INTRODUCTION

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The -factor mating pheromone receptor that promotes the conjugation of the yeast Saccharomyces cerevisiae belongs to the large family of G protein-coupled receptors (GPCRs) (13). Receptors in this family transmit the signals for a wide range of stimuli, including light, hormones, and neurotransmitters (63). GPCRs function in a similar manner because they transduce their signal across the plasma membrane by activating a heterotrimeric guanine nucleotide binding protein (G protein) (6). In the case of the pheromone pathway, identification of the components that transduce the signal has been greatly facilitated by the genetic accessibility of yeast (15, 36, 37). Interestingly, analysis of these components has demonstrated that they are remarkably similar to the signaling components in mammalian cells including the receptors, G protein subunits, an RGS protein that regulates G protein signaling, protein kinases that form a mitogen-activated protein kinase (MAP kinase) cascade, and transcription factors (13, 22, 37).

Although the -factor receptor and other GPCRs respond to a wide range of diverse stimuli, it is interesting that they share a common structural organization in that they are all composed of seven transmembrane domains (TMDs) that are connected by intracellular and extracellular loops (6). The functional domains of the -factor receptor are also similar to other GPCRs. For example, the core region of the -factor receptor encompassing the seven TMDs carries out ligand binding and G protein activation (32, 47, 53). In addition, the third intracellular loop functions in G protein coupling (11, 49, 58), and the cytoplasmic C terminus of the -factor receptor is a target for negative regulation by phosphorylation (10, 16, 24). Unfortunately, GPCRs do not contain significant sequence similarity across the whole receptor family to help identify the functionally important residues. In spite of this lack of sequence similarity, many mammalian GPCRs can activate the pheromone signal pathway if they are expressed in yeast (28, 43, 44). The ability of GPCRs to activate G protein signaling in such heterologous cell types indicates that there are underlying similarities in the mechanisms of GPCR activation that can be explored with the help of the genetic approaches possible in yeast.

The mechanisms of GPCR activation are poorly understood, but several different approaches indicate that the TMDs play an important role in signaling. Mutagenesis studies aimed at identifying the functional domains of GPCRs have shown that the TMDs play a key role in transducing the signal across the plasma membrane (6, 13). Biochemical studies indicate that ligand binding induces specific conformational changes in the TMDs that are associated with the activated state of the receptors (14, 17, 25). In the case of the -factor receptor, a ligand-induced conformational change was identified by studies which showed that the binding of -factor to the receptor increased the accessibility of the third intracellular loop to protease digestion (7). Analysis of constitutively active GPCR mutants that signal in a ligand-independent manner suggests that the effect of ligand binding may be to relieve constraints on receptor structure and allow for isomerization to the activated conformation (30, 38, 48, 50). Altogether, these studies indicate that receptor activation results from a change in the organization and packing of TMDs (6). However, the hydrophobic nature of the TMDs has made it difficult to analyze specific structural changes, and the key changes that promote G protein activation are not known.

Polar residues in TMDs are given special emphasis in models for receptor function because of their potential to form intramolecular contacts that can determine receptor conformation (2). Polar residues are not usually found in hydrophobic membrane spanning segments, but the polar residues in GPCRs can be shielded from the nonpolar membrane environment by facing inward toward the other TMDs, since the seven TMDs of GPCRs are thought to be clustered in a bundle (46, 61). This arrangement allows some of the polar residues to form intramolecular contacts between TMDs that can play a key role in receptor structure. As an example, a salt bridge between residues in TMD3 and TMD7 of rhodopsin helps to maintain photoreceptors in the inactive state (48). A polar region at the base of TMD3, termed the DRY motif, has also been implicated in the function of some GPCRs (42, 50). However, these domains are not conserved in all receptors and are not present in the -factor receptor. In general, there are only limited data on the function of polar residues in TMDs, and their function in the -factor receptor has not been analyzed. Therefore, we investigated the effects of mutating polar residues in TMD6 of the -factor receptor. The polar residues in TMD6 were specifically targeted for analysis, because a previous study showed that a P258L mutation in TMD6 caused constitutive receptor activity, indicating that this region of the -factor receptor is important for receptor function (33). Furthermore, these polar residues are mainly located in the cytoplasmic half of TMD6 and are adjacent to the third intracellular loop of the receptor that functions in G protein activation. The analysis of the effects of these mutations on ligand binding and G protein signaling in this study identified a novel polar region of TMD6 that plays an important role in receptor signaling.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains and media. The yeast strains used are described in Table 1. Cells were grown in media described by Sherman (56). Plasmid-containing cells were grown in synthetic medium containing adenine and amino acid additives but lacking uracil to select for plasmid maintenance. Yeast transformations were performed by the lithium acetate method (52). Deletion of the -factor genes in strain JKY127-36-1 was accomplished by introducing a mf1::LEU2 mutation (35) and a mf2::his5⁺ mutation. In order to create the mf2::his5⁺ allele, primers that contained 50 bp of homology to the sequences flanking the M F 2 gene followed by 17 bp flanking the Schizosaccharomyces pombe his5⁺ gene in plasmid pME3 (kindly provided by N. Dean) were used in a PCR with pME3 as the template. The resulting mf2::his5⁺ DNA was used to transform his3 S. cerevisiae cells, and the replacement of MF2 with his5⁺ sequences was selected for by plating cells on medium lacking histidine, since the S. pombe his5⁺ gene will complement a his3 mutation in S. cerevisiae. Deletions were confirmed by PCR analysis of the genomic DNA and also by a backcross of this strain which showed that the combination of the mf1::LEU2 and the mf2::his5⁺ mutations blocked the ability of MAT cells to produce -factor.

