Epidermal Growth Factor Receptor Tyrosine Kinase Mediates Ras Activation by Gonadotropin-releasing Hormone

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 ␤-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In ␣T3-1 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 G␤␥ subunits but could be mimicked by shortterm 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 ␣T3-1 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-␤ receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases. In many cells proliferation and differentiation is regulated by the extracellular signal-regulated kinase (ERK)1 subfamily of MAP kinases. ERKs represent distal members of a threecomponent kinase module through which extracellular stimuli are transmitted into the cell (1). The best studied signaling cascade is initiated by ligand-bound receptor tyrosine kinases (RTKs) which upon autophosphorylation and tyrosine phosphorylation of adaptor proteins like Shc and Grb2 recruit guanine nucleotide exchange factors for the monomeric GTPase Ras. Activated, i.e. GTP-loaded, Ras subsequently engages the ERK-MAPK cascade involving Raf, MAPK/ERK kinase (MEK), and finally ERKs. It has only recently been appreciated that besides classical growth factors like EGF and PDGF, agonists acting at Gprotein-coupled receptors (GPCRs) also 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. In many cells, Ras-dependent ERK activation via GPCRs appears to be mediated by G␤␥ dimers released from PTX-sensitive G-proteins (4). However, G␤␥ subunits derived from PTX-insensitive G␣ q proteins ha...

Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation

“…However, studies on the role of the EGF-R in GnRH action have not given consistent results (9, 10). Thus, whereas Grosse et al (10) implicated transactivation of the EGF-R in GnRH-induced stimulation of ERK1/2 phosphorylation in ␣T3-1 pituitary gonadotrophs, Benard et al (9) subsequently reported that the major pathway of ERK1/2 activation was through PKC and activation of Raf-1 and did not involve the EGF-R. Since GT1-7 cells express receptors for both EGF and GnRH, we evaluated the role of the EGF-R in GnRH-induced MAPK signaling.…”

“…Depending on the cell type, EGF-R transactivation has been reported to be mediated by G i protein ␤␥-subunits (40,41), Ca 2ϩ (26,30), PKC (10,37,42), and heparinbinding EGF (HB-EGF) released by matrix metalloproteinases (43,44). However, the latter does not appear to be a universal mechanism for transactivation of the EGF-R by GPCRs (17,45).…”

“…GPCR-mediated activation of Pyk2 and ERK1/2 is reported to be dependent on both Ca 2ϩ (26,30,41,50) and PKC (19,37,39,51). PKC is also known to cause activation of Src (24) and the EGF-R (10,24,44,45) in several cell types. In fact, PKC␣ and -␦ undergo direct physical and functional interactions with the EGF-R and Pyk2, respectively (56,57).…”

“…Endogenous EGF-Rs are expressed in several model systems, including ␣T3-1 gonadotrophs, COS-7 cells, and HEK-293 cells, that have been used in studies on GnRH signaling. However, the role of EGF-R transactivation in GnRH-induced ERK activation has been a subject of controversy and is not clearly defined (9,10,23). Also, the signaling molecules involved in cross-talk between the neuronal GnRH-R and the EGF-R have not been identified.…”

“…The major signal transduction pathways in cells expressing GnRH receptors are initiated by activation of phospholipase C. The consequent calcium (Ca 2ϩ ) mobilization and activation of protein kinase C (PKC) by GnRH are key elements in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland (1,3,5). Activation of PKC and Ca 2ϩ mobilization during GnRH receptor stimulation are also responsible for mediating downstream signals leading to activation of extracellularly regulated mitogen-activated protein kinases (ERK1/2 MAPKs) that transmit signals from the cell surface to the nucleus to regulate transcriptional and other processes (7)(8)(9)(10)(11)(12)(13). However, the specific PKC isoforms that are involved in GnRH-induced ERK1/2 activation in GT1-7 cells are not known.…”