Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor

The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.     

Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor

The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis.     

Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor

Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.     

Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor

The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains. NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule. The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies.     

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.     

Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding

The Saccharomyces cerevisiae pheromone, alpha-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 alpha-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 alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors

Mating in Saccharomyces cerevisiae is initiated by the secretion of diffusible peptide pheromones that are recognized by G protein-coupled receptors (GPCR). This review summarizes the use of the alpha-factor (WHWLQLKPGQPMY)--GPCR (Ste2p) interaction as a paradigm to understand the recognition between medium-sized peptide hormones and their cognate receptors. Studies over the past 15 years have indicated that the alpha-factor is bent around the center of the pheromone and that residues near the amine terminus play a central role in triggering signal transduction. The bend in the center appears not to be rigid and this flexibility is likely necessary for conformational changes that occur as the receptor switches from the inactive to active state. The results of synthetic, biological, biochemical, molecular biological, and biophysical analyses have led to a preliminary model for the structure of the peptide bound to its receptor. Antagonists for Ste2p have changes near the N-terminus of alpha-factor, and mutated forms of Ste2p were discovered that appear to favor binding of these antagonists relative to agonists. Many features of this yeast recognition system are relevant to and have counterparts in mammalian cells.     

The chain length dependence of helix formation of the second transmembrane domain of a G protein-coupled receptor of Saccharomyces cerevisiae

The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the alpha-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disordered, and all of the other peptides assumed partially alpha-helical secondary structures in 99% trifluoroethanol (TFE)/H(2)O. In 50% TFE/H(2)O, M2-30 assumed a beta-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H(2)O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed alpha-helical structures, whereas the other peptides formed beta-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed beta-structures in this environment. Another homologous 30-residue peptide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were alpha-helical and oriented at angles of 12 degrees, 13 degrees, 36 degrees, and 34 degrees, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.     

Structural determinants for ligand-receptor conformational selection in a peptide G protein-coupled receptor

G protein coupled receptors (GPCRs) modulate the majority of physiological processes through specific intermolecular interactions with structurally diverse ligands and activation of differential intracellular signaling. A key issue yet to be resolved is how GPCRs developed selectivity and diversity of ligand binding and intracellular signaling during evolution. We have explored the structural basis of selectivity of naturally occurring gonadotropin-releasing hormones (GnRHs) from different species in the single functional human GnRH receptor. We found that the highly variable amino acids in position 8 of the naturally occurring isoforms of GnRH play a discriminating role in selecting receptor conformational states. The human GnRH receptor has a higher affinity for the cognate GnRH I but a lower affinity for GnRH II and GnRHs from other species possessing substitutions for Arg(8). The latter were partial agonists in the human GnRH receptor. Mutation of Asn(7.45) in transmembrane domain (TM) 7 had no effect on GnRH I affinity but specifically increased affinity for other GnRHs and converted them to full agonists. Using molecular modeling and site-directed mutagenesis, we demonstrated that the highly conserved Asn(7.45) makes intramolecular interactions with a highly conserved Cys(6.47) in TM 6, suggesting that disruption of this intramolecular interaction induces a receptor conformational change which allosterically alters ligand specific binding sites and changes ligand selectivity and signaling efficacy. These results reveal GnRH ligand and receptor structural elements for conformational selection, and support co-evolution of GnRH ligand and receptor conformations.     

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.     

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.     

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product. Received: 15 January 1999 / Accepted: 25 March 1999     

Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.     

Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.     

Molecular characterization of a new leucine-rich repeat-containing G protein-coupled receptor from a bivalve mollusc: evolutionary implications

The family of leucine-rich repeat-containing G protein-coupled receptors (LGRs) shows members in both vertebrates and invertebrates including the most ancestral ones. Although this suggests an early evolutionary origin of this family of receptors, little is known about their diversity in molluscs, a major phylum of bilaterian invertebrates. Based on sequences of mammalian and insect LGRs, we have cloned and characterized a new typical LGR in the bivalve mollusc Crassostrea gigas. This receptor named Cg-LGRB exhibits high degree of amino acid sequence identity with both mammalian and Drosophila LGRs. Phylogenetic analysis indicates that Cg-LGRB belongs to the cluster of type B orphan LGRs and suggests that molluscs likely express the three LGR subgroups identified previously in other animals. Quantitative RT-PCR shows that Cg-LGRB is expressed mainly in the digestive gland and only at moderate levels in other organs and developmental stages. A possible involvement in the control of cytological changes occurring in bivalve mollusc digestive gland is discussed.     

Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae

Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae. The enzyme was purified 472-fold to apparent homogeneity. The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P. The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+. Yeast MAP failed to cleave methionine p-nitroanilide. Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser. MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g. Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg. His, Leu, Met, or Tyr). Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Werlen, R. C., Garber, P., and Movva, N. R. (1987) Proc. Natl, Acad. Sci. U.S.A. 84, 2718-2722). In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S. M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984).     

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.