R.B. Price G.W. Couve A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar, 40Pagano A. Rovelli G. Mosbacher J. Lohmann T. Duthey B. Stauffer D. Ristig D. Schuler V. Meigel I. Lampert C. Stein T. Prezeau L. Blahos J. Pin J. Froestl W. Kuhn R. Heid J. Kaupmann K. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar), and the extracellular N-terminal domain of GABAB-R1 heterodimerizes with that of GABAB-R2 (41Schwarz D.A. Barry G. Eliasof S.D. Petroski R.E. Conlon P.J. Maki R.A. J. Biol. Chem. 2000; 275: 32174-32181Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Whether GPCRs generally dimerize in the endoplasmic reticulum is unknown, although this would seem likely. We have used the α-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae as a model system to investigate the mechanisms of GPCR oligomerization. We have shown previously that this receptor constitutively forms homo-oligomers, as detected in living yeast cells by performing fluorescence resonance energy transfer (FRET) experiments between CFP- and YFP-tagged receptors (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). α-Factor receptor oligomers can also be detected by showing that GFP-tagged endocytosis-defective receptors interact with untagged wild type receptors to be recruited into the endocytic pathway (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 43Yesilaltay A. Jenness D.D. Mol. Biol. Cell. 2000; 11: 2873-2884Crossref PubMed Scopus (60) Google Scholar). Analysis of dominant-negative mutants of the α-factor receptor suggests that oligomerization may be important for signal transduction (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). To elucidate mechanisms of α-factor receptor oligomerization, we have characterized intracellular compartments where oligomerization occurs and identified receptor domains that mediate oligomerization in living cells. The results implicate which domains of α-factor receptors are involved in oligomerization, address whether α-factor receptors interact by monomer-monomer contact or by exchange of complementary TM domains (domain swapping), and suggest whether α-factor receptors and certain mammalian GPCRs oligomerize by employing similar mechanisms. The S. cerevisiaestrain used in this study was KBY58 (MATa ura3-52 leu2-3,112 his3Δ-1 trp1 ste2Δ) (44Stefan C.J. Blumer K.J. J. Biol. Chem. 1999; 274: 1835-1841Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), which carries a complete deletion of the α-factor receptor structural gene (STE2) that allows various combinations of receptor mutants to be expressed from plasmids. Unless stated otherwise, all receptor constructs used in this study lacked sequences encoding the C-terminal cytoplasmic domain, which is dispensable for agonist binding and signaling but which is required for receptor internalization and desensitization (45Reneke J.E. Blumer K.J. Courchesne W.E. Thorner J. Cell. 1988; 55: 221-234Abstract Full Text PDF PubMed Scopus (239) Google Scholar). Use of receptors lacking their C-terminal domain is necessary to detect FRET between CFP- and YFP-tagged α-factor receptors (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), because FRET is exquisitely sensitive to interfluorophore distance, orientation, and mobility. Because preliminary experiments indicated that deletions removing various transmembrane domains of the α-factor caused significant expression defects, it was necessary to express deletion mutants from theSTE2 promoter on high copy plasmids. To generate these overexpression plasmids, we cloned BamHI fragments containing the STE2 promoter and coding region for Ste2Δtail-YFP and -CFP fusions (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) into theBamHI sites of the high copy plasmids pRS423 and pRS424, yielding pRS423STE2Δtail-YFP and pRS424STE2Δtail-CFP. The following method was used to delete coding sequences for various transmembrane domains. First, pRS423STE2Δtail-YFP and pRS424STE2Δtail-CFP were subjected to two separate rounds of site-directed mutagenesis (Stratagene QuikChangeTMmutagenesis kit) to create a series of constructs with all possible pairwise