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.
Henry
L Keith
LK
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Khare
Sanjay
S
Son
Cagdas
C
Babu
V V Suresh
VV
Naider
Fred
F
Becker
Jeffrey M
JM
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D000072235:Mating Factor; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000072235:Mating Factor; D019204:GTP-Binding Proteins