Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis.In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation.From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.ERK mitogen-activated protein (MAP) kinases are commonly activated by 7TMRs, which leads to a wide array of cellular processes including cell proliferation and cell differentiation. In the last decade, a tremendous amount of works have been dedicated to elucidate the cell signaling mechanisms whereby 7TMRs activate ERK. To achieve ERK activation, some 7TMRs, such as the lutropin receptor [1], rely solely on G protein activation and to second messenger production. Besides, several reports support the view that MAP kinase activation requires receptor internalization, mediated by β-arrestins [2]. Originally, β-arrestins have been viewed as responsible for receptor desensitization, by uncoupling an agonist-activated receptor from its effector G proteins, and then by driving the uncoupled receptor to clathrin-coated pits [3,4]. β-arrestin-dependent internaliza