Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands

Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle(12)]-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle(12)]-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle(12)]-alpha-factor or [Orn7(NBD),Nle(12)]-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.     

A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)?Ala(3)?lpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.     

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.     

A covalently constrained congener of the Saccharomyces cerevisiae tridecapeptide mating pheromone is an agonist

An analog of alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), in which the side chains of Lys7 and Gln10 were covalently linked, was synthesized using solid phase methodologies. The yield of the purified cyclic analog cyclo7,10[Nle12]alpha-factor was 30%, and its structure was verified by amino acid analysis, peptide sequencing, fast atom bombardment-mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Cyclo7,10[Nle12]alpha-factor caused growth arrest and morphological alterations in S. cerevisiae MATa cells qualitatively identical to those induced by linear pheromone and was one-fourth to one-twentieth as active as the linear alpha-factor depending upon the S. cerevisiae strain tested. Consistent with the relative activities of the linear and cyclic peptides, binding competition studies indicated that cyclo7,10[Nle12]alpha-factor had approximately 20-40-fold less affinity for the alpha-factor receptor. Hydrolysis of the cyclic peptide by the target cells did not lead to opening of the ring and was less rapid than that of linear alpha-factor. The alpha-factor antagonist des-Trp1-[Ala3,Nle12]alpha-factor reversed the activity of the cyclic analog, and cyclo7,10[Nle12]alpha-factor was not active at the restrictive temperature in a temperature-sensitive receptor mutant. These results support the conclusion that the cyclic alpha-factor occupies the same binding site within the receptor as is occupied by the natural pheromone. The cyclic alpha-factor represents a rare example of an agonist among covalently constrained congeners of small linear peptide messengers.     

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.     

Subunits of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers

G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast alpha-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding- and G protein coupling-defective alpha-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding- and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the alpha-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.     

Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.     

Receptor binding of NBD-labeled fluorescent estrogens and progestins in whole cells and cell-free preparations

We have studied the interactions of four fluorescent steroid conjugates with either the estrogen or progesterone receptor, both in whole cells and cell-free receptor preparations. The fluorophore, nitrobenzoxadiazole (NBD), was conjugated with a synthetic progestin, with a steroidal estrogen, a non-steroidal estrogen, and with an antiestrogen. With all compounds, receptor-specific binding could be detected by fluorescence measurements following extraction from the protein into an organic solvent. In the native state, however, the NBD-ligand-receptor complex is essentially non-emissive, although these ligands fluoresce strongly when associated with non-specific binders such as albumin. The binding site concentrations and relative affinities determined by fluorescence (after extraction) correspond well with those determined by [3H]estradiol or [3H]R5020 binding to their respective receptors. In T47D breast cancer cells, the NBD-progestin showed receptor-mediated uptake and nuclear localization. These compounds have provided valuable information about the interactions of low and medium affinity ligands with their receptors; however, the successful use of fluorescent ligands for detecting steroid receptors under native-bound conditions, by "imaging" modalities (fluorescence microscopy and flow cytometry) will require the development of fluorophores that are emissive while receptor bound or assay protocols that enable the environment of ligands associated with the receptor to be controlled.     

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.     

Determinants of high-affinity binding and receptor activation in the N-terminus of CCL-19 (MIP-3 beta)

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.     

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.     

Oligomerization of the yeast alpha-factor receptor: implications for dominant negative effects of mutant receptors

Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the alpha-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated alpha-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated alpha-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of alpha-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers.     

Synthetic probes for the alpha-factor receptor

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae

alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.

The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.     

Double-mutant cycle scanning of the interaction of a peptide ligand and its G protein-coupled receptor

The interaction between the yeast G protein-coupled receptor (GPCR), Ste2p, and its alpha-factor tridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwise interaction energy of each ligand residue with two receptor residues, N205 and Y266, was determined. The mutations N205A and Y266A were previously shown to result in deficient signaling but cause only a 2.5-fold and 6-fold decrease, respectively, in the affinity for alpha-factor. The analysis shows that residues at the amine terminus of alpha-factor interact strongly with N205 and Y266 whereas residues in the center and at the carboxyl terminus of the peptide interact only weakly if at all with these receptor residues. Multiple-mutant thermodynamic cycle analysis was used to assess whether the energies of selected pairwise interactions between residues of the alpha-factor peptide changed upon binding to Ste2p. Strong positive cooperativity between residues 1 through 4 of alpha-factor was observed during receptor binding. In contrast, no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminus of alpha-factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutant cycle analyses of the binding of an alanine-scanned peptide to wild-type and mutant GPCRs can provide detailed information on contributions of inter- and intramolecular interactions to the binding energy and potentially prove useful in developing 3D models of ligand docked to its receptor.     

Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.