Abstract
BackgroundThe 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.MethodsHuman 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.ResultsIn 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.ConclusionFrom 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.
Highlights
The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction
In Human embryonic kidney (HEK) 293 cells stimulated by isoproterenol, overexpression of β-arrestin V53D, or of a β-arrestin (319–418) peptide, both impaired in their receptor-binding ability [9], reduces the β2adrenergic receptor internalization level, and decreases extracellular signal-regulated kinases (ERK) activation [10]
ERK phosphorylation peaked around 6 min, declined slowly, since 44% of the maximum phosphorylation was still observed by 15 min of FSH stimulation (Figure 1C, 1D)
Summary
The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. We examined the ability of the FSHactivated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Several reports support the view that MAP kinase activation requires receptor internalization, mediated by β-arrestins [2]. In HEK 293 cells stimulated by isoproterenol, overexpression of β-arrestin V53D, or of a β-arrestin (319–418) peptide, both impaired in their receptor-binding ability [9], reduces the β2adrenergic receptor internalization level, and decreases ERK activation [10]. Overexpression of a defective K44A dynamin mutated in its catalytic domain [12] impairs both receptor internalization as well as ERK stimulation transduced by the δ-opioid receptor [13]. Whether 7TMR-mediated ERK activation will depend on β-arrestinpromoted internalization or not seems to be a receptorrelated issue
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