Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11.
The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.
Yu
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Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Hinkle
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PM
D000818:Animals; D019390:Arrestins / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002707:Chlordiazepoxide / Q000494:pharmacology; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D000071557:beta-Arrestins
D019390:Arrestins; D018025:Receptors, Thyrotropin-Releasing Hormone; D014168:Transferrin; D000071557:beta-Arrestins; D013973:Thyrotropin-Releasing Hormone; D002707:Chlordiazepoxide; D019204:GTP-Binding Proteins; D001920:Bradykinin; D002118:Calcium