title: Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells.
abstract: GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.
authors.affiliation: Department of Biomedical Sciences, University of Edinburgh, United Kingdom.
authors.forename: M S
authors.initials: MS
authors.lastname: Johnson
authors.forename: E M
authors.initials: EM
authors.lastname: Lutz
authors.forename: C J
authors.initials: CJ
authors.lastname: MacKenzie
authors.forename: W B
authors.initials: WB
authors.lastname: Wolbers
authors.forename: D N
authors.initials: DN
authors.lastname: Robertson
authors.forename: P J
authors.initials: PJ
authors.lastname: Holland
authors.forename: R
authors.initials: R
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D007295:Inositol Phosphates / Q000096:biosynthesis; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000276:immunology / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011966:Receptors, LHRH / Q000187:drug effects* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection / Q000235:genetics
chemical_list: D007295:Inositol Phosphates; D012333:RNA, Messenger; D011966:Receptors, LHRH; D019000:Phosphotyrosine; D007987:Gonadotropin-Releasing Hormone; D011388:Prolactin; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases