Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor

The alpha-factor pheromone receptor stimulates MATa yeast tells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing signal to the cytoplasmic receptor sequences to mediate G protein activation, A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor, The Pro-258 --> Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor, Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to greater than or equal to 90% of the maximum level, Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6, The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling, These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor, Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptor contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.