Epithermal Growth Factor Receptor Research Articles

Epidermal Growth Factor Receptor (EGFR) Inhibitors Screened from Autodisplayed Fv-Antibody Library.

Inhibitors of the epithermal growth factor receptor (EGFR) were screened from an autodisplayed Fv-antibody library using an anti-EGF antibody. The Fv-antibody library was expressed on the outer membrane of Escherichia coli, which corresponds to the heavy chain VH region of immunoglobulin G. The library was constructed by randomizing the CDR3 region of expressed VH regions (11 amino acid residues) by site-directed mutagenesis. Using an anti-EGF antibody as a screening probe, amino acid sequences (CDR3 region) with antibody binding affinity were screened from the Fv-antibody library. These amino acid sequences were considered to have similar chemical properties to EGF, which can bind to EGFR. Two autodisplayed clones with Fv-antibodies against EGFR were screened from the Fv-antibody library, and the screened Fv-antibodies were expressed as soluble proteins. The binding affinity (KD) was estimated using an SPR biosensor, and the inhibitory activity of expressed Fv-antibodies was observed for PANC-1 pancreatic tumor cells and T98G glioblastoma cells using Western blot analysis of proteins in the EGFR-mediated signaling pathway. The viability of PANC-1 and T98G cells was observed to decrease via the inhibitory activity of expressed Fv-antibodies. Finally, interactions between Fv-antibodies and EGFR were analyzed by using molecular docking simulations.

Modifications of IGF2 and EGFR plasma protein concentrations in NAFLD patients after bariatric surgery.

Nonalcoholic fatty liver disease (NAFLD) is strictly associated with the epidemic of obesity and is becoming the most prevalent liver disease worldwide. In severe obesity, bariatric surgery (BS) is the most effective treatment not only for obesity but also for the associated metabolic co-morbidities, NAFLD, among others. To date, noninvasive diagnostic/prognostic methods cannot evaluate hepatic improvements following surgery. We aimed to measure plasma level of insulin-growth factor-2 protein (IGF2) and epithermal growth factor receptor (EGFR), and to assess their relationship with clinical and biochemical parameters during the 12 months follow-up. Demographic, clinical-biochemical data, and plasma IGF2 and EGFR were measured in 69 patients preoperatively (T0) and 6 and 12 months (T6M and T12M, respectively) after BS. Liver biopsy was performed at T0. Relationships between IGF2, EGFR, and several biochemical parameters were performed using Pearson or Spearman correlation analysis. IGF2 plasma level increases during follow-up, passing from 2.5 (1.8-15.5) at baseline to 13.3 (8.6-19.1) at T12M, p < 0.001. Conversely, EGFR showed a not significant reduction. At T12M, the plasma level of both markers was comparable to those of lean subjects. The clinical-biochemical parameters (BMI, glycated hemoglobin, HOMA-IR) also return to the normal range at T12M. Correlation analysis demonstrated that IGF2 was significantly associated with total bilirubin, direct bilirubin, and albumin at T0 while with blood glucose, ALT, GGT, and AST/ALT ratio at T6M and T12M. IGF2 plasma levels increase after bariatric surgery, and these changes are associated with the modification of hepatic biochemical parameters, even if other clinic or metabolic improvements cannot be excluded.

Synergistic effects of Bcl-2 inhibitors with AZD9291 on overcoming the acquired resistance of AZD9291 in H1975 cells.

