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The Role of Protein Kinases A and C Pathways in the Regulation of Mitogen-Activated Protein Kinase Activation in Response to Gonadotropin-Releasing Hormone Receptor Activation

Oregon Regional Primate Research Center (X.-B.H., P.M.C.), Oregon Health Sciences University, Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (P.M.C.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. P. Michael Conn, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}OHSU.edu

There is convincing evidence that mitogen-activated protein kinase (MAPK) activation is coupled to both receptor tyrosine kinase and G protein-coupled receptors. The presence of the epidermal growth factor (EGF) receptor and the GnRH receptor on the surface of GGH₃1' cells makes this cell line a good model for the assessment of MAPK activation by receptor tyrosine kinases and G protein-coupled receptors. In this study, to assess the activated and total (i.e. activated plus inactivated) MAPK, the phosphorylation state of p44 and p42 MAPKs was examined using antisera that distinguish phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) from p44/42 MAPK (phosphorylation state independent). The data show that both EGF (200 ng/ml) and Buserelin (a GnRH agonist; 10 ng/ml) provoke rapid activation of MAPK (within 5 and 15 min, respectively) after binding to their receptors. The role of protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways in mediating MAPK activation was also assessed. Both phorbol ester (phorbol 12-myristate 13-acetate; 10 ng/ml) and (Bu)₂cAMP (1 mM) trigger the phosphorylation of MAPK, suggesting potential roles for PKC and PKA signaling events in MAPK activation in GGH₃1' cells. Treatment of PKC-depleted cells with Buserelin activated MAPK, suggesting involvement of PKC-independent signal transduction pathways in MAPK activation in response to GnRH. Similarly, treatment of PKC-depleted cells with forskolin (50 µM) or cholera toxin (100 ng/ml) stimulated MAPK activation, whereas pertussis toxin (100 ng/ml) had no measurable effect. To further assess the role of PKA in response to EGF and Buserelin, cells were treated with EGF (200 ng/ml) for 3 min or with Buserelin (10 ng/ml) for 10 min after pretreatment with 3-isobutyl-1-methylxanthine (0.5 mM), forskolin (50 µM), or (Bu)₂cAMP (1 mM) for 15 min. The results show that MAPK can be activated in a PKA-dependent manner in GGH₃1' cells. Consistent with previous reports, the current data support the view that MAPK activation can be achieved via both PKC- and PKA-dependent signaling pathways triggered by the GnRH receptor that couples to G_q/11 and G_s -subunit proteins. In contrast, G_i/o does not appear to participate in MAPK activation in GGH₃1' cells.

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