Abstract
Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the α2B-adrenoceptor (AR) more sensitive to agonist-induced down-regulation (J Pharmacol Exp Ther312:767–773, 2005). However, an absolute requirement of GRK3 and GRK2 for α2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous α2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the α2B-AR. Pretreatment of NG108 cells with 20 μM epinephrine (EPI) begins down-regulating the α2B-AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in α2B-AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in α2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gβγ subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the α2B-AR caused by 24-h pretreatment with 20 μM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the α2B-AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonist-induced down-regulation of the α2-AR.
Footnotes
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This work was supported in part by a Grant to Entrance and Advance Research grant from the University of Houston, Grant 0555032Y from the American Heart Association, Texas Affiliate, awarded to D.C.E., and National Institutes of Health Grant DA017380 awarded to K.M.S.
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doi:10.1124/jpet.105.098996.
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ABBREVIATIONS: AR, adrenoceptor; GPCR, G protein-coupled receptor; NE, norepinephrine; EPI, epinephrine; GRK, G protein-coupled receptor kinase; CHO, Chinese hamster ovary; PGE1, prostaglandin E1; DMEM, Dulbecco's modified Eagle's medium; [3H]RX821002, (1,4-[6,7(n)-3H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride; CC, chelerythrine chloride; TEMED, N,N,N′,N′-tetramethylethylenediamine; GAPDH, glyceraldehyde phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; PBSS, phosphate-buffered saline with 1.2% sucrose; PKC, protein kinase C.
- Received November 29, 2005.
- Accepted March 9, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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