Identification of Residues of the Saccharomyces cerevisiae G Protein-coupled Receptor Contributing to α-Factor Pheromone Binding

Abstract

The Saccharomyces cerevisiaepheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 α-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by α-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]α-factor, whereas the affinity forSaccharomyces kluyveri α-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]α-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveriα-factor was abolished. The affinity of [Lys10]- and [Orn10]α-factor for the S47E,T48E receptor dropped 4–6-fold in the presence of 1 m NaCl, whereas the affinity of α-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae α-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of α-factors from two Saccharomycesspecies is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of α-factor binding to its receptor. The Saccharomyces cerevisiaepheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 α-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by α-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]α-factor, whereas the affinity forSaccharomyces kluyveri α-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]α-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveriα-factor was abolished. The affinity of [Lys10]- and [Orn10]α-factor for the S47E,T48E receptor dropped 4–6-fold in the presence of 1 m NaCl, whereas the affinity of α-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae α-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of α-factors from two Saccharomycesspecies is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of α-factor binding to its receptor. G protein-coupled receptor transmembrane domain high performance liquid chromatography [3H-Pro8,Nle12]α-factor trifluoroacetic acid wild type G protein-coupled receptors (GPCRs)1 comprise a broadly distributed class of transmembrane proteins that mediate cellular responses to extracellular signals such as light, hormones, and neurotransmitters. Upon activation, GPCRs interact with a cytoplasmic heterotrimeric G protein, leading to the exchange of GTP for bound GDP and dissociation into its subunits, which in turn activates diverse pathways mediated by molecules such as protein kinases, adenylate cyclase, phospholipases, and ion channels (1Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1132) Google Scholar, 2Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 167-183Crossref PubMed Google Scholar). GPCRs contain a common structural pattern consisting of seven hydrophobic segments that are predicted to form transmembrane helices. A detailed knowledge of the structure of GPCRs will be required to understand the determinants of ligand binding and the mechanism by which binding leads to activation of the cytoplasmic G protein. Except for a recent crystallographic structure of rhodopsin (3Placzewski 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 (5022) Google Scholar), the difficulty of obtaining, purifying, solubilizing, and crystallizing integral membrane proteins has hindered the application of x-ray crystallography to all other GPCRs. The size of the GPCR membrane complex prevents application of high resolution NMR procedures to solve the three-dimensional structure of the receptor. However, insights into the structural basis underlying the biological function of the GPCRs have come from the analysis of mutant receptors, either site-directed or chimeric, where large stretches of residues are swapped for those of a related receptor. Mutant receptors have been used to examine several aspects of receptor functions including post-translational modifications, G protein coupling, and, of most relevance to us, ligand binding. The tridecapeptide α-factor cerevisiae in by binding to Ste2p, its cognate a G protein signal that is with pathways M. PubMed Scopus Google Scholar). peptide has been studied as a model for the of peptide NMR on this peptide that the residues a structure and that this is in the of the PubMed Scopus Google Scholar, 32: PubMed Scopus Google Scholar). structural has been by of PubMed Scopus Google and analogs PubMed Scopus Google Scholar). In a recent of the of the of α-factor and of its residues or segments with in the of α-factor in receptor binding and in receptor activation and signal transduction M.G. J. PubMed Scopus Google Scholar). The structure and function of the α-factor receptor have been studied of that residues in the transmembrane domain a in receptor activation M. S. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). of the of the receptor have that the is for G protein activation T. J. PubMed Google Scholar). that the cytoplasmic is in of receptors by PubMed Google and of receptors by PubMed Google Scholar). In to the results of the results from and J. 2000; PubMed Scopus Google the of these of the receptor, and a of synthetic to of Ste2p H. 2000; PubMed Scopus Google for the of this the a of the GPCR is a in the structure and function of these receptors. the structure and function of α-factor or its receptor, have on the of the ligand binding has been on determinants in ligand binding to the α-factor receptor, a recent for the α-factor binding In α-factor receptor a in was with a binding whereas the affinities of α-factor and several or at the of were by M.G. PubMed Scopus Google Scholar). not to the wild-type receptor M.G. J. PubMed Scopus Google Scholar), we that of the binding of α-factor to the receptor of the of the with the extracellular of for α-factor binding has come from the of of the S. cerevisiae kluyveri α-factor receptors. The α-factor receptors from these two related to other I. S. PubMed Scopus Google Scholar). The S. kluyveri α-factor (WHWLSFSKGEPMY) to the S. at of residues M. PubMed Scopus Google Scholar). of these two α-factor receptors, M. J. PubMed Google Scholar, M. PubMed Scopus Google extracellular that the of ligand binding receptor in S. cerevisiae and in S. kluyveri α-factor at the the domain and were to binding and were to be a with or of the residues of the two α-factors and In of these a turn structure in residues of and the of α-factor and we that the 10th residue of α-factor with residues of the receptor. this we receptors with mutations in this and the binding affinities and biological activities of these with α-factor and its analogs in which the 10th residue was by various The used in the has been by and M. J. PubMed Google Scholar). The is α-factor receptor was used as the of the wild-type and site-directed mutant and used to and gene and to binding. 