Mutagenesis of the -factor receptor gene. Mutations were introduced into the -factor receptor gene (STE2) by PCR. Taq DNA polymerase and all other PCR reagents were purchased from Boehringer Mannheim. Plasmid pDB02 (STE2 CEN3 URA3 ARS1) (33) was used as template for all PCRs. Plasmid pDB02 was constructed by using PCR to add an SphI site 5' of the STE2 coding region (minus 837 bp) and a SacI site 3' of the coding region (695 bp downstream) and then inserting the modified STE2 fragment into pJK67, which was derived from YCplac33 (18) by destroying the endogenous AatII site by deleting the 3' overhang. The PCR primers were complementary to the STE2 sequence, except for the noted codon change required to introduce the mutation. After PCR, the 276-bp AatII-ClaI fragment containing the desired change was subcloned into pDB02 to create ste2-S251L (TCACTA), ste2-C252L (TGTCTT), ste2-Q253L (CAACTA), and ste2-S254L (TCTCTT). ste2-S259L was created by first subcloning the 605-bp AatII-PstI fragment of STE2 into the AatII-PstI sites of pBR322 to create pPD03. A 283-bp AatII-SspI PCR fragment containing the S259L (TCGCTG) change was subcloned into pPD03, and then the 605-bp AatII-PstI fragment of pPD03 was subcloned into pDB02 to create ste2-S259L. ste2-S254F was isolated by genetic screening based on the ability of this mutant to activate a FUS1-lacZ reporter gene in the absence of -factor, and then this mutant receptor gene was sequenced to identify the mutation. The methods used in this genetic screen were similar to those used previously to isolate the constitutive mutant STE2-P258L (33). To verify that the S254F mutation accounted for the constitutive activity, an AatII-ClaI fragment carrying this mutation was subcloned into pDB02. Receptor genes containing other mutations at position 254 were constructed with a heterogeneous PCR primer that randomly introduced all four bases at the three positions of codon 254. After PCR, the 276-bp AatII-ClaI fragment was subcloned into pDB02. This pool of mutant plasmids was introduced into yeast strain JKY78, and then the cells were tested for mating ability. DNA sequence analysis of receptor plasmids recovered from 18 mating-competent strains that showed constitutive activity identified nine different substitution mutations at position 254. Mutations that resulted in the substitution of Ser²⁸⁸, Ser²⁹², and Ser²⁹³ with alanine were constructed with the SURE CHANGE mutagenesis kit (Stratagene). Plasmid pDB02 was used as the template for single mutants, and a pSTE2-Q253L plasmid was used as the template for the double mutants. Mutagenic oligonucleotides were designed according to manufacturer's instructions and were complementary to the STE2 sequence, except for the indicated changes required to change the appropriate serine codon to alanine: STE2-S288A (TCTGCT), STE2-S292A (TCAGCA), and STE2-S293A (TCAGCA). Plasmids with STE2 under the control of the galactose-inducible GAL1 promoter were created by subcloning the 605-bp AatII-PstI fragment from each of the mutants into pJK57. pJK57 was constructed by using PCR to add a BamHI site to the 5' end of the STE2 open reading frame and a SacI site to the 3' end so that it could be cloned into pCTG2 downstream of the GAL1 promoter. pCTG2 was a gift from Phil James and contains a 690-bp EcoRI-BamHI fragment containing the GAL1 promoter inserted into pRS314 (57). All of the mutations constructed in this study were confirmed by dideoxy sequencing of the double-stranded DNA with Sequenase (U.S. Biochemical).

