Calmodulin binding to G protein-coupling domain of opioid receptors.
The ubiquitous intracellular Ca(2+) sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the mu-opioid (OP(3)) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP(3) receptor. Functionally, CaM reduced basal and agonist-stimulated (35)S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP(3) receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP(3) further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP(3) receptor mutant with a Lys(273) --> Ala substitution (K273A-OP(3)), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP(1) (delta-opioid) and OP(3) wild-type receptors, but not the K273A-OP(3) mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation.
Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry and the Center for the Neurobiology of Drug Addiction, University of California San Francisco, San Francisco, California 94143-0446, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009020:Morphine / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009270:Naloxone / Q000493:pharmacokinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D002147:Calmodulin; D010446:Peptide Fragments; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D009270:Naloxone; D009020:Morphine; D019204:GTP-Binding Proteins