| Title: | Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking |
| Author(s): | Son CD; Sargsyan H; Naider F; Becker JM; |
| Address: | "Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0845, USA" |
| ISSN/ISBN: | 0006-2960 (Print) 0006-2960 (Linking) |
| Abstract: | "Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor" |
| Keywords: | "Binding, Competitive Biotin/metabolism Cross-Linking Reagents/*metabolism Hydrolysis Ligands Mating Factor Peptide Fragments/chemistry/metabolism Peptides/chemical synthesis/*metabolism Phenylalanine/*analogs & derivatives/metabolism Pheromones/chemical s;" |
| Notes: | "MedlineSon, Cagdas D Sargsyan, Hasmik Naider, Fred Becker, Jeffrey M eng GM22086/GM/NIGMS NIH HHS/ GM22087/GM/NIGMS NIH HHS/ Research Support, U.S. Gov't, P.H.S. 2004/10/13 Biochemistry. 2004 Oct 19; 43(41):13193-203. doi: 10.1021/bi0496889" |