Non-small cell lung cancer (NSCLC) patients with epithermal growth factor receptor (EGFR) mutations can be treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), however, development of acquired resistance could significantly limit curative effects of EGFR-TKIs. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been widely reported, but there were few reports on the resistant mechanism of third-generation EGFR-TKI such as osimertinib (AZD9291). In the present study, significant upregulation of Bcl-2 was found in AZD9291-resistant H1975 cells (H1975AR) compared with H1975, which may constitute an important resistant mechanism of acquired resistance to AZD9291. More importantly, our study showed that synergism between AZD9291 and Bcl-2 inhibitor ABT263 (0.25μM) or ABT199 (1μM) could effectively overcome the acquired resistance of AZD9291 in H1975AR in vitro. Flow cytometry analyses demonstrated that AZD9291 + ABT263/ABT199 caused a significantly different cell cycle distribution and produced significantly more apoptosis compared with either AZD9291 or ABT263/ABT199 treatment alone. Further multiscreen/Western blot analyses revealed that NF-κB was significantly downregulated in AZD9291 + ABT263/ABT199 treatment groups compared with AZD9291 or ABT263/ABT199 treatment alone, with a more significant reduction of NF-κB in AZD9291 + ABT199 compared with AZD9291 + ABT263. It is also noticeable that AZD9291 + ABT263 specifically caused a significantly reduced expression of p21 compared with AZD9291 or ABT263 treatment alone while AZD9291 + ABT199 specifically caused significantly reduced expressions of SQSTM1 and survivin, but increased expression of autophagosome marker LC3-II compared with AZD9291 or ABT199 treatment alone. Furthermore, cytotoxicity of AZD9291 + ABT199 could be partially reversed by autophagy inhibitor chloroquine. These results suggest that ABT263 and ABT199 may work through different signaling pathways to achieve synergistic cytotoxicity with AZD9291 in H1975AR. These findings suggest that Bcl-2 inhibitor may provide an effective option in combination therapy with EGFR-TKIs to treat NSCLC with EGFR-TKI acquired resistance.

Secretome Analysis of Host Cells Infected with Toxoplasma gondii after Treatment of Human Epidermal Growth Factor Receptor 2/4 Inhibitors.

Toxoplasma gondii, a ubiquitous, intracellular parasite of the phylum Apicomplexa, infects an estimated one-third of the human population as well as a broad range of warm-blooded animals. We have observed that some tyrosine kinase inhibitors suppressed the growth of T. gondii within host ARPE-10 cells. Among them, afatinib, human epithermal growth factor receptor 2 and 4 (HER2/4) inhibitor, may be used as a therapeutic agent for inhibiting parasite growth with minimal adverse effects on host. In this report, we conducted a proteomic analysis to observe changes in host proteins that were altered via infection with T. gondii and the treatment of HER2/4 inhibitors. Secreting proteins were subjected to a procedure of micor basic reverse phase liquid chromatography, nano-liquid chromatography-mass spectrometry, and ingenuity pathway analysis serially. As a result, the expression level of heterogeneous nuclear ribonucleoprotein K, semaphorin 7A, a GPI membrane anchor, serine/threonine-protein phosphatase 2A, and calpain small subunit 1 proteins were significantly changed, and which were confirmed further by western blot analysis. Changes in various proteins, including these 4 proteins, can be used as a basis for explaining the effects of T. gondii infections and HER2/4 inhibitors.

Potential Mechanistic Links between Cancer Treatment and Alzheimer’s Disease: An Insight from the Study of Extracellular Vesicles

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV–visible spectroscopy (UV–visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at − 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL−1 to 50 ng mL−1. The detection limit was 1.37 pg mL−1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

EGFR detection in extracellular vesicles of breast cancer patients through immunosensor based on silica-chitosan nanoplatform

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV–visible spectroscopy (UV–visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at − 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL−1 to 50 ng mL−1. The detection limit was 1.37 pg mL−1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

Multiplexed SERS Detection of Soluble Cancer Protein Biomarkers with Gold-Silver Alloy Nanoboxes and Nanoyeast Single-Chain Variable Fragments.

Highly sensitive, multiplexed detection of soluble cancer protein biomarkers can facilitate early cancer screening as well as enable real-time monitoring of patients' sensitivity and resistance to therapy. Current technologies for detection of soluble cancer protein biomarkers, e.g., enzyme-linked immunosorbent assay,however, suffer from limited sensitivity, as well as the requirement of expensive monoclonal antibodies, which undergo the quality variability. Herein, we propose a sensitive, cheap, and robust surface-enhanced Raman scattering technology to detect a panel of soluble cancer protein biomarkers, including soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1) and soluble epithermal growth factor receptor (sEGFR), which are related to disease progression and treatment efficacy. In this assay, gold-silver alloy nanoboxes that have strong Raman signal enhancement capability were used as plasmonic nanostructures to facilitate highly sensitive detection. In addition, nanoyeast single-chain variable fragments were utilized as mAb alternatives to allow specific and stable protein capture performance. We successfully detected sPD-1, sPD-L1, and sEGFR with a limit of detection of 6.17 pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. We further tested the detection of these three soluble cancer protein biomarkers in human serum and achieved recovery rates between 82.99% and 101.67%. We believe our novel platform that achieves sensitive, multiplexed, and specific detection of soluble cancer protein biomarkers could greatly benefit cancer treatment and improve patient outcome.

