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


RESULTS
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.


CONCLUSIONS
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.
Piketty
Vincent
V
Laboratoire de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Centre National pour la Recherche Scientifique, Université de Tours, France. piketty@tours.inra.fr
Kara
Elodie
E
Guillou
Florian
F
Reiter
Eric
E
Crepieux
Pascale
P
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034281:Dynamins / Q000235:genetics / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D005799:Genes, Dominant; D006801:Humans; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011962:Receptors, FSH / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605256:Arrb1 protein, rat; D019390:Arrestins; D011962:Receptors, FSH; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D005640:Follicle Stimulating Hormone; D048049:Extracellular Signal-Regulated MAP Kinases; D034281:Dynamins