| Desensitization of the mu-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Desensitization of G-protein coupled receptor (GPCR) is thought to involve receptor phosphorylation and subsequent recruitment of betaArrestins (betaArrs). However, the roles of receptor phosphorylation and betaArr in morphine-induced MOR desensitization remain to be demonstrated; this may result from the insensitivity of the methods used to study receptor function. Using MOR-induced intracellular Ca2+ ([Ca2+] i) release to monitor receptor activation, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) induced MOR desensitization in a receptor phosphorylation- and betaArr-dependent manner. DAMGO-induced desensitization was blunted in HEK293 cells expressing the MORS375A mutant and was eliminated in MEF cells isolated from betaArr2 knockout mice expressing the wild type MOR. However, although morphine induced a more rapid desensitization of [Ca2+]i release than DAMGO did and could induce the phosphorylation of the Ser375 residue of MOR, morphine-induced desensitization was not influenced by mutating MOR phosphorylation sites or in MEF cells lacking betaArr1 and 2. In contrast, morphine induced MOR desensitization via protein kinase C (PKC). By using subtype-specific inhibitors, PKCepsilon was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. Meanwhile, DAMGO did not increase PKCepsilon activity and DAMGO-induced MOR desensitization was not affected by a PKCepsilon inhibitor. Among the various proteins within the receptor signaling complex, Galphai2 was phosphorylated by morphine-activated PKCepsilon. Moreover, mutating three putative PKC phosphorylation sites, Ser44, Ser144 and Ser302 on Galphai2 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 with a PKCepsilon inhibitor or overexpressing of Galphai2 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 Galphai-coupled receptors such as the CB1 receptor. |
