Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization

Desensitization of the p-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca2+ ([Ca2+](i)), we reported that [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of beta-arrestins (beta Arrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKC epsilon was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKC epsilon activity and DAMGO-induced MOR desensitization was not affected by modulating PKC epsilon activity. Among the various proteins within the receptor signaling complex, G alpha i2 was phosphorylated by morphine-activated PKC epsilon. Moreover, mutating three putative PKC phosphorylation sites, ser(44), Ser(144) and Ser(302) on G alpha i2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca2+](i) release, and this desensitization could be reversed by pretreating the cells with PKC epsilon inhibitor or overexpressing G alpha i2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other G alpha i-coupled receptors, such as the CB1. (C) 2009 Elsevier Inc. All rights reserved.