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G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. Theα-factor receptor (Ste2p) fromSaccharomyces cerevisiaeis a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatableα-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to anα-factor analog with the photoaffinity groupp-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 ofα-factor and a region of Ste2p covering residues 251–294. [ABSTRACT FROM AUTHOR]

Mating in Saccharomyces cerevisiae is induced by the interaction of α-factor (W¹H²W³L[sup 4]Q[sup 5]L[sup 6]K[sup 7]P[sup 8]G[sup 9]Q[sup 10]P[sup 11]M[sup 12]Y[sup 13]) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of α-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the αN-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 α-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 α-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 λ[sub 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 λ[sub max] (≈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 (λ[sub 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... [ABSTRACT FROM AUTHOR]

Abstract: Analogs of the α-factor tridecapeptide mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which Tyr[sup 13] was replaced with Phe, p-F-Phe, m-F-Phe, p-NO[sub 2]-Phe, p-NH[sub 2]-Phe or Ser were synthesized and purified to > 99% homogeneity. These analogs were bioassayed using a growth arrest assay and a gene induction assay and evaluated for their ability to compete with binding of tritiated α-factor to its receptor Ste2p. The results showed that the phenolic OH of Tyr[sup 13] is not required for either biological activity or receptor recognition. Analogs containing fluorine, amino, nitro or a hydrogen in place of OH had 80–120% of the biological activity of the parent pheromone in the gene induction assay and had receptor affinities from nearly equal to 6-fold lower than that of α-factor. In contrast, substitution of Ser or Ala at position 13 resulted in a > 100-fold decrease in receptor affinity suggesting that the aromatic ring is involved in binding to the receptor. The lack of a strict requirement for Tyr[sup 13] allowed the design of several multiple replacement analogs in which Phe or p-F-Phe were substituted at position 13 and Tyr was placed in other positions of the peptide. These analogs could then be iodinated and used in the development of a highly sensitive receptor-binding assay. One potential receptor ligand [Tyr([sup 125]I)[sup 1],Nle[sup 12], Phe[sup 13]]α-factor exhibited saturable binding with a K[sub D] of 81 n m and was competed by α-factor for binding in a whole-cell assay. Thus a new family of radioactive ligands for the α-factor receptor has been revealed. These ligands should be extremely useful in defining active site residues during mutagenesis and cross-linking studies. [ABSTRACT FROM AUTHOR]

Abstract: Previous biochemical investigations on the Saccharomyces cerevisiaea‐factor indicated that this lipopeptide pheromone [YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II β‐turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a‐factor, in which residues at positions 4 and 5 were replaced with: l‐Pro[sup 4](I); d‐Pro[sup 4](II); l‐Pro[sup 4]‐ d‐Ala[sup 5](III); d‐Pro[sup 4]‐ l‐Ala[sup 5](IV); or Nle[sup 4](V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a‐factor analogs I and III were found to be almost 50‐fold more active than the diastereometric homologs II and IV and were equally active to wild‐type a‐factor. Replacement of Lys[sup 4] with the isosteric Nle[sup 4] almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II β‐turn compensated for the loss of the favorable contribution of the Lys[sup 4] side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H[sub 2]O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a‐factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity. [ABSTRACT FROM AUTHOR]