-Factor receptor analysis. Western immunoblots were carried out essentially as described elsewhere (32). Mid-logarithmic-phase cells (2.5 × 10⁸) were harvested and lysed by agitation with glass beads in 250 µl of lysis buffer (2% sodium dodecyl sulfate [SDS], 100 mM Tris [pH 7.5], 8 M urea). A 100-µg amount of protein extract, as determined by the bicinchoninic acid protein assay kit (Pierce), was separated by electrophoresis on an 10% SDS-polyacrylamide gel, electrophoretically transferred to nitrocellulose, and probed with rabbit anti-Ste2p antibodies (32). Immunoreactive proteins were detected by chemiluminescence with an ECL kit (Amersham). -Factor binding assays were conducted essentially as described elsewhere (49). Logarithmic-phase cells were collected by centrifugation, washed twice with ice-cold inhibitor medium (IM; YEPD medium containing 10 mM KF and 10 mM NaN₃), and resuspended at a density of 10⁹ cells/ml. A 50-µl volume of cells was mixed with 50 µl of ³⁵S--factor and incubated for 30 min, and then the cells were collected on a Whatman GF/C filter and the unbound -factor was removed by washing. Nonspecific binding was determined by performing reactions in the presence of a 100-fold excess of cold -factor. Scatchard plots represent the averages of three to six independent assays done in duplicate. Similar results were obtained when the data were plotted by the method of Klotz (31). ³⁵S-labeled -factor was purified from the supernatant of MAT cells labeled with [³⁵S]SO₄ by chromatography on a Bio-Rex 70 column as described previously (49).

-Factor-induced responses. To examine the ability of cells to mate, patches of yeast strain JKY78 carrying the indicated STE2 allele on a plasmid pJK67 were replica plated to YPD plates containing a lawn of MAT cells (lys1). The cells were incubated at 30°C for 12 h to allow mating and were then replica plated to minimal plates lacking amino acids, incubated at 30°C for 48 h to select for the growth of diploids, and then photographed. Quantitative mating assays were performed by mixing 3 × 10⁶ lys1 cells with various dilutions of MATa cells containing the indicated mutant receptor plasmids on a synthetic medium plate lacking lysine. The plates were incubated at 30°C for 72 h, and then efficiency of mating was determined by counting the number of diploid colonies that were formed at each dilution of cells. To assay induction of FUS1-lacZ in far1 cells, cultures were grown overnight to logarithmic phase in selective medium, diluted to 4 × 10⁶ cells/ml and incubated with the indicated concentrations of synthetic -factor (Bachem) for 2 h. Inductions were stopped by adding cycloheximide (final concentration, 10 µg/ml), and then -galactosidase assays were performed by using the colorimetric substrate O-nitrophenyl--D-galactopyranoside (ONPG) as described elsewhere (41). Basal levels of FUS1-lacZ expression were determined as described above, except that the cells were incubated in the absence of -factor. To assay the effects of galactose-induced expression of receptor mutants, the cells were first grown overnight in synthetic medium containing raffinose and diluted to 4 × 10⁶ cells/ml, galactose was added to the medium at a final concentration of 2%, and then after a 5-h incubation the cells were assayed for FUS1-lacZ induction as described above. Induction of FUS1-lacZ was assayed in ste5-3^ts cells (JKY79) that were grown overnight to logarithmic phase in selective medium at 34°C which was adjusted to 2.5 × 10⁶ cells/ml and then shifted to 23°C for 8 h. -Factor was added to a final concentration of 10⁶M to induce signaling in the wild-type cells. Inductions were stopped by adding cycloheximide (final concentration, 10 µg/ml), and then -galactosidase assays were performed in duplicate with the colorimetric substrate chlorophenyl red--D-galactopyranoside (CPRG). The results of at least two independent assays, each done in duplicate, are reported.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Mutation of polar residues in TMD6. Our previous studies implicated TMD6 in -factor receptor activation because a mutation of Pro²⁵⁸ to Leu (P258L) in TMD6 caused constitutive receptor activity in the absence of -factor (33). To analyze the mechanisms of receptor activation further, we screened for additional constitutive mutations and identified another mutation in TMD6, S254F, that also caused constitutive signaling (see Materials and Methods). Interestingly, Ser²⁵⁴ is one of five polar residues found in TMD6 that may interact with the other TMDs to influence receptor structure. Therefore, the function of the polar amino acids in TMD6 of the -factor receptor was examined by mutating the corresponding codons in the receptor gene (STE2) to leucine codons by site-directed mutagenesis (see Materials and Methods). As shown in Fig. 1, the most polar amino acids in TMD6 include Ser²⁵¹, Cys²⁵², Gln²⁵³, Ser²⁵⁴, and Ser²⁵⁹. In addition, we also studied the ste2-S254F mutant that was isolated by genetic screening for constitutive -factor receptor mutants. To confirm that the mutant receptor proteins were produced, the plasmids carrying the mutant receptor genes were introduced into a yeast strain (JKY78) lacking the wild-type STE2 gene, and then cell extracts were analyzed by Western blotting (Fig. 2). As expected, the wild-type receptor protein (Ste2p) was detected as multiple bands on Western blots due to the N-linked glycosylation of the receptor (4). The mutant receptor proteins displayed a similar heterogeneous pattern, indicating that they entered the secretory pathway efficiently. All of the mutants appeared to produce similar levels of receptor protein, which was slightly less than the wild-type level.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Polar residues in TMD6 of the -factor receptor. The predicted two-dimensional topology of the receptor in the plasma membrane, with the extracellular domain at the top, is shown. The expanded view of TMD6 details the relative positions of amino acid residues. The polar amino acid residues investigated in this study are in boldface.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Immunoblot analysis of mutant receptor proteins. Extracts of yeast strain JKY78 carrying the indicated wild-type or mutant STE2 allele on plasmid vector pJK67 were separated by electrophoresis on an SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with affinity-purified rabbit anti-Ste2p antibodies (32). Immunoreactive proteins were detected by chemiluminescence. The relative molecular weights of prestained molecular weight markers (BioRad) are noted to the left.