Variations in EGFR ctDNA Correlates to the Clinical Efficacy of Afatinib in Non Small Cell Lung Cancer with Acquired Resistance.

Monitoring of non small cell lung cancer (NSCLC) patients on afatinib after acquired resistance to 1st generation tyrosine kinase inhibitors is important. Circulating tumor DNA (ctDNA) offers an attractive means other than conventional tissue biopsy to characterize real time dynamic changes of the disease. In our study, we aim to ascertain the clinical value for ctDNA monitoring of NSCLC patients with acquired resistance for afatinib treatment. 200 patients positive for the activating epithermal growth factor receptor (EGFR) mutations were recruited for the study. Baseline molecular profiling for L858R, Exon 19 deletion and T790M were performed. Thereafter, serial blood samples were taken and patients were assessed by overall survival (OS) to determine the usefulness of ctDNA monitoring. At baseline, matched tumor biopsy and ctDNA analysis had a concordance agreement of 93.5% for L858R and exon 19 deletion. We also determined that a large proportion of patients had the drug resistance mutation T790M prior to starting afatinib and these patients were linked to a worse survival outcome. For patients that registered a drop in ctDNA levels after afatinib was administered, we observed that their survival outcome was more favorable (hazard ratio 1.56, (95% CI 1.04 to 2.43). ctDNA levels were mostly elevated after the 3rd sampling cycle. Our results suggest that ctDNA can be used to predict the clinical benefits of afatinib treatment. Pre and post blood sampling aids to identify patient groups that may benefit most from the treatment and ctDNA is relatively sensitive to address the dynamic changes of the disease at the molecular level.

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition.

Hepatocellular carcinoma (HCC) is the third-most lethal cancer worldwide. Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients' prognosis. In this study, we report that protein tyrosine phosphatase receptor S (PTPRS) is significantly down-regulated in nearly 80% of HCCs, and its expression negatively correlates with aggressive pathological features, such as larger tumor size and advanced stage. In addition, PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients, although 16.7% of HCCs show intratumor heterogeneous expression of PTPRS. Restoration of wild-type, but not mutant, PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo, whereas knockdown of its expression significantly promotes invasion and metastasis. Notably, PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition (EMT). Moreover, PTPRS forms a complex with epithermal growth factor receptor (EGFR) and regulates its tyrosine residues' phosphorylation. Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS, whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation. Meanwhile, promoter hypermethylation of PTPRS is frequently detected in HCC samples and cell lines. Treatment with a demethylation agent, 5-aza-2'-deoxycytidine, recovers PTPRS expression in a dose-dependent manner. Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC.

Highly sensitive detection of epidermal growth factor receptor expression levels using a capacitance sensor

We developed a capacitance sensor with parallel plate geometry to measure epithermal growth factor receptor (EGFR) expression levels on cell membrane in real-time. We first proved correlations between capacitance changes and cell numbers settled down between electrodes, and then observed capacitance changes elicited by interactions between EGFR on membrane and EGF proteins in real time. Consequently, we confirmed that the EGFR expression levels of varied typed cells were successfully quantified. This approach can effectively distinguish differences of EGFR levels of cancer cells and normal cells in real-time. Also, up to 600% sensitivity enhancements and around 2.2h on average sensing time saving were achieved by using the capacitance sensor over a conventional immunoassay technique. Such a capacitance biosensor can be extended to broad fields where the receptor–antibody reactions, the receptor–virus reactions or DNA hybridizations are involved.

Abstract 320: Long-term Atorvastatin, But Not Pravastatin, Treatment Leads To Repressed Mitochondrial Gene Expression And Altered Cardiac Ultrastructure

