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Characterization of G protein-coupled receptor regulation in antisense mRNA-expressing cells with reduced arrestin
S J Mundell1, R P Loudon, J L Benovic
1Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.
Insights
Reducing nonvisual arrestin levels impairs beta2-adrenergic receptor (beta2AR) regulation and desensitization of other G protein-coupled receptors (GPCRs). This study validates arrestin
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Biology
Background:
- Nonvisual arrestins play a role in the regulation of beta2-adrenergic receptors (beta2ARs).
- Previous studies utilized overexpression of arrestins to understand their function.
- The specific roles of arrestin-2 and arrestin-3 in G protein-coupled receptor (GPCR) signaling require further investigation.
Purpose of the Study:
- To validate and extend findings on the role of nonvisual arrestins in GPCR regulation.
- To investigate the effects of reduced arrestin-2 and arrestin-3 levels on GPCR signaling using an antisense strategy.
- To assess the impact of reduced arrestin levels on the internalization, desensitization, and resensitization of beta2ARs and other endogenous GPCRs.
Main Methods:
- HEK293 cells were stably transfected with antisense constructs targeting arrestin-2 and/or arrestin-3.
- Arrestin levels were quantified to confirm knockdown efficiency.
- Agonist-induced internalization, desensitization, and resensitization of exogenously expressed and endogenous GPCRs (beta2AR, m2/m3 muscarinic acetylcholine receptors, A2b adenosine, and prostaglandin E2-stimulated receptors) were assessed.
Main Results:
- Antisense targeting resulted in significant reductions in arrestin-2 and/or arrestin-3 levels.
- Reduced arrestin levels decreased beta2AR internalization and significantly altered the rate of endogenous beta2AR internalization, desensitization, and resensitization.
- Desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels.
Conclusions:
- Nonvisual arrestins are critical for the regulation of beta2AR internalization, desensitization, and resensitization.
- Reduced arrestin levels impact the desensitization of multiple endogenous GPCRs.
- Antisense approaches are effective tools for studying arrestin-mediated GPCR regulation.