Regulation of D-1 dopamine receptors with mutations of protein kinase phosphorylation sites: Attenuation of the rate of agonist-induced desensitization

Investigations of D-1 receptor regulation have suggested a role for cAMP-de pendent protein kinase (PKA) in agonist-induced desensitization and down-re gulation of receptor expression. Given the presence of at least four possib le consensus recognition sites for PKA on the D-1 receptor protein, a reaso nable hypothesis is that some of these PKA-mediated effects are caused by p hosphorylation of the receptor. As an initial test of this hypothesis, we u sed site-directed mutagenesis to create a mutant D-1 receptor with substitu tions at each of its four potential PKA phosphorylation sites. The modified amino acids are as follows: Thr135 to Val, Ser229 to Ala, Thr268 to Val, a nd Ser380 to Ala. Characterization of the wild-type and mutant receptors st ably expressed in C6 glioma cells suggests that the mutations have no effec t on receptor expression, antagonist or agonist affinities, or on functiona l coupling with respect to cAMP generation. Similarly, dopamine preincubati on of the stably trans-fected C6 cells expressing either the wild-type or m utated D-1 receptors results in an agonist-induced loss of ligand binding a ctivity (down-regulation) in an identical fashion. In contrast, the time of onset of dopamine-induced desensitization is greatly attenuated in the qua druple mutant receptor. After 1 h of dopamine pretreatment, the wild-type r eceptor exhibits similar to 80% desensitization of the cAMP response, where as the mutant receptor is desensitized by only similar to 20%. Further anal yses of single mutated receptors, in which only one of the four putative ph osphorylation sites is modified, reveals that Thr268 in the third cytoplasm ic loop of the receptor protein is primarily responsible for regulating the desensitization kinetics. These results are consistent with the hypothesis that phosphorylation of the D-1 receptor on Thr268 is important for rapid agonist-induced homologous desensitization.