| Title: | Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling |
| Author(s): | Henry LK; Khare S; Son C; Babu VV; Naider F; Becker JM; |
| Address: | "Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA" |
| ISSN/ISBN: | 0006-2960 (Print) 0006-2960 (Linking) |
| Abstract: | "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" |
| Keywords: | "Affinity Labels Amino Acid Sequence Binding Sites Binding, Competitive Cross-Linking Reagents GTP-Binding Proteins/*metabolism Kinetics Mating Factor Models, Molecular Molecular Sequence Data Peptides/*chemistry/genetics/*metabolism Photochemistry Recepto;" |
| Notes: | "MedlineHenry, L Keith Khare, Sanjay Son, Cagdas Babu, V V Suresh Naider, Fred Becker, Jeffrey M eng GM22086/GM/NIGMS NIH HHS/ GM22087/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 2002/05/08 Biochemistry. 2002 May 14; 41(19):6128-39. doi: 10.1021/bi015863z" |
