AGONIST-SPECIFIC CONFORMATIONAL-CHANGES IN THE YEAST ALPHA-FACTOR PHEROMONE RECEPTOR

The yeast alpha-factor pheromone receptor is a member of the G-protein -coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the thir d intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-facto r, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. alpha-Factor inhi bited cleavage weakly at a second site near the cytoplasmic terminus o f the seventh transmembrane helix, whereas the antagonist showed a str onger inhibition of cleavage at this site and at another site in the C -terminal domain of the receptor. The alpha-factor-induced conformatio nal changes appeared to be inherent properties of the receptor, as the y were retained in G-protein-deficient mutants. Moreover, a mutant rec eptor (ste2-L236H) that affects the third loop and is defective for G- protein coupling retained the ability to undergo the agonist-induced c onformational changes. These results are consistent with a model in wh ich G-protein activation is limited by the availability of specific co ntacts between the G protein and the third intracellular loop of the r eceptor. The antagonist appears to promote a distinct conformational s tate that differs from either the unoccupied or the agonist occupied s tate.