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

Abstract

Background: The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Methods: 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 1251-FSH binding at the cell surface when compared to internalized 1251-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. Results: In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Cotransfection 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. Conclusion: 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. © 2006 Piketty et al; licensee BioMed Central Ltd.