Mutagenesis of residues adjacent to transmembrane prolines alters D1 dopamine receptor binding and signal transduction.

Proline is highly conserved in the presumed transmembrane alpha-helices of seven-transmembrane helix-containing, G protein coupled receptors. Unique properties of this imino acid have led to speculations of structural and perhaps dynamic importance for seven-transmembrane helix-containing receptor function. To avoid potentially deleterious consequences of proline-directed mutagenesis, substitutions were made in the X residue of X-Pro peptide bonds (where X is the residue on the amino-terminal side of proline), which may influence static geometries and potential agonist-induced conformational changes at the X-Pro peptide bond. In the fifth helix, Ile205 was substituted with either an alanine (I205A) or a tyrosine (I205Y). Similarly, in the sixth helix, Leu286 was substituted with either an alanine (L286A) or a tyrosine (L286Y). Mutant I205A demonstrated subtle changes in D1 pharmacology and signal transduction. The I205Y and L286Y mutations produced comparatively drastic impairments in both binding and signal transduction. Remarkably, the L286A mutation resulted in constitutive activity characterized by elevated basal signal transduction and increased agonist potencies. In addition, (R)-(+)-SCH23390, a classical antagonist at the wild-type D1 receptor, behaved as a partial agonist at L286A. This is the first report of a constitutively active receptor resulting from this point mutation and the first report of a constitutively active mutant dopamine receptor. These results are discussed in terms of binding pocket geometry and potential mechanisms of signal transduction.