Abstract
The α1D-adrenergic receptor (α1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote α1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of α1D-AR at the cell surface: α1B-AR and β2-AR. Confocal imaging confirmed that coexpression with β2-AR resulted in translocation of α1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that α1D-AR and β2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total α1D-AR binding sites upon coexpression with β2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with β2-AR significantly enhanced the coupling of α1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of β2-AR with α1D-AR also conferred the ability of α1D-AR to cointernalize upon β2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of α1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between α1- and β2-ARs that is mediated through heterodimerization and cross-internalization.
Footnotes
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This work was supported by grants from the National Institutes of Health to R.A.H. and K.P.M., by grants from the American Heart Association to K.P.M and C.H., and by a Distinguished Young Scholar in Medical Research award from the W.M. Keck Foundation to R.A.H.
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doi:10.1124/jpet.104.079541.
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ABBREVIATIONS: AR, adrenergic receptor; NE, norepinephrine; GPCR, G protein-coupled receptor; HA, hemagglutinin; GFP, green fluorescent protein; HEK, human embryonic kidney; DβM, n-dodecyl-β-d-maltoside; BMY 7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; HRP, horseradish peroxidase; ECL, enzyme-linked chemiluminescence; PBS, phosphate-buffered saline; BE, 2-[β-(4-hydroxyphenyl)-ethylaminomethyl]-tetralone; CGP-12177, 4-[3-[(1,1-dimethyethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; CGP-20712A, (±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(methylethyl)amino-2-butanol.
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↵1 Current address: Synaptic Pharmaceutical Corp., Paramus, NJ.
- Received October 20, 2004.
- Accepted December 16, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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