combinations of in-frame SphI sites that introduced either a 6-bp insertion or substitution encoding an Ala-Cys dipeptide at the following positions within theSTE2-coding region (Fig. 1): bp 4–9 (substitution of amino acids Ser-2 and Asp-3); bp 137–142 (substitution of Ser-47 and Thr-48 immediately preceding TM1); bp 236–241 (insertion between Pro-79 and Ile-80 in intracellular loop 1); bp 341–346 (insertion between Thr-114 and Gly-115 in extracellular loop 1); bp 485–490 (insertion between Thr-155 and Glu-156 in intracellular loop 2); bp 593–598 (insertion between Ala-198 and Thr-199 in extracellular loop 2); bp 707–712 (insertion between Leu-236 and Gly-237 intracellular loop 3); bp 818–823 (insertion between Gly-273 and Thr-274 in extracellular loop 3); bp 897–902 (substitution of Ala-297 and Ala-298 immediately following TM7). Introduction of the Ala-Cys substitution or insertion encoded by the SphI sites at these positions preserved receptor function as indicated by quantitative assays of agonist-induced growth arrest (data not shown). These constructs were then digested with SphI and ligated to generate deletion mutations (Fig. 1), all of which were non-functional, as expected. Subcellular fractionation of yeast cell lysates was performed as described previously by equilibrium density gradient centrifugation (46Stefan C.J. Overton M.C. Blumer K.J. Mol. Biol. Cell. 1998; 9: 885-899Crossref PubMed Scopus (51) Google Scholar). Briefly, cells were grown in selective medium to a density of 107 cells/ml and were killed by addition of 10 mm NaN3 and 10 mm KF. Cells were harvested and washed once with 25 ml of sorbitol buffer (10 mm Tris, pH 7.6, 0.8 m sorbitol, 10 mm NaN3, 10 mm KF, 1 mmEDTA, pH 8.0), once with 1 ml of sorbitol buffer, once with 1 ml of sucrose buffer (10 mm Tris, pH 7.6, 1 mm EDTA, pH 8.0, 10% (w/v) sucrose), and suspended in 1 ml of sucrose buffer containing protease inhibitors 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm benzamidine, 20 μmtosylphenylalanine chloromethyl ketone, 5 μmpepstatin A, 5 μm leupeptin, and 1 mm N α-p-tosyl-l-arginine methyl ester. Cells were lysed by mechanical disruption and cleared by centrifugation at 300 × g for 5 min. The supernatant fraction was mixed with 0.5 ml of 50% (w/v) sucrose in 10 mm Tris, pH 7.6, 1 mm EDTA, and layered on top of a 4-ml, 35–60% linear sucrose gradient prepared in 10 mm Tris, pH 7.6, 1 mm EDTA. Gradients were centrifuged at 150,000 × g in an SW50.1 rotor at 4 °C for 20 h. Fractions (350 μl) were collected from the top of the gradient. Aliquots (150 μl) were assayed in FRET experiments as described below. Aliquots (50 μl) of gradient fractions were diluted 1:2 with 2× Laemmli sample buffer containing 8m urea for use in immunoblotting analysis with antibodies directed against tagged receptors and markers of various organelles (Vph1 (vacuole), Gda1 (Golgi), Dpm1 (ER), and Pma1 (plasma membrane)). Scanning fluorometry of intact yeast cells or subcellular fractions co-expressing CFP- and YFP-tagged α-factor receptors was used to detect FRET between oligomerized receptors in vivo andin vitro as described previously (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In each FRET experiment, fluorescence emission spectra were recorded from four samples that contained the same number of cells (or membrane protein for in vitro experiments) as follows: (a) control cells that do not express tagged receptors; (b) cells that express only CFP-tagged receptors; (c) cells that express only YFP-tagged receptors; and (d) cells that co-express CFP- and YFP-tagged receptors. Fluorescence emission due to FRET between CFP- and YFP-tagged receptors was detected by employing the following three-step procedure. Briefly, this involved irradiation of cells at the optimized λmax for excitation of CFP, recording emission spectra, and subtracting the components of the emission spectra due to cell autofluorescence, CFP emission, and YPF emission due to direct excitation; this resulted in the YFP emission spectrum due only to FRET. Step 1 involved data acquisition and correction for cell autofluorescence. Control cells and