Calmodulin binding to G protein-coupling domain of opioid receptors

被引:99
作者
Wang, DX
Sadée, W [1 ]
Quillan, JM
机构
[1] Univ Calif San Francisco, Sch Pharm, Dept Biopharmaceut Sci, San Francisco, CA 94143 USA
[2] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
[3] Univ Calif San Francisco, Ctr Neurobiol Drug Addict, San Francisco, CA 94143 USA
关键词
D O I
10.1074/jbc.274.31.22081
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mu-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 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 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 Lys(273) --> 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 (delta-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.
引用
收藏
页码:22081 / 22088
页数:8
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