Statins reduce low-density lipoprotein cholesterol (LDL-C) and decrease cardiovascular events. Although statins are generally well tolerated, the FDA warns that statins may induce skeletal muscle side effects, cognitive changes and increased fasting glucose levels. Skeletal muscle biopsies from patients with statin myopathy have revealed defects in mitochondrial ultrastructure. Impaired mitochondrial function has been postulated as a key cause of statin-induced myopathy and hepatotoxicity. Long-term statin effects on cardiac muscle are currently unknown. Wild type mice received atorvastatin, pravastatin or vehicle daily for seven months by oral gavage. Atorvastatin and pravastatin reduced LDL-C compared to vehicle. Echocardiography at two-week intervals showed no differences in %FS, %EF, circumferential fiber shortening and ventricular wall thicknesses between atorvastatin, pravastatin and vehicle treated mice. After seven months of atorvastatin, pravastatin or vehicle administration cardiac muscles (n=21-29) were analyzed, and only atorvastatin treated hearts revealed: A) swollen and misaligned mitochondria and accumulation of protein aggregates by transmission electron microscopy (n=4, each), and B) repression of mitochondrial and endoplasmatic reticulum related genes by genome-wide expression profiling. In cultured ventricular myocytes, atorvastatin, but not pravastatin; 1) down-regulated survival pathways via inhibition of ERK1/2T202/Y204, AktSer473 and mTOR signaling (p70 S6 kinaseThy421/Ser4240 and S6 RPSER 235/236), 2) reduced protein expression of caveolin-1, dystrophin, epithermal growth factor receptor and insulin receptor β, 3) decreased RhoA activation, and 4) induced apoptosis. LDL-C reduction by atorvastatin, but not pravastatin, was associated with a repression of mitochondrial and endoplasmic reticulum related genes, an accumulation of protein aggregates, and swollen mitochondria. This is the first report demonstrating that long-term atorvastatin treatment causes adverse effects on cardiac muscle with preserved cardiac function. Whether these changes predispose atorvastatin treated hearts to contractile dysfunction after hemodynamic stress needs further investigation.

Positron emission tomography-computed tomography on predicting the efficacy of targeted therapy for lung adenocarcinoma.

In this study, positron emission tomography-computed tomography (PET-CT) was used to monitor the maximal standard uptake value (SUVmax) in advanced lung adenocarcinoma patients with epithermal growth factor receptor (EGFR) mutation to prove its role in predicting the prognosis of targeted therapy. A total of 46 patients with advanced lung adenocarcinoma (IIIb-IV stage) were enrolled in the current study. They were positive for EGFR mutation. All patients received gefitinib (250 mg per day, administered orally). PET-CT was conducted prior to (at baseline) and six months after gefitinib administration for the lesion size and SUVmax. The recommendations of the European Organization for Research and Treatment of Cancer criteria were chosen for PET assessment. Metabolic response (SUV decline < -25%) was compared with morphologic response evaluated by CT scan and overall survival. Compared to patients with △SUV% ≥ 25% (progressive metabolic disease), the survival time was significantly prolonged in △SUV% < -25% (including complete metabolic response and progressive metabolic disease) (10.6/18.4, P = 0.000), but was not in -25% ≤ △SUV% < 25% (stable metabolic disease) (10.6/10.7, P = 0.088). Patients who achieved △SUV% < -25% after treatment were associated with a longer median survival, higher control rate, and better prognosis. There was a strong correlation between SUV changes (△SUV%) and CT size change (△lesion size%) (R(2) = 0.891, P = 0.000). Changes in the SUV could be used to predict the prognosis of targeted therapy in advanced lung adenocarcinoma.

Association of epithermal growth factor receptor expression and its downstream gene mutation status with radiosensitivity of colorectal carcinoma cell lines in vitro

To investigate the effect of epithermal growth factor receptor (EGFR) expression and K-ras, B-raf and PIK3CA mutation status on the radiosensitivity of human colorectal carcinoma (CRC) cell lines in vitro. Real-time RT-PCR was used to measure EGFR mRNA expression in nine human CRC cell lines, and K-ras, B-raf and PIK3CA mutation status of each CRC cell line was also identified respectively. After treatment with irradiation at graded dose, the cell viability was measured by clonogenic survival assay. The rate of cell apoptosis and cell cycle distribution were tested by flow cytometry. The cell morphology was observed with hoechst 33258 staining to analyze the correlation between EGFR mRNA expression and radiosensitivity of CRC cell lines. A positive correlation between EGFR mRNA expression and survival fraction of 2 Gy(SF2) was observed (r=0.717, P=0.030). Association was also identified between the mutation status of PIK3CA and radiosensitivity (t=2.401, P=0.047), while mutation status of K-ras and B-raf was not associated with radiosensitivity. At 48-hour after exposing to irradiation, the apoptosis rate of radiosensitive cell line (HCT116) was significantly increased in a dose-dependent manner (P<0.05), while the apoptosis rate of radioresistant cell line (HT29) was significantly increased only when radiation dose increased to 6 Gy. The ratio of G0/G1 phase was reduced significantly with the increase of radiation dose in radiosensitive cell line (HCT116, P<0.05), while this trend was not observed in radioresistant cell line (HT29, P>0.05). Over-expression of EGFR mRNA is correlated to radioresistance of human CRC cell lines, and mutation status of PIK3CA is closely related with radiosensitivity of CRC cells. The inhibition of apoptosis and G0/G1 arrest may induce the radioresistance of CRC cell lines.

The role of EGFR MAbs C225 in breast cancer stem cells

e22093 Background: The epithermal growth factor receptor(EGFR) signaling pathways has been implicated in self-renewal of breast cancer stem cells. It has been proved that the EGFR tyrosine kinase inhibitor-gefitinib significantly decreased the mammosphere- forming efficiency(MFE) in ductal carcinoma in situ (DCIS) derived cancer cells. We investigated the response of breast cancer stem cells in breast cancer cell line MCF-7 to EGFR MAbs C225(cetuximab). Methods: Cells were used for nonadherent (i.e.,mammosphere) culture and were divided into four groups according to the different culture medium whether or not including exogenous EGF and MAbs C225. MFE was calculated as the number of mammospheres (≥60um) and was expressed as a percentage. We Compared the percentage of CD44+/CD24- cells from the four groups, and measured the levels of the Wnt and Notch-3 gene expression of the four groups. Results: There was no significant difference of MFE between groups. In the presence of exogenous EGF, MAbs C225 significantly decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with exogenous EGF alone(0.7% versus 1.5%, P &lt; 0.001 and 4% versus 11%P &lt; 0.001;respectively). In the absence of exogenous EGF, MAbs C225 aslo statistically decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with that of lacking exogenous EGF and MAbs C225(0.53% versus 1.3%,P = 0.003 and 9% versus 3%, P &lt; 0.01;respectively). And we observed that the Notch-3 gene expression was compensativly increased in the two groups of addition of MAb C225. No significant differences of Wnt gene expression were observed between groups. Conclusions: EGFR MAbs C225 could disrupt mammoshpere formation and decrease the percentage of CD44+/CD24- cells of mammoshpere cells. The expression of Notch-3 is increased with the EGFR antagonistics in breast cancer stem cells. No significant financial relationships to disclose.

Epidermal growth factor differentially augments G(i)-mediated stimulation of c-Jun N-terminal kinase activity.

1. Signaling networks involving different receptor systems allow extracellular signals to be integrated and transformed into various biological activities. In this report, we studied the activity of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (MAPKs), in response to stimulation by G protein-coupled receptors (GPCRs) and co-activation with epithermal growth factor receptor (EGFR). 2. Stimulation of exogenous GPCRs in Cos-7 cells induced JNK activation of different magnitudes depending on their G-protein coupling specificities (G(q)>G(i)>G(s)), and a moderate JNK activation was linked to stimulation of endogenous EGFR by EGF. 3. Co-stimulation with GPCR agonists and EGF resulted in differential augmentation of JNK activities, with G(i)-coupled receptors associated with a synergistic JNK activation upon co-stimulation with EGF, while G(q)- and G(s)-coupled receptors were incapable of triggering this effect. 4. This G(i)/EGF-induced synergistic JNK activation was inhibited by pertussis toxin and AG1478, and may involve Src family tyrosine kinases, PI3 K, Ca(2+)/calmodulin and small GTPases as important intermediates, while Ca(2+) mobilization was triggered by the stimulation of G(q)-coupled receptor or EGF treatment, but not by the G(i)- or G(s)-coupled receptors. 5. Transient expression of Gbetagamma subunits with EGF treatment, or co-activation of exogenous G(i)-coupled receptor with thapsigargin also resulted in a synergistic JNK activation. Activation of G(i)-coupled receptor accompanied with EGF treatment enhanced the expression level and activity of MAPK phosphatase type I, which occurred after the maximal synergistic JNK activation. 6. Our results support a mechanistic model where EGF signaling may differentially regulate the JNK activities triggered by GPCRs of different coupling specificities.