cells that co-express CFP- and YFP-tagged receptors were irradiated at 425 nm, near the λmax for excitation of CFP, which gives reduced direct excitation of YFP. The emission spectrum obtained from control cells (no tagged receptors expressed) was subtracted from that obtained with an equivalent number of cells co-expressing CFP- and YFP-tagged receptors, resulting in the CFP + YFP emission spectrum. This emission spectrum is a composite of CFP emission, YFP emission due to direct excitation, and YFP emission due to FRET. Step 2 involved subtraction of CFP emission from the CFP + YFP emission spectrum. Cells expressing only CFP-tagged receptors were irradiated at the optimized λmax for excitation of CFP (425 nm). This spectrum was normalized to give a CFP emission peak value identical to the CFP emission peak value of the CFP + YFP spectrum obtained from the preceding step. After normalization, the CFP spectrum was subtracted from the CFP + YPF emission spectrum. This resulted in a YFP emission spectrum composed of a FRET component and a direct excitation of YFP component. Step 3 involved obtaining the YFP emission spectrum due to FRET. To obtain the YFP emission spectrum due only to FRET, it was necessary to subtract the component of the total YFP emission that was due to direct excitation of YFP. To accomplish this, the YFP emission spectra of cells co-expressing CFP- and YFP-tagged receptorsversus cells expressing only YFP-tagged receptors were normalized for small differences in YFP expression level. This was achieved by irradiating these two types of cells at the λmax for YFP (510 nm) and recording their respective YFP emission spectra. Because CFP was not excited at this wavelength, these emission spectra quantify only the level of YFP-tagged receptors. The ratio of the YFP emission peak heights of these two spectra was then used as a scaling factor to normalize the emission spectrum obtained when cells expressing YFP-tagged receptors were irradiated at 425 nm, near the λmax for CFP. This normalized emission spectrum was then subtracted from the total YFP emission spectrum obtained in step 2 from cells co-expressing CFP- and YFP-tagged receptors. The result was a YFP emission spectrum due solely to FRET. To calculate the apparent efficiency of FRET, we used the following two spectra obtained during the process of generating the FRET emission spectrum described above: the YFP emission spectrum due specifically to FRET, and the YFP emission spectrum obtained by irradiating cells co-expressing CFP- and YFP-tagged receptors at the λmaxfor YFP. Apparent FRET efficiency was calculated by dividing the integrated area of the FRET spectrum by the integrated area of the YFP emission spectrum obtained by excitation at the λmax for YFP. Fluorescence and Nomarski images of cells expressing wild type and various receptor fragments tagged with YFP were captured by using a DAGE cooled CCD camera mounted on an Olympus BH-2 microscope equipped with a DPlanApo100UV 100× objective, as described previously (44Stefan C.J. Blumer K.J. J. Biol. Chem. 1999; 274: 1835-1841Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Endocytosis assays were performed as described previously (42Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Briefly, cells co-expressing full-length untagged wild type receptors overexpressed from thePGK1 promoter on the high copy plasmid pPGK (47Kang Y. Kane J. Kurjan J. Stadel J.M. Tipper D.J. Mol. Cell. Biol. 1990; 10: 2582-2590Crossref PubMed Google Scholar) and various tailless receptor fragments tagged with YFP expressed from theSTE2 promoter on high copy plasmids were treated with agonist (5 μm α-factor). Images were collected before and at the indicated times after agonist treatment. To identify domains of the yeast α-factor receptor involved in oligomerization, we used deletion mutagenesis to identify receptor fragments that could oligomerize as indicated by FRET. Deletion mutants rather than chimeric receptors were used to map oligomerization domains because α-factor receptors from sufficiently closely related species of yeast have not yet been cloned. The utility of deletion mutants was suggested by studies of Dumont and colleagues (48Martin N.P. Leavitt L.M. Sommers C.M. Dumont M.E. Biochemistry. 