A Preformed Signaling Complex Mediates GnRH-Activated ERK Phosphorylation of Paxillin and FAK at Focal Adhesions in LβT2 Gonadotrope Cells

Abstract

Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of L beta T2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase C delta, -epsilon, and -alpha, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

Gq Protein-induced Apoptosis Is Mediated by AKT Kinase Inhibition That Leads to Protein Kinase C-induced c-Jun N-terminal Kinase Activation

“…5) and also ERK (36,37) downstream of G q PCRs. These differences in c-Src effects before and after stimulation could be a result of an alternative complex formation, as recently reported upon GnRH-a nonapoptotic stimulation of L␤T2 cells (38). Alternatively, it can also be due to interference by interacting downstream processes, such as phosphatases or additional inhibitory phosphorylations.…”

Gq-Induced Apoptosis is Mediated by AKT Inhibition That Leads to PKC-Induced JNK Activation

“…5) and also ERK [42,43] downstream of GqPCRs. These differences in c-Src effects before and after stimulation could be a result of an alternative complex formation, as recently reported upon GnRH-a non-apoptotic stimulation of LβT2 cells [44]. Alternatively, it can also be due to interference by interacting downstream processes, such as phosphatases or additional inhibitory phosphorylations.…”

Paxillin Regulates Androgen- and Epidermal Growth Factor-induced MAPK Signaling and Cell Proliferation in Prostate Cancer Cells

“…4). Previous studies (40,41,61) also suggested that paxillin might function as a scaffold to hold Raf, MEK, and Erk1/2 in a signaling complex. Most of these studies (17, 40 -42, 61, 62) focused on the association of paxillin with focal adhesion molecules, using overexpression and co-precipitation studies to show interactions.…”