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Gonadotropin-Releasing Hormone Receptor Activation of Extracellular Signal-Regulated Kinase and Tyrosine Kinases in Transfected GH3 Cells and in T3–1 Cells¹

Medical Research Council Membrane and Adapter Proteins Cooperative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom EH8 9XD; and Medical Research Council Brain Metabolism Unit, Edinburgh, United Kingdom EH8 9JZ

Address all correspondence and requests for reprints to: Dr. Melanie S. Johnson, Medical Research Council Membrane and Adapter Proteins Cooperative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, United Kingdom EH8 9XD.

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 T3–1 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 T3–1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in T3–1 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 T3–1 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 T3–1 cells.

In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in T3–1 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.

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