1999; 38: 682-695Crossref PubMed Scopus (63) Google Scholar), which indicated that a functional receptor can be generated by co-expressing complementary receptor fragments (e.g. TM1 expressed from one plasmid and TM2–7 expressed from another in the same cell). These findings and similar studies of mammalian GPCRs suggested that receptor fragments can be expressed in a relatively native form (34Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1084) Google Scholar, 49Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (602) Google Scholar, 50Ridge K.D. Lee S.S. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar). Consistent with this hypothesis, synthetic peptides corresponding to certain individual transmembrane segments of the α-factor receptor adopt α-helical conformations when reconstituted in lipid vesicles (51Xie H. Ding F.X. Schreiber D. Eng G. Liu S.F. Arshava B. Arevalo E. Becker J.M. Naider F. Biochemistry. 2000; 39: 15462-15474Crossref PubMed Scopus (45) Google Scholar, 52Ding F.X. Xie H. Arshava B. Becker J.M. Naider F. Biochemistry. 2001; 40: 8945-8954Crossref PubMed Scopus (38) Google Scholar). We generated deletions that removed sequences encoding the N-terminal extracellular domain or various transmembrane domains to produce all possible receptor fragments that allow a CFP or YFP tag appended at the C terminus of the molecule to be located intracellularly (Fig.1). With the exception of deletions removing TM1 or TM7, deletions of single TM domains could not be analyzed because they would disrupt the topology of the receptor. To analyze receptor mutants by FRET, all of the mutant receptors lacked a C-terminal cytoplasmic domain (which is dispensable for signaling but is required for endocytosis (45Reneke J.E. Blumer K.J. Courchesne W.E. Thorner J. Cell. 1988; 55: 221-234Abstract Full Text PDF PubMed Scopus (239) Google Scholar)) to bring CFP and YFP within sufficient proximity. Each mutant receptor tagged at the C terminus of its last TM with CFP or YFP was expressed from its normal promoter on a high copy vector, which was necessary to express receptor fragments at levels similar to that of wild type receptors (Fig.2 and data not shown). As expected, when expressed alone none of these fragments produced a functional receptor (data not shown). To determine whether receptor fragments could be useful to identify domains involved in α-factor receptor oligomerization, we determined whether FRET could detect association between pairs of complementary receptor fragments that are known to reconstitute a functional receptor (48Martin N.P. Leavitt L.M. Sommers C.M. Dumont M.E. Biochemistry. 1999; 38: 682-695Crossref PubMed Scopus (63) Google Scholar). For this purpose, we analyzed cells that co-expressed a YFP-tagged receptor fragment containing the N terminus and TM1–5 with a CFP-tagged receptor fragment containing TM6–7 (Fig. 1), or that co-expressed a YFP-tagged receptor fragment containing the N-terminal domain and TM1–3 with a CFP-tagged receptor fragment containing TM4–7 (Fig. 1). Cells were excited at 440 nm (CFP excitation maximum), and fluorescence emission spectra were recorded. After correcting spectra for differences in protein expression levels, we subtracted the emission spectra obtained with control cells (expressing only the CFP- or YFP-tagged receptor fragment fusion) from the spectrum obtained upon co-expressing the relevant pair of receptor fragments. This procedure yielded the fluorescence emission spectrum due to FRET (indicated by the solid curve in all" @default.
• W2116764040 created "2016-06-24" @default.
• W2116764040 creator A5031831888 @default.
• W2116764040 creator A5084194276 @default.
• W2116764040 date "2002-11-01" @default.
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• W2116764040 title "The Extracellular N-terminal Domain and Transmembrane Domains 1 and 2 Mediate Oligomerization of a Yeast G Protein-coupled Receptor" @default.
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