Calmodulin binding to G protein-coupling domain of opioid receptors

The ubiquitous intracellular Ca2+ sensor calmodulin (CaM) regulates numerou s proteins involved in cellular signaling of G protein-coupled receptors, b ut most known interactions between GPCRs and CaM occur downstream of the re ceptor. Using a sequence-based motif search, we have identified the third i ntracellular loop of the opioid receptor family as a possible direct contac t point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of t he mu-opioid (OP3) receptor strongly bound CaM and were able to reduce bind ing interactions observed between CaM and immunopurified OP3 receptor. Func tionally, CaM reduced basal and agonist-stimulated S-35-labeled guanosine 5 '-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP3 receptor. Changes in CaM membrane l evels 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 OP3 further suppor ts the hypothesis of a direct interaction between CaM and opioid receptors, An OP3 receptor mutant with a Lys(273) --> Ala substitution (K273A-OP3), a n amino acid predicted to play a critical role in CaM binding based on moti f structure, was found to be unaffected by changes in CaM levels but couple d more efficiently to G proteins than the wild-type receptor, Stimulation o f both the OP1 (delta-opioid) and OP3 wild-type receptors, but not the K273 A-OP3 mutant, induced release of CaM from the plasma membrane. These result s 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.