Regulation of dopamine D1-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D-2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D-1 receptor activation that occurs without changes in D-1 receptor expression levels. This raises the possibility that, in brains of D-2 mutants, a substantial portion of D-1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D-1-agonist-stimulated [S-35]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D-2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D-2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [S-35]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D-2 mutants. Furthermore, for D-2 mutants, immunoprecipitation experiments revealed increased basal andD(1)-agonist-stimulated phosphorylation of D-1-receptor proteins at serine residues. Finally, D-1 immunoprecipitates of both wild type and D-2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D-2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D-1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D-1 receptors in brains of D-2 mutants is due to defective dephosphorylation of the receptor rather than increasedkinase-mediated phosphorylation.