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BIOPHYSICAL AND MUTAGENIC ANALYSIS OF A G PROTEIN-COUPLED RECEPTOR: PHOTOCROSSLINKING OF THE TRIDECAPEPTIDE ALPHA-FACTOR INTO STE2P OF SACCHAROMYCES CEREVISIAE REVEALS CONTACT POINTS BETWEEN THE PEPTIDE AND ITS RECEPTOR BINDING SITE

2004-01-01
Naider, F.
Son, Çağdaş Devrim
Sargsyan, H.
Becker, J. M.
Mating of Saccharomyces cerevisiae is initiated by binding of a peptide pheromone, alpha-factor, to its cognate G protein-coupled receptor (Ste2p). Analogs of alpha-factor (WHWLQLKPGQPMY) containing the photoactivatable group p-benzoyl-L-phenylalanine (Bpa) and biotin as a tag were synthesized using solid-phase methodologies. Bpa was inserted at positions one, three, five, eight, and thirteen of alpha-factor to obtain a set of cross-linkable analogs spanning the pheromone. The biological activity and binding affinities of all analogs for Ste2p were determined. Two of the analogs tested, [Bpa1]alpha-factor and [Bpa5]alpha-factor, showed three- to four-fold lower affinity compared to alpha-factor, whereas [Bpa3]alpha-factor and [Bpa13]alpha-factor had seven- to twelve-fold lower affinities, respectively. [Bpa8]alpha-factor competed poorly with [3H]alpha-factor for Ste2p. All of the analogs tested except [Bpa8]alpha-factor were alpha-factor agonists. Cross-linking studies demonstrated that [Bpa1]alpha-factor, [Bpa3]alpha-factor, [Bpa5]alpha-factor and [Bpa13]alpha-factor cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of receptors crosslinked to [Bpax]alpha-factor (x = 1, 3, 13) 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 (transmembrane 5), TM6, and TM7 and portions of EL2 (extracellular loop 2) 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. Site-directed mutagenesis results correlated with the crosslinking studies. The results have been incorporated into a model for alpha-factor bound to its receptor.