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

M. S. Johnson, Eve M. Lutz, Christopher J MacKenzie, W. Bart Wolbers, Derek N. Robertson, Pamela J. Holland, Rory Mitchell

Research output: Contribution to journalArticlepeer-review


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 αT3–1 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 αT3–1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in αT3–1 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 inα 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 αT3–1 cells.

In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in αT3–1 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.
Original languageEnglish
Pages (from-to)3087-3097
Issue number9
Publication statusPublished - 1 Sep 2000


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