G Protein-coupled Receptor Kinase Epidermal Growth Factor Signal-regulated Molecular Mechanisms Discussed

Receptor tyrosine kinases (RTK), G protein-coupled receptors (GPCR) and ion channel receptors are main cell surface receptors. RTKs are a subgroup of transmembrane proteins with intrinsic tyrosine kinase activity, which determines various cellular functions as diverse as cell cycle, cell mobility, cell metabolism, ceil growth and cell differentiation. Most of RTK family members are characterized by a modular structure consisting of an extracellular domain, a single hydrophobic transmembrane helix and an intracellular domain, which includes a region with protein tyrosine kinase activity. Signal from RTKs is mediated by their intrinsic tyrosine kinases. A number of mechanisms have been demonstrated to modulate RTK signaling, including receptor tyrosine dephosphorylation, receptor internalization, receptor degradation and Ser/Thr phosphorylation on its intracelluar domain. Epidermal Growth Factor Receptor (EGFR) is an important RTK family member. It plays critical roles in embryo development, cell growth, cell differentiation and cell apoptosis. Disregulation of EGFR by overexpression or mutation frequently links to hyperproliferative diseases, such as cancer.G protein-coupled receptors (GPCRs) constitute another superfamily of plasma membrane receptors. GPCRs contain seven membrane-spanning regions with their N-terminal segment on the exoplasmic face and their C-terminal segment on the cytosolic face of the plasma membrane. GPCRs tranduce a huge number of extracellular signals from hormones, neurotransmitters, chemokines and other environmental stimuli to the interior of cells and play fundamental roles in regulating a variety of cellular functions. GPCR signal is modulated by Ser/Thr phosphorylation on the carboxyl terminus, which leads to receptor desensitization by uncoupling the receptor from its cognate G proteins and GPCR internalizaiton and down-regulation. G protein-coupled receptor kinases (GRKs) catalyze phosphorylation of agonist-occupied GPCR, and this phosphorylation is the initial step of GPCR desensitization, thus GRK is the major factor regulating GPCR signaling. Seven members of the GRK family have been identified to date, and they have been divided on the basis of structural and functional similarities into rhodopsin kinase, GRK2 and GRK4 subfamilies.RTKs and GPCRs do not operate in isolation, but often function as partners to initiate complex interactions between downstream signaling molecules generated by each receptor. There is wide spread crosstalk between RTK signaling pathway and GPCR signaling pathway. Previous studies have demonstrated that overexpression of GRK2 may enhance EGF-induced ERK/MAPK phosphorylation, suggesting that GRK2 may play a modulatory role in EGF signaling pathway. But the underlying mechanisms remain to be elucidated.In this study we investigated the molecular mechanisms underlying the modulatory role of GRK2 in EGF signaling pathway. The present study showed that: (1) Overexpression of GRK2 in HEK 293 cells enhanced EGF-induced ERK/MAPK phosphorylation. Phosphorylated-ERK/MAPK was significantly enhanced in cells overexpressing GRK2 as compared with control cells overexpressing Pgal at the time point 30, 60 and 120 min of EGF stimulation (p<0.05). (2) GRK2 resided in EGFR immunoprecipitation complex upon EGF stimulation in cells coexpressing EGFR and GRK2, while there was barely GRK2 in the EGFR immunoprecipitation complex before EGF stimulation. Moreover the EGF-induced binding of GRK2 and EGFR was time-dependent. There was GRK2 in EGFR immunoprecipitation complex at 1 min of EGF stimulation and the GRK2 level in the EGFR immunoprecipitation complex reached peak at 5 min of EGF stimulation. Laser confocal microscopy studies showed that GRK2GFP translocated to cell memrane and colocalized with EGFR upon EGF stimulation. (3) Pretreatment of HEK 293 cells coexpressing EGFR and GRK2 with EGFR tyrosine kinase specific inhibitor Typhostins AG1478 inhibited EGFR tyrosine phosphorylation and the binding of GRK2 to EGFR. Coimmunoprecipitation experiments from HEK 293 cells coexpressing EGFR kinase dead mutant (EGFR KD) or wild type EGFR and GRK2 have shown that EGF stimulation could not induce EGFR KD tyrosine phosphorylation and the binding of GRK2 to EGFR KD. GRK2 kinase dead mutant (GRK2 K220R) could bind with EGFR even without EGF stimulation, upon EGF stimulation the binding of GRK2 K220R to EGFR was enhanced. Moreover GRK2 K220R has shown enhanced binding with EGFR as compared with wild type GRK2. (4) GRK2 N-terminus (~ 185 amino acids) deletion mutant (GRK2 Del N) could bind...