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 Ca(2+) oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca(2+) and the D(2) receptor. By activation of calmodulin (CaM), Ca(2+) directly inhibits the D(2) receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH(2)-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 Ca(2+)-dependent manner (K(D) approximately 0.1 micrometer). (ii) Activation of purified Galpha(i1) by D2N, and D(2) receptor-promoted GTPgammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca(2+)/CaM (IC(50) approximately 0.1 micrometer). (iii) If Ca(2+) 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.
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
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Bofill-Cardona
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Kudlacek
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Yang
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Ahorn
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Freissmuth
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Nanoff
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000502:physiology*; D044094:Receptors, Melatonin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D002147:Calmodulin; D010446:Peptide Fragments; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D017448:Receptors, Dopamine D2; D044094:Receptors, Melatonin; D011994:Recombinant Proteins; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium