C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

Sarah Kraus,Outhiriaradjou Benard,Rony Seger,Zvi Naor

doi:10.3390/ijms21228575

Sarah Kraus, Outhiriaradjou Benard + Show 2 more

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https://doi.org/10.3390/ijms21228575

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Abstract

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gβγ and β-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

Highlights

G-protein-coupled receptors (GPCRs) are the largest group of membranal receptors, which transmit signals from a diverse array of external stimuli, including neurotransmitters, hormones, phospholipids, and mitogens
To determine the cell-type specificity of Gonadotropin-releasing hormone (GnRH) signaling and to study the effect of various signaling inhibitors on this activation, we used COS7 cells that do not express endogenous gonadotropin-releasing hormone (GnRHR). These cells were transfected with a plasmid-containing mouse GnRHR, which yielded a considerable amount of expression of the GnRHR in most cells, as demonstrated by Western blot analysis with an anti-GnRHR antibody and expression of an unrelated green fluorescent protein (GFP)
Genistein, wortmannin, AG1478, and PP1 significantly prevented Jun N-terminal kinase (JNK) activation by GnRH analog (GnRH-a), while GF109203X had no inhibitory effect. This pattern of inhibition is markedly different from that obtained in αT3-1 cells, where stimulation of JNK activity with GnRH-a was inhibited by GF109203X but not by wortmannin or AG1478 [32], indicating that the JNK activation by GnRH may differ in different cell lines

Summary

Introduction

G-protein-coupled receptors (GPCRs) are the largest group of membranal receptors, which transmit signals from a diverse array of external stimuli, including neurotransmitters, hormones, phospholipids, and mitogens. Each of these extracellular agents binds to a specific GPCR, which interacts with a G protein to induce downstream signaling. Many isoforms of the different subunits have been identified and classified according to the subtype of their α subunit into four groups (Gs, Gi, Gq, and G12) All of these Gα subunits, as well as the dissociated βγ dimer and other receptor-interacting proteins, are capable of initiating diverse downstream signaling pathways [1,2,3,4]. Gαi signals can be transmitted via transactivation of receptor tyrosine kinases (RTKs; [17]) or association or activation of Rap-GAP [18]

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