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PubMed Google Scholar). peptide in was in in and by a on a The peptide was into the and with a from to peptide from the to α-factor at were and for in a All were in trifluoroacetic and and the and activity of the peptide were with and was with of of from in were with of peptide at various and the The were at for and for the The were expressed as the of the including the of the All were at with a in size at a for The were as size the of peptide and by the activities of with receptors, the of peptide a size of a size was from the was to the and peptide to the of high on the size was of the of the in the presence of S. a gene that is by were in at to by and for at was by of peptide at various to of The were and at with for this were by and was and for activity as in by a Google of a PubMed Scopus Google Scholar, in Scholar). and were with a was at with the results similar in and binding were α-factor. In were at and at 1 by at at The were two in M.G. J. 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PubMed Scopus Google Scholar), where The of this was to the of the 10th residue of α-factor (Gln) to Ste2p with residues and of this The used was to by site-directed of Ser47 and Thr48 in the receptor and synthetic of of the α-factor The of the of the receptor and synthetic α-factor analogs are in of α-factor and and and and of were procedures on a All were by and were in two The synthetic had the acid the and residues as by site-directed Ste2p mutant receptors either mutant receptor is to the in the and receptor receptor or S47E,T48E receptor in of of the wild-type receptor were All mutations were by and other in the receptor were The binding of the wild-type and mutant receptors were by a binding The for the receptor was to that of the wild-type receptor and The affinity of was in the receptor and and in the and receptors, by the all receptors were expressed at the in These results that in this of the receptor not the binding of α-factor to the receptors or the of the receptor to the membrane and a structure for ligand for wild-type and mutant α-factor receptors with binding were wild-type or mutant receptors a are as the of at was in in a binding were wild-type or mutant receptors a are as the of at was in The induction of of S. cerevisiae by α-factor was a on The were and the of α-factor to a was compared with wild-type Ste2p, the mutant receptor was to α-factor The receptor was as to a with wild-type Ste2p the and the receptors were reduced in to In a that gene induction by the the of α-factor required to induction of gene was for the wild-type and receptor and was and for the and receptors, and The and receptors all a gene induction to that of wild-type The that all mutations in receptors that signal as as wild-type Ste2p and that the potency (EC50) of by these receptors with activity of α-factor and α-factor analogs for wild-type and mutant of α-factor were required for a in the wild-type are the of the of peptide required for a compared to the of α-factor required for a in the wild-type receptor. not of α-factor were required for a in the wild-type receptor. in a gene induction of α-factor and analogs for wild-type and mutant and were from by the are expressed as of at two of which was in activity over in a The are the of the of peptide required for a compared to the of α-factor required for a in the wild-type receptor. not and were from by the are expressed as of at two of which was in activity over The binding affinities of a of analogs of α-factor in which residue was to other residues and residues or were a binding in the were a of and the in which were of the α-factor was from the receptor. The binding that the affinity of α-factor for the receptor was that of α-factor for wild-type Ste2p The and were and These affinities differed in as compared with the for the various receptors by binding the receptor affinities the in we that binding be used to α-factor analogs bound to the various of wild-type and mutant receptors for α-factor and of α-factor and its analogs were in binding by of All are the not in a The of α-factor and its analogs were in binding by of All are the not of of the α-factor structure with a of or acid residues in a to a in affinity for wild-type Ste2p The in affinity was for the whereas the the the position-10 the bound the the bound α-factor. These results that the binding of Ste2p for the position-10 are the the biological activity of the position-10 analogs not in all with the binding be predicted by the binding affinities was and [Orn10]α-factor was However, and were and which bound the wild-type was α-factor in the The which bound as activity in the is that of the of be by the that was used and the in of the various analogs in the have this in and that of the in the not for biological the used in the is and the which α-factor. in not the in two of the analogs the gene induction in The results that the the its was compared with α-factor that a had a biological