Abstract
Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.
Highlights
It has only recently been appreciated that besides classical growth factors like EGF and PDGF, agonists acting at Gprotein-coupled receptors (GPCRs) play a role in differentiation, proliferation, and even cellular transformation (2, 3)
Apart from non-receptor tyrosine kinases, RTKs like the PDGF and EGF receptors have been invoked to participate in the signal transmission from GPCRs to extracellular signal-regulated kinase (ERK) (9, 10), and we have provided evidence that the receptor cross-talk is realized in a cell-specific manner (11)
We recently demonstrated that in several different cell lines agonist-bound human and murine Gonadotropin releasing hormone (GnRH) receptors exclusively interact with Gq/11 proteins
Summary
It has only recently been appreciated that besides classical growth factors like EGF and PDGF, agonists acting at Gprotein-coupled receptors (GPCRs) play a role in differentiation, proliferation, and even cellular transformation (2, 3). Both pertussis toxin (PTX)-sensitive and -insensitive heterotrimeric G-proteins mediate ERK activation, and distinct signaling pathways are ascribed to GTP-bound ␣ and free ␥ Gprotein subunits. Besides terminating receptor/G-protein coupling, -arrestins have been implicated in Src recruitment to the agonist-occupied receptor, thereby initiating Ras-dependent ERK activation in a second wave of signal transduction emanating from various GPCRs (13)
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