Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking.
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.
Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0845, USA.
Cagdas D
CD
Son
Hasmik
H
Sargsyan
Fred
F
Naider
Jeffrey M
JM
Becker
D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D000072235:Mating Factor; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D010675:Pheromones / Q000138:chemical synthesis / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D010675:Pheromones; D019920:Photoaffinity Labels; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010649:Phenylalanine; D000072235:Mating Factor; D001710:Biotin; C053764:benzoylphenylalanine; D014357:Trypsin