activity be on its binding binding the affinity of the was for all receptors by site-directed In the of the position-10 α-factor analogs and were in the affinities for the and receptors in to wild-type the α-factor analogs with and at the 10th in in the receptor the affinities of the and [Orn10]α-factor by a of compared with Ste2p, whereas the affinity of by and that of increased by a of at The in the affinities for the receptor were in the In this the affinity of the by a of whereas bound two to the wild-type receptor. The receptor bound as the whereas the bound this In the affinities of the analogs for the and receptors the with and [Orn10]α-factor binding and the binding was in on the α-factor from S. has in in other residues in the of the peptide The S. kluyveri α-factor has reduced binding to Ste2p in to the S. cerevisiae α-factor However, a 20-fold in affinity in the receptor and as as the S. cerevisiae α-factor. In binding to the receptor, and its binding to the and receptors was to that with the wild-type and receptors. The S. kluyveri had activity in the for all receptors the S. kluyveriα-factor and binding to the wild-type and receptors. of these these receptors be that the binding of to the receptor was not to whereas binding of to the was at high the of the the receptor, whereas the gene induction in a expressing the receptor and α-factor as a the had biological activities that were on the receptor affinities of these to the of the of various with the receptor binding we the affinities of several to the wild-type and of Ste2p in the presence of 1 m The binding of α-factor to Ste2p or the mutant receptor was not affected by high and The affinities of [Orn10]α-factor, and for wild-type receptor by a of the affinities of and analogs for the receptor by in the presence of NaCl, the affinity of on binding of α-factor and α-factor analogs to the wild-type and mutant 1 m affinity was in the presence or of 1 as were the of two in a The affinity was in the presence or of 1 as were the of two high 2000; PubMed Google Scholar), the size of the in the presence of 1 m was the size of the the activity of was compared with α-factor for the and receptors with or 1 m The activity of α-factor for the mutant receptor was from to that for receptor in the presence of 1 m by [Lys10]- and [Orn10]α-factor for and mutant receptor was greatly in the presence of NaCl, and this was for the mutant receptor. The activity of for wild-type and mutant receptors in the presence of high These results in with those of the binding that the of residue with acid residues and in the binding of the on 1 m of α-factor that a in the of was of α-factor that a in the presence of was was in the presence of 1 m in the and as The are activity of α-factor analogs for the of α-factor to a in the presence or a in the of 1 m The of α-factor that a in the of was The of α-factor that a in the presence of was in a The was in the presence of 1 m in the and as The are activity of α-factor analogs for the of α-factor to a in the presence or a in the of 1 m The was to two acid residues at the the transmembrane domain and the domain of Ste2p and the 10th residue of α-factor. The of this of the receptor was in M. J. PubMed Google Scholar, M. PubMed Scopus Google of the S. cerevisiae and S. kluyveriα-factor receptors. of the receptor was as responsible for ligand selectivity the cerevisiae and S. kluyveri α-factors not for the receptor activation Based on analysis of the of the S. cerevisiae and S. we that the 10th residue of these α-factors was responsible for ligand the residue of the S. kluyveri α-factor interact with the and residues of the S. kluyveri α-factor receptor. was by results from the of these residues into the S. receptor that high ligand selectivity for S. kluyveri α-factor M. J. PubMed Google Scholar). this in the we site-directed on Ste2p cerevisiae and the binding affinity and biological activity of a of synthetic α-factor analogs that were at the 10th that analysis of in the receptor and ligand of either receptor or binding The mutant receptors in this all binding affinities for α-factor a of of the receptor, were all expressed at the as the wild-type receptor, and were all for and gene induction by the wild-type These that the mutations not in in the receptor that are in binding of the mutant receptors insights into the of the binding of The results of the that the of residue of α-factor with the of residues of of the wild-type residues in the and the receptor that the most the of or The of was by the of of on the binding affinity of α-factor to wild-type Ste2p when the wild-type Ser47 and Thr48 residues in Ste2p were by or and the position-10 residue was for the of these was the binding affinity for with was in the whereas was increased in the mutant was not with the was with the and the S. kluyveriα-factor 1 the binding of the and α-factor analogs to the receptor by the of residues of the receptor and the peptide is to that the binding of Ste2p is of the of residues and and that these interact with the of of α-factor. the in the 10th residue of α-factor. analogs from to over binding affinity for the wild-type receptor compared with that of that the structural at the 10th of α-factor for high affinity binding to Ste2p is to to or to with dramatically the affinity [Orn10]α-factor had affinity and at affinity in affinity was when the residue of α-factor was by The of the various are and for and and H. and Scholar). The with binding affinity is with a that of the analysis of the of the size of the 10th residue of α-factor to its affinity for the mutant receptors and a complex size and of the residues of the and and residues of the receptor. with and at the 10th a of their affinities to the of of the and residues and with affinities for the receptor in with affinities