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.
Wang
D
D
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.
Sadée
W
W
Quillan
J M
JM
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