Ligand responsiveness of mutant receptors. The overall ability of the mutant receptors to function was assessed by examining the mating ability of JKY78 cells carrying the mutant STE2 genes on plasmids. As shown in Fig. 3, MATa cells carrying the wild-type STE2 gene mated with MAT cells, as evidenced by formation of diploid cells that grew on selective medium. Similarly, all of the cells carrying mutant receptor genes mated. Some of the mutant strains appeared to mate poorly compared to wild type; thus, quantitative mating assays were carried out (Fig. 3). Of the group, the ste2-S259L mutant mated the most poorly, with an efficiency of 1.4% of that of the wild type. The ste2-S251L and -S254F mutants mated with slightly better efficiency (7.5 and 5.4%, respectively), and the other mutants (ste2-C252L, -Q253L, and -S254L) mated essentially as well as the wild type. Thus, all of the mutants are at least partially functional to carry out the full range of receptor activities.

View larger version (82K): [in this window] [in a new window]   FIG. 3.   Mating efficiencies of receptor mutants. Mating abilities were tested by replica plating patches of MATa strain JKY78 carrying the indicated wild-type or mutant STE2 allele on plasmid vector pJK67 to a lawn of MAT cells (lys1). After the cells were allowed to mate, they were replica plated to selective plates for 2 days at 30°C, and then the growth of diploid cells that were formed by mating was photographed. Mating efficiencies are expressed as a percentages of wild type as determined by quantitative mating analysis.

7

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Ligand-dependent activation of a FUS1-lacZ reporter gene by mutant receptors; dose-response assays for yeast strain JKY78 carrying the indicated wild-type (WT) or mutant STE2 allele on plasmid vector pJK67. Logarithmic-phase cells were incubated with the indicated concentrations of -factor for 2 h and then assayed for -galactosidase activity to measure the induction of the pheromone-responsive FUS1-lacZ reporter gene. The results represent two to six independent assays, each done in duplicate. The standard deviation was less than 8% for all data points. (A) Results for the ste2-Q253L and -S254F mutants; (B) results for the ste2-S251L, -C252L, -S254L, and -S259L mutants.

Ligand-binding properties of mutant receptors. Radioligand-binding assays were performed to determine whether mutations affecting the polar residues altered the ligand-binding properties. Ligand binding is a sensitive measure of the structure of mutant receptors, because the -factor binding pocket is formed by the region of the receptor that contains the seven TMDs. Equilibrium-binding assays were carried out on whole cells with ³⁵S-labeled -factor, and then the data were analyzed by the Scatchard method. Scatchard plots for the wild type and the mutants that had the strongest effect on binding (ste2-Q253L and -S254F) are shown in Fig. 5. Binding data for the wild type and for all of the mutants are summarized in Table 2. The K_d for wild-type receptors was 10 nM, which is in agreement with previous studies (27, 33). Interestingly, the mutants all bound -factor with an affinity equal to or greater than that of the wild-type receptors. The mutation affecting the most polar residue in TMD6, Q253L, had the strongest effect on binding. The K_d for the binding of -factor to ste2-Q253L cells was 25-fold lower than that for wild-type cells, indicating that these cells have significantly increased affinity for -factor. The mutations affecting Ser²⁵⁴ also increased binding affinity: the ste2-S254F and the ste2-S254L mutants showed 11- and 5-fold increased affinities, respectively. The ste2-S259L mutant displayed 3.7-fold increased affinity, and the ste2-S251L and -C252L mutants bound -factor with about the same affinity as that of the wild type. Thus, none of the polar residues were essential for ligand binding, and, in fact, binding affinity was improved in some mutants. Increased binding affinity for the mutant receptors is interesting, because an increase in binding affinity is often associated with the activated conformation of GPCRs (5, 38, 50).

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Radioligand-binding studies. Scatchard plot analysis of wild-type (A), Q253L (B), and S254F (C) receptors. Equilibrium binding studies were carried out by measuring the binding of 35S--factor to strain JKY78 carrying the indicated wild-type or mutant STE2 allele on plasmid vector pJK67. The small upper panels are plots of the bound versus the free -factor under the conditions used. The lower panels show the results plotted by the Scatchard method. Lines, best fits of the data determined by the least-squares method; data points, averages of three to eight independent assays, each done in duplicate.

Constitutive receptor activity. The dose-response assays shown in Fig. 4 indicated that at least two of the mutants, ste2-Q253L and -S254F, displayed elevated levels of basal signaling. To facilitate the analysis of this ligand-independent signaling activity, plasmids containing mutant receptor genes were transformed into a yeast strain (JKY79) that carries a temperature-sensitive mutation in a postreceptor component of the pheromone signaling pathway, ste5-3^ts (21). This strain also carries a pheromone-responsive FUS1-lacZ reporter gene and lacks the chromosomal copy of the receptor gene to prevent interference from the wild-type receptors. The advantage of incorporating the ste5-3^ts mutation into the strain is that it allows for propagation of the cells without activation of the pheromone pathway by growing the cells at the restrictive temperature (34°C), at which there is essentially no basal activation of the FUS1-lacZ reporter gene. The pheromone signal pathway can then be made competent by shifting the cells to the permissive temperature (23°C). When the ligand-independent activation of the FUS1-lacZ reporter gene was determined at 23°C, two of the mutants, ste2-Q253L and -S254F, displayed significantly elevated basal levels of signaling (Table 3). The basal activity of ste2-Q253L was elevated by about 5-fold, and that of ste2-S254F was elevated 2.4-fold compared to the wild type.

View this table: [in this window] [in a new window]   TABLE 3.   Constitutive activities of -factor receptor mutants

6

Basal levels of signaling in an sst2 adaptation-defective strain. In order to gain increased sensitivity for the analysis of the basal signaling by the mutant receptors, we analyzed the effects of the mutants in an adaptation-defective sst2-1 strain. These cells are more sensitive to -factor because of the inability of Sst2p to regulate the pheromone-responsive G subunit (1, 8). To help maintain a low basal level of signaling in these cells, the genes encoding -factor were deleted. This prevents the rare cells that switch mating type from producing -factor that could otherwise stimulate this supersensitive strain. The cells also carry a mutation in the FAR1 gene to prevent pheromone-induced cell division arrest (9). Analysis of the ste2-Q253L and -S254F mutants in this strain showed that they both caused greater than a 10-fold increase in the basal level of signaling (Fig. 6). These results demonstrate that the elevated basal level of signaling by these mutants is not due to autocrine signaling, because the -factor genes are deleted from this strain. Furthermore, the detection of elevated basal signaling by the ste2 mutants in an sst2 strain shows that the constitutive receptor activity is independent of SST2 and is not a consequence of a defect in SST2-mediated adaptation. Interestingly, the ste2-S254L mutant showed a readily detectable sixfold increase in basal signaling in this sst2 strain that was not obvious in the SST2⁺ strain. The elevated level of basal signaling detected for the ste2-S254L mutant in this strain demonstrates that the sst2-1 mutation had the predicted effect of increasing the sensitivity of the assay. In spite of this, the other receptor mutants (ste2-S251L, -C252L, and -S259L) did not show a significant increase in basal activity. These results demonstrate that in comparison to the other polar residues in TMD6, Gln²⁵³ and Ser²⁵⁴ play a special role in receptor function.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Analysis of the basal signaling levels by receptor mutants in an sst2 adaptation-defective strain. Supersensitive sst2-1 strain JKY127-36-1 carrying the indicated wild-type or mutant STE2 allele on plasmid vector pJK67 was assayed for -galactosidase activity to measure the basal level of the pheromone-responsive FUS1-lacZ reporter gene in the absence of added -factor. The results are averages of two independent assays done in duplicate (± standard deviations).

Effects of different substitution mutations at residue 254. The observation that substitution of Ser²⁵⁴ with Phe caused greater constitutive activity than substitution with Leu suggested that the type of amino acid substituted at position 254 could influence the degree of constitutive activity. To examine this in more detail, site-directed mutagenesis was used to introduce all possible codon combinations at this position, and then the pool of mutant receptor plasmids was introduced into yeast cells for analysis (see Materials and Methods). Yeast colonies that contained functional receptors were identified by testing for mating ability, and then nine different constitutive mutations were identified after sequencing 18 independent isolates. The basal level of signaling was at least slightly increased in all of the mutant strains (Fig. 7). Substitution of Ser²⁵⁴ with Ala, Asp, Val, Gln, or Tyr all caused about a 2-fold increase in the basal level of signaling, indicating that the loss of serine at position 254 promoted higher basal levels of signaling. Higher levels of basal signaling (3- to 7.5-fold) were observed when Ser²⁵⁴ was substituted with Gly, Leu, Phe, or Trp, indicating that the insertion of these residues caused an additional effect on constitutive signaling. All of the mutants with nonaromatic amino acids at residue 254 (Gly, Ser, Ala, Asp, Val, Gln, and Leu) displayed the ability to maximally induce a reporter gene (Fig. 4 and data not shown), indicating that these receptors were not obviously impaired in signaling. However, substitution of Ser²⁵⁴ with aromatic amino acids (Phe, Tyr, or Trp) resulted in a partial defect in -factor-induced signaling (Fig. 4 and data not shown), indicating that the substitution of Ser²⁵⁴ with these larger amino acids altered receptor structure in a way that also adversely affected G protein activation. Thus, the basal signaling activity of this latter group of mutants may be slightly lower than might otherwise be expected based on the correlation with the type of amino acid substituted at position 254 (see Discussion). Altogether, the differential abilities of the various substitution mutants to promote ligand-independent activation of the receptor indicate that Ser²⁵⁴ is located within a region of the receptor that can alter the structure of the TMDs in a way that mimics the activated state of the receptor.

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Constitutive signaling activity of Ser254 substitution mutants. Strain JKY127-36-1 carrying the wild-type STE2 allele or the indicated substitution mutations affecting Ser254 on plasmid vector pJK67 was assayed for -galactosidase activity in the absence of -factor to determine the basal levels of the pheromone-responsive FUS1-lacZ reporter gene. The different mutants are listed in order of increasing size of the residue substituted at position 254. Results are fold increases in basal activity over that of the wild type and are the averages of two independent assays, each done in duplicate (± standard deviations).

Analysis of potential contact residues. The phenotypes caused by mutations affecting Gln²⁵³ and Ser²⁵⁴ suggest that these residues may form important contacts with the other TMDs. Inspection of the -factor receptor sequence indicates that there are more than 40 residues in the predicted TMDs that may be capable of forming hydrogen bond interactions. As an approach to identifying potential contact residues for Gln²⁵³ and Ser²⁵⁴, we reasoned that mutations affecting the contact residues should cause constitutive activity similar to the effects of mutating Gln²⁵³ and Ser²⁵⁴. To focus our efforts on identifying residues that may contact TMD6, we analyzed the effects of mutating the polar residues in TMD5 and TMD7, since they are likely to be adjacent to TMD6 in the receptor (2). In addition, we further narrowed our analysis to examining the effects of mutating the residues in the cytoplasmic halves of TMD5 and -7 that are likely to be in a position to interact with either Gln²⁵³ or Ser²⁵⁴ in TMD6.

