Morphine-induced μ-opioid receptor rapid desensitization is independent of receptor phosphorylation and β-arrestins

Receptor desensitization involving receptor phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca²⁺ ([Ca²⁺]_i)release to monitor receptor activation, as predicted, [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca²⁺]_i release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser³⁷⁵ residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr.