Characterization of G Protein-Coupled Receptor Regulation in Antisense mRNA-Expressing Cells with Reduced Arrestin Levels
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- 作者：Stuart J. Mundell ; Robert P. Loudon ; and Jeffrey L. Benovic
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：July 6, 1999
- 年：1999
- 卷：38
- 期：27
- 页码：8723 - 8732
- 全文大小：151K
- 年卷期：v.38,no.27(July 6, 1999)
- ISSN：1520-4995
文摘
Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins haveestablished a role for these proteins in 
le">2-adrenergic receptor (le">2AR) internalization, desensitization, andresensitization. To validate and extend such findings, we employed an antisense strategy to target thenonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of Gprotein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targetingarrestin-2 exhibited a selective reduction (~50%) in arrestin-2 levels, while arrestin-3 antisense constructsresulted in reductions (
50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cellsdemonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent ofagonist-induced internalization of exogenously expressed le">2ARs, but had no effect on internalization ofeither m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing therole of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantlydecreased the rate of endogenous le">2AR internalization, desensitization, and resensitization. Further analysisdemonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptorswas also attenuated in cells with reduced arrestin levels. The effects on the le">2-adrenergic, A2b adenosine,and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction inarrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility ofantisense approaches in the examination of arrestin-mediated GPCR regulation.
le">2-adrenergic receptor (le">2AR) internalization, desensitization, andresensitization. To validate and extend such findings, we employed an antisense strategy to target thenonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of Gprotein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targetingarrestin-2 exhibited a selective reduction (~50%) in arrestin-2 levels, while arrestin-3 antisense constructsresulted in reductions (50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cellsdemonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent ofagonist-induced internalization of exogenously expressed le">2ARs, but had no effect on internalization ofeither m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing therole of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantlydecreased the rate of endogenous le">2AR internalization, desensitization, and resensitization. Further analysisdemonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptorswas also attenuated in cells with reduced arrestin levels. The effects on the le">2-adrenergic, A2b adenosine,and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction inarrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility ofantisense approaches in the examination of arrestin-mediated GPCR regulation.