Phosphorylation of unique C‐terminal sites of the mu‐opioid receptor variants 1B2 and 1C1 influences their Gs association following chronic morphine

We recently demonstrated in rat spinal cord that a regimen of escalating doses of systemic morphine, analogous to regimens used clinically for chronic pain management, selectively up-regulates the mu-opioid receptor (MOR) splice variants MOR-1B2 and MOR-1C1 mRNA and functional protein. This study investigated the potential relevance of up-regulating MOR-1B2 and MOR-1C1 to the ability of chronic morphine to shift MOR signaling from predominantly Gi /Go inhibitory to Gs stimulatory. Specifically, we tested the hypotheses that chronic morphine induces phosphorylation of carboxyl terminal sites unique to MOR-1B2 and MOR-1C1, and that this phosphorylation is causally related to augmented association of these variants with Gs alpha. Hypotheses were validated by (i) abolition of the chronic morphine-induced increment in MOR-1C1 and MOR-1B2 association with Gs alpha by inhibitors of protein kinase A and Casein kinase 2, respectively; (ii) failure of chronic morphine to augment MOR variant Gs alpha interactions in Chinese hamster ovary cells transiently transfected with either rat MOR-1C1 or MOR-1B2 in which targeted protein kinase A and Casein kinase 2 serine phosphorylation sites, respectively, were mutated to alanine; (iii) abrogation of chronic morphine-induced augmented MOR Gs alpha association in spinal cord of male rats following intrathecal administration of dicer substrate small interfering RNAs targeting MOR-1B2/MOR-1C1 mRNA. The ability of chronic morphine to not only up-regulate-specific MOR variants but also their carboxyl terminal phosphorylation and consequent augmented association with Gs alpha may represent a novel component of opioid tolerance mechanisms, suggesting novel potential targets for tolerance abatement.