View larger version (25K): [in this window] [in a new window]   FIG. 8.   Mutational analysis of polar residues in TMD7. Strain JKY127-36-1 carrying the indicated wild-type or mutant STE2 allele on plasmid vector pJK67 was assayed for -galactosidase activity to determine the relative levels of the pheromone-responsive FUS1-lacZ reporter gene. Cells were assayed in the absence of -factor (A) and in the presence of 106 M -factor (B) to determine the basal and pheromone-induced levels, respectively. The results are the averages of two independent assays, each done in duplicate (± standard deviations).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In order to gain insight into the mechanisms of GPCR activation, we investigated the effects of mutating the polar residues in TMD6 of -factor receptor. The function of polar residues was examined because they have the ability to form contacts between TMDs that can play a key role in receptor structure. We focused on the polar residues in TMD6 because of the previous identification of constitutive receptor mutations in this domain. Also, TMD6 is adjacent to the third intracellular loop that plays a key role in G protein activation. The mutant receptor proteins were produced at levels similar to that for the wild type and were glycosylated, indicating that they enter the secretory pathway efficiently. However, the mutant cells contained fewer surface receptors for -factor than wild-type cells. This decrease in cell surface receptors suggests that the mutations affecting the polar residues altered receptor structure in a way that was recognized by the mechanisms that control the trafficking of receptors to or from the plasma membrane (23, 26, 59). The decrease in receptor number was not so high as to prevent detection of receptor signaling, because all of the mutant receptors showed at least partial ability to respond to -factor. Evolutionarily conserved polar residues in TMDs are often given special emphasis in molecular models for receptor function (2, 3), so it is worth noting that two of the polar residues in TMD6 (Ser²⁵¹ and Gln²⁵³) are conserved in the pheromone receptors from Saccharomyces kluyveri (40) and Schizosaccharomyces pombe (29). Mutations affecting Gln²⁵³ had significant effects on ligand binding and signaling. In contrast, mutations affecting Ser²⁵¹ did not appear to alter receptor function aside from lowering the number of cell surface binding sites. This indicates that some conserved polar residues, such as Ser²⁵¹, may be more important for proper receptor production than for signal transduction.

Effects on ligand binding. Previous studies have shown that -factor binding is mediated by the central region of the -factor receptor that contains the TMDs (32, 47). Studies of chimeric receptors formed between the -factor receptors from S. cerevisiae and S. kluyveri further indicate that ligand binding is carried out by noncontiguous domains within this central region (39, 53). It was, therefore, very interesting that mutating the most polar residue in TMD6 to Leu (Q253L) resulted in 25-fold increased binding affinity. A mutation affecting the adjacent Ser²⁵⁴ residue (S254L) also caused a significant increase in affinity (5-fold). The mutated residues are unlikely to be directly involved in ligand binding, because two different substitutions at position 254 (S254L and S254F) both increased binding affinity. Instead, these results suggest that the mutated residues influence binding affinity indirectly by altering the packing of the TMDs. Increased binding affinity may result from the abilities of these mutants to mimic the activated state of the receptor, because they also displayed constitutive receptor activity. Many GPCRs display increased affinity for ligand when they are coupled to a G protein; thus, it has been suggested that the increased affinity of constitutive mutants is due to their ability to mimic the activated state of the receptor (38). Altogether, these results suggest that the polar residues in TMD6, particularly Gln²⁵³ and Ser²⁵⁴, influence the packing of the TMDs in a way that affects the ligand binding pocket.

Effects on constitutive signaling. Mutations affecting a subset of the polar residues in TMD6 also altered receptor structure in a way that caused higher basal levels of signaling in the absence of -factor. In particular, mutation of the Ser²⁵⁴ and Gln²⁵³ residues increased basal signaling. To examine the strength of this constitutive receptor activity, we made use of an ste5^ts strain (Table 3) so that we could compare the levels of constitutive signaling by the mutants with the maximum levels of -factor-induced signaling by wild-type receptors. In these studies, we found that, in the absence of -factor, ste2-Q253L signaled at 19% and ste2-S254F signaled at about 9% of the ligand-induced maximum. The ste2-S254L mutant displayed weaker constitutive activity that was not readily detected in the ste5^ts strain, but in a supersensitive sst2 strain the ste2-S254L receptors increased basal signaling by about fivefold above that of background. In contrast, the basal signaling levels were not significantly increased by the ste2-S251L, -C252L, or -S259L mutant receptors, even in a supersensitive strain. These results indicate that Gln²⁵³ and Ser²⁵⁴ in TMD6 play a special role in stabilizing receptors in the inactive conformation and in preventing constitutive activity in the absence of ligand.

