Calmodulin binding to G protein-coupling domain of opioid receptors

The ubiquitous intracellular Ca²⁺ 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 μ-opioid (OP₃) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP₃ receptor. Functionally, CaM reduced basal and agonist-stimulated ³⁵S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP₃ 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 OP₃ further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP₃ receptor mutant with a Lys²⁷3 → Ala substitution (K273A-OP₃), 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 OP₁ (δ-opioid) and OP₃ wild-type receptors, but not the K273A-OP₃ 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.