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Subcellular Localization of μ-Opioid Receptor Gs Signaling

In membranes obtained from μ-opioid receptor (MOR) expressing Chinese hamster ovary (CHO) cells (MOR-CHO), the MOR-selective 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 Gs inactivation (via cholera toxin) abolished both the increased trafficking of Gsα into the Triton-insoluble membrane fraction of MOR-CHO and the augmented co-IP from spinal cord membranes of Gsα 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 Gsα 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.