Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch

Signaling by D-2-dopamine receptors in neurons likely proceeds in the presence of Ca2+ oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca2+ and the D-2 receptor. By activation of calmodulin (CaM), Ca2+ directly inhibits the D-2 receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH2-terminal end of the third loop, a domain involved in activating G(i/o). A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca2+-dependent manner (K-D similar to 0.1 muM). (ii) Activation of purified G alpha (il) by D2N, and D-2 receptor-promoted GTP gammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca2+/CaM (IC50 similar to 0.1 muM), (iii) If Ca2+ influx was elicited in D-2 receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other G(i)-coupled receptors (A(1)-adenosine and Mel(1A)-melatonin receptor). (iv) The D-2 receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D-2 receptor from its cognate G protein(s); rather, CaM directly targets the D-2 receptor to block the receptor-operated G protein activation switch.