Orientation of TMD6. The effects of mutating Gln²⁵³ and Ser²⁵⁴ described above, as well as the previously described effects of mutating Pro²⁵⁸ (33), indicate that these residues in TMD6 play a special role in the structure of the -factor receptor. To examine the potential significance of these residues further, the amino acids of TMD6 were displayed on a helical wheel projection to predict their orientation in an -helix, as shown in Fig. 9. TMD6 of the -factor receptor is assumed to form an -helix similar to those of the TMDs in rhodopsin and other GPCRs (2, 61). Consistent with this, a synthetic peptide corresponding to the sequence of TMD6 exhibited helical character (45). Interestingly, the residues in TMD6 that cause constitutive activity when mutated (Gln²⁵³, Ser²⁵⁴, and Pro²⁵⁸) are predicted to reside on one side of the transmembrane helix. In contrast, the polar residues that did not cause constitutive activity when mutated to leucine (Ser²⁵¹, Cys²⁵², and Ser²⁵⁹) all lie on the opposite side of TMD6. This alignment indicates that the side of TMD6 containing Gln²⁵³ and Ser²⁵⁴ is likely to be involved in interactions with other TMDs. Furthermore, Gln²⁵³ is likely to be facing inward toward the other TMDs, since it is the most polar residue in TMD6. Thus, the polar nature of Gln²⁵³ and Ser²⁵⁴ and the phenotypes caused by mutations that affect these residues indicate that they are likely to face inward toward the other TMDs and to be in a position to have cooperative effects on the packing arrangement of the other TMDs.

View larger version (35K): [in this window] [in a new window]   FIG. 9.   Helical wheel diagram of TMD6. The residues of TMD6 are plotted on a helical wheel diagram to display their relative orientations in an -helix as viewed from the cytoplasmic side of the plasma membrane. Polar residues that were mutated as part of this study are boxed. Pro258 is also boxed, because a ste2-P258L mutation was identified previously as causing constitutive activity (33). The black boxes with white letters identify the residues that caused constitutive activation of the receptor when they were mutated to leucine. Interestingly, these residues were all located on one side of the helical wheel as shown. The white boxes with black letters on the opposite side of the wheel identify the residues that did not cause constitutive activity when they were mutated to leucine. Although proline residues can perturb the structure of an -helix, Pro258 is not expected to cause a significant change in the relative orientations of the residues in this model, because proline residues are well tolerated in the transmembrane helices of membrane proteins (19, 62).

Mechanism of receptor activation. Polar residues have been implicated in the activation of GPCRs because of their ability to form intramolecular contacts. In the case of rhodopsin, a salt bridge between TMD3 and TMD7 is thought to keep rhodopsin in the inactive conformation until photoactivation of the retinal chromophore leads to disruption of the salt bridge and subsequent receptor activation. This mechanism, which is specific for the photoreceptors, was confirmed in part by showing that mutations affecting the residues that form the salt bridge caused constitutive signaling in the dark (48). Another polar region, which is known as the DRY motif, is thought to play a key role in activation of a broader range of GPCRs, including the members of the rhodopsin-adrenergic receptor family (42, 50). The DRY motif, which is found at the cytoplasmic end of TMD3, is proposed to form a polar pocket in conjunction with polar residues from other TMDs until protonation of the Asp (or Glu) residue in the DRY motif causes it to move out of the polar pocket and to permit receptor activation. Consistent with this, certain mutations in this sequence cause constitutive receptor activity and increased affinity for ligand (51). Although this motif is found in many GPCRs, its absence from the pheromone receptors and other receptors indicates that additional mechanisms must exist to regulate receptor activation. In this study, we identified a novel polar region in TMD6 of the -factor receptor that influences ligand binding and G protein activation. Since this domain occurs in the cytoplasmic half of TMD6 adjacent to the third intracellular loop and since the third intracellular loop is involved in receptor activation, it is expected that conformational changes in this part of TMD6 can play a significant role in receptor activation. This aspect of receptor regulation may be conserved in some of the other GPCRs, because constitutively active mutations in the luteinizing hormone (55) and the thyrotropin receptors (34) that are caused by mutation of polar residues in TMD6 have been discovered. The constitutive activity caused by these mutations has been implicated in their ability to cause precocious puberty and hyperfunctioning thyroid adenomas in humans (34, 55). Thus, analogous polar pockets in TMD6 of other GPCRs may function in regulation of signaling and consequently may also be important sites for mutations that cause human diseases.