for the receptor. However, the at of α-factor and not a their size and the of the of the and residues of the and receptors. in the binding of α-factor analogs with at with the and receptors. with the analysis of and their affinities with the wild-type receptor, these the that residue of α-factor with residues and of its receptor. In α-factor a in and a in binding affinity as compared with α-factor M.G. J. PubMed Scopus Google Scholar). In the of α-factor analogs we that a in was of α-factor activity PubMed Scopus Google Scholar). The the of the of at the 10th residue of α-factor for high affinity binding on a in affinity of compared with α-factor In is that the size and at the of the 10th residue are for the binding of α-factor. The for the the Ser47 and Thr48 residues of cerevisiae α-factor receptor and the of α-factor. However, is not these residues in the α-factor receptor are responsible for binding or are structural of the binding for this the of has a for that the in the interact with the binding in the receptor and with the of the receptor. The that the binding affinity of by at most a of when the wild-type and mutant receptors was However, be that in is to the in binding compared with that of the wild-type α-factor to receptor. the with a affinity to Ste2p α-factor. The binding to the and receptors are and α-factor affinity for a receptor as the the of residues at leads to in the binding affinity and the of residues leads to binding for the A similar for the binding of for wild-type Ste2p and for the receptor, that the of the binding of to that of α-factor. similar binding be for the and receptors, the binding affinity was to be this analysis the affinity of for the receptor that receptor this of the is not to that the binding for of α-factor the of Ser47 and Thr48 and in from residues the extracellular of and at the extracellular of be to the results in the the residue is to the binding for the residue of either in residues and of the receptor or in the 10th residue of α-factor be to to the binding that such of the of the of the receptor. the activity for the receptor in the and the high biological activities of for and mutant receptors compared with those of other were to the binding for the be to the when to the mutant receptor. is with that the affinity of the for the mutant receptor is the residue interact with in the or the receptor, is to dramatically the activation of the receptor by this is was However, the the receptor and the 1 m has on the affinity of this complex the of in the binding is to be to the in the receptor. The results that the receptor has biological activity with all is that the α-factor analogs with residues in interact with the membrane we this is a not the affinity and biological activity for are to the that the residue in receptor activation and α-factor binding. for the of of α-factor to residues and the extracellular domain to a model for α-factor binding and M. PubMed Scopus Google Scholar). In this the and of α-factor to the of whereas the including a turn with the extracellular In to the results of the two results this A structure in α-factor 32: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M.G. J. PubMed Scopus Google Scholar), and The high affinity and activity of α-factor at the of the K. and J. M. the of at the extracellular M. PubMed Scopus Google that residues the of and the of of the receptor are responsible for cerevisiae and S. kluyveri model is with this the residues of these into with the of the receptor. that this is we that the 1 and are for of with residues of α-factor. of the model is the of the of α-factor with and A in GPCR activation is of these binding of Scopus Google Scholar). the residue of α-factor is for receptor activation M.G. J. PubMed Scopus Google Scholar), the and with the of α-factor be in the of the signal transduction and which were as α-factor binding PubMed Google Scholar, M. PubMed Google Scholar), at the extracellular of and are for the with hydrophobic residues at the of α-factor. that binding be a that several in a K. S. J. Scopus Google Scholar, S. T. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the binding of structural of the to a binding of in a as a of and of to that with these a ligand binding was and its GPCR J. PubMed Scopus Google Scholar). In this the that the of the bound to the domain of the receptor by of the of the with the domain of the receptor. of the of or turn are common to peptide with the of acid in the a turn structure the residue is for the of activation domain and a binding domain in H. Rev. PubMed Scopus Google Scholar). The was to have a turn structure at its that is required for ligand binding M. M. PubMed Scopus Google Scholar). The of a structure was for the J. J. J. PubMed Scopus Google Scholar). the of a turn structure and the of in be to a binding of α-factor that is with the The binding domain of α-factor is by Ste2p, and the turn the of the domain to the binding model a for that at the of α-factor and whereas at the analogs that their receptor binding and activity and M.G. J. PubMed Scopus Google Scholar, J. PubMed Google Scholar, 1992; PubMed Scopus Google Scholar). this binding be with the high affinity binding and activity for the S. kluyveri α-factor and the mutant receptor and the affinity of was increased by the of and residues in the mutant receptor, the of not the for the of the of the which is for receptor In results that residues and of Ste2p interact with the of the 10th residue of cerevisiae α-factor. we that of the S. kluyveri α-factor interact with and of the S. kluyveri receptor. In we detailed insights into the for of the α-factor model is and is of most of the results in this will for and for the of as a for site-directed and ligand are to for the in the presence of high and to for