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 G i /G o inhibitory to G s 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 G s α. Hypotheses were validated by (i) abolition of the chronic morphine‐induced increment in MOR‐1C1 and MOR‐1B2 association with G s α by inhibitors of protein kinase A and Casein kinase 2, respectively; (ii) failure of chronic morphine to augment MOR variant G s α 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 G s α 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 G s α may represent a novel component of opioid tolerance mechanisms, suggesting novel potential targets for tolerance abatement.