Synergistic Regulation of β2-Adrenergic Receptor Sequestration: Intracellular Complement of β-Adrenergic Receptor Kinase and β-Arrestin Determine Kinetics of Internalization
- Luc Ménard1,1,2,
- Stephen S. G. Ferguson1,2,
- Jie Zhang1,2,
- Fang-Tsyr Lin1,
- Robert J. Lefkowitz1,3,4,
- Marc G. Caron1,2,3 and
- Larry S. Barak1,2
- 1Howard Hughes Medical Institute Laboratories (L.M., S.S.G.F., J.Z., F.-T.L., R.J.L., M.G.C., L.S.B.), Departments of 2Cell Biology (L.M., S.S.G.F., J.Z., M.G.C., L.S.B.), 3Medicine (R.J.L., M.G.C.), and4Biochemistry (R.J.L.), Duke University Medical Center, Durham, North Carolina 27710
Abstract
Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the β-adrenergic receptor kinase (βARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the β2-adrenergic receptor (β2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that βARK and arrestin proteins (β-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the β2AR to be phosphorylated by βARK or to interact normally with β-arrestin substantially reduces agonist-mediated sequestration. To evaluate βARK and β-arrestin regulation of β2AR sequestration, we examined the relationship between βARK and/or β-arrestin expression and β2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous β-arrestin expression and β2AR sequestration, whereas HEK 293 had the highest. Overexpression of β-arrestin, but not βARK, in COS cells increased the extent of wild-type β2AR sequestration to levels observed in HEK 293 cells. However, a βARK phosphorylation-impaired β2AR mutant (Y326A) required the simultaneous overexpression of both βARK and β-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of βARK and β-arrestin expression. Moreover, an agonist-mediated translocation of wild-type β2AR and endogenous β-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular βARK and arrestin proteins synergistically regulate β2AR sequestration but also that β-arrestins directly regulate β2AR trafficking as well as desensitization.
Footnotes
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Send reprint requests to: Dr. Larry Barak, Box 3287, Department of Cell Biology, Duke University, Durham, NC 27710. E-mail:lbarak{at}cellbio.duke.edu
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↵1 Current affiliation: BioSignal Inc., Montreal, Quebec, Canada H3J IR4.
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↵2 The use of purified β-arrestin in preliminary experiments indicates endogenous cell β-arrestin is in excess of receptor by 100-fold, which is in agreement with the observations of others (35).
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↵3 J. Zhang and M. G. Caron, unpublished observations.
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This work was supported in part by National Institutes of Health Grant NS19576, a Bristol Myers Squibb Unrestricted Grant Award (M.G.C.), a K-08 award HL03422 (L.S.B.), and a Michael Smith Postdoctoral Fellowship from the MRC Canada (S.S.G.F.).
- Abbreviations:
- β2AR
- β2-adrenergic receptor
- GPCR
- G protein-coupled receptor
- βARK
- β-adrenergic receptor kinase
- PKA
- protein kinase A
- PBS
- phosphate-buffered saline
- HEK
- human embryonic kidney
- CHO
- Chinese hamster ovary
- CHW
- Chinese hamster fibroblasts
- COS
- African Green Monkey fibroblasts
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
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- Received November 21, 1996.
- Accepted February 12, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
