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• W2031835913 abstract "Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine2A, lysophosphatidic acid, angiotensin AT1, bradykinin B2) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-κB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative β-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis. Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine2A, lysophosphatidic acid, angiotensin AT1, bradykinin B2) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-κB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative β-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis. receptor tyrosine kinase epidermal growth factor epidermal growthfactor receptor green fluorescent protein Gprotein-coupledreceptor protein kinase C analysis of variance 5-hydroxytryptamine extracellular signal-regulated kinase mitogen-activated protein kinase/ERK kinase cAMP-response element-binding protein phorbol 12-myristate 13-acetate phosphate-buffered saline concanavalin A Receptor tyrosine kinases (RTKs)1 and G protein-coupled receptors (GPCRs) are the two major families of receptors that convert extracellular signals into cellular physiological and mitogenic responses. Previously, the signals generated by RTKs and GPCRs were thought to be neatly compartmentalized, with very little cross-talk between or sharing of the signaling pathways. There is a new awareness that RTKs, such as the EGF receptor, and GPCRs possess the capacity for cross-talk during signal initiation and propagation. Cross-talk can take the form of using shared signaling pathways (1Piiper A. Stryjek-Kaminska D. Zeuzem S. Am. J. Physiol. 1997; 272: G1276-G1284PubMed Google Scholar, 2Ramirez I. Tebar F. Grau M. Soley M. Cell. Signal. 1995; 7: 303-311Crossref PubMed Scopus (29) Google Scholar, 3van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) or, for GPCRs, using RTKs themselves as signaling platforms (4Della Rocca G. Maudsley S. Daaka Y. Lefkowitz R. Luttrell L. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 5Moriguchi Y. Matsubara H. Mori Y. Murasawa S. Masaki H. Maruyama K. Tsutsumi Y. Shibasaki Y. Tanaka Y. Nakajima T. Oda K. Iwasaka T. Circ. Res. 1999; 84: 1073-1084Crossref PubMed Scopus (109) Google Scholar, 6Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar, 7Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar, 8Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1324) Google Scholar, 10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar, 12Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Thus, contrary to relatively recent dogma, it is now abundantly clear that RTKs and GPCRs engage in extensive cross-talk with each other. Just as there are similarities in the mechanisms that initiate the signaling pathways of GPCRs and RTKs, there might also be similarities in the mechanisms by which those signals are terminated or desensitized. Indeed, there is a growing body of evidence that GPCRs and RTKs share mechanisms that regulate signal desensitization. Desensitization is a group of processes through which receptors or components of their signaling pathways become less responsive after previous exposures to receptor ligands. Homologous desensitization occurs when cells become unresponsive only to subsequent activation of the receptor that was previously stimulated. This type of desensitization is usually mediated by receptor specific kinases (GRKs). Heterologous desensitization refers to attenuