G_i/o-Coupled Receptors Compete for Signaling to Adenylyl Cyclase in SH-SY5Y Cells and Reduce Opioid-Mediated cAMP Overshoot

Abstract

Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of G i/o -coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the -opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to ␦-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and ␣ 2 AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal.

Molecular Pharmacology ofδ-Opioid Receptors

“…In SK-N-SH neuroblastoma cells endogenously expressing MOPr and DOPr, the combined effects of DOPr and MOPr agonists were also additive, which is also consistent with activation of distinct pools of G proteins (Shapira et al, 2000). By contrast, in studies completed in transfected COS-7 cells and in SH-SY5Y neuroblastoma cells, DOPr and MOPr, as well as other GPCRs, compete for G protein activation and AC modulation, with no evidence of additive responses (Shapira et al, 2000;Levitt et al, 2011). This signaling output is consistent with a translocation-collision model in which different receptors travel laterally within the membrane to activate a shared pool of downstream effectors (Rimon et al, 1978).…”

Heterologous Regulation of Mu-Opioid (MOP) Receptor Mobility in the Membrane of SH-SY5Y Cells

“…In contrast, clonidine was not found to phosphorylate the MOP receptor on Ser 377 in SH-SY5Y cells (19). The cross-talk between ␣ 2 -adrenergic and MOP receptors involves a mutual cross-desensitization of receptors (13,75), not sensitive to PTX (15) and shown to implicate ␤-arrestin and p38 MAP kinase in DRG neurons (14). It has been also shown by intramolecular FRET that a rapid and direct, PTX-insensitive, transconformational switch between MOP and ␣ 2 -adrenergic receptors occurs upon agonist stimulation and could stabilize an inactive conformation of receptors (15).…”

Modulation of μ-Opioid Receptor Signaling by RGS19 in SH-SY5Y Cells

“…This cannot be ascribed to GPCR specificity for different Ga subunits since both MOR and DOR activate the same Gai/o protein subtypes (Prather et al, 1994a,b;Chakrabarti et al, 1995) and in SH-SY5Y cells, MOR, DOR, and NOPR share the same pool of heterotrimeric G proteins (Alt et al, 2002;Levitt et al, 2011). RGS19 and RGS4 are both small RGS proteins that lack large proteinprotein binding domains and have approximately 60% homology in the RH domains (Hollinger and Hepler, 2002).…”

“…Therefore, we re-examined the GAP activity of RGS19 on opioid receptor-G protein-mediated signaling. Human neuroblastoma SH-SY5Y cells endogenously express MOR, DOR, and NOPR (Wang et al, 2009;Levitt et al, 2011), which couple to Gai/o proteins. Here we show that these cells also abundantly express RGS19.…”