Subcellular localization of μ-opioid receptor Gs signaling

Abstract

In membranes obtained from μ-opioid receptor (MOR) expressing Chinese hamster ovary (CHO) cells (MOR-CHO), the MORselective agonist sufentanil produced a concentration-dependent stimulation of guanosine 5′-O-(3-[ 35S]thio)triphosphate binding to Gsα that was abolished by blocking MOR with naloxone. This unequivocally demonstrates the long-debated functionality of the previously described association of MOR with Gsα. Several complementary observations indicate the relevance of caveolae to MOR-coupled Gsα signaling. 1) In MOR-CHO membranes, sufentanil stimulated the translocation of Gsα into Triton-insoluble membrane compartments. 2) Sufentanil enhanced the coimmunoprecipitation (co-IP) of Gsα and adenylyl cyclase (AC) with caveolin-1 (a marker for caveolae) from the Triton-insoluble membrane fraction of spinal cord and MOR-CHO. 3) MOR blockade (via naloxone) or G s inactivation (via cholera toxin) abolished both the increased trafficking of Gsαinto the Triton-insoluble membrane fraction of MORCHO and the augmented co-IP from spinal cord membranes of G sα and AC with caveolin-1. This indicates that these events occurred subsequent to activation of MOR and Gsα. Strikingly, lesser-phosphorylated Gsα, which preferentially couple to MOR (Mol Brain Res 135:217-224, 2005; Mol Pharmacol 72:753-760, 2007; Mol Pharmacol 73:868-879, 2008), are concentrated in caveolae, underscoring their relevance to MOR Gsα signaling. MOR-stimulated trafficking of G sα and AC into caveolae and the likelihood of increased MOR Gsα coupling within caveolae could suggest that they contain the downstream effectors for MOR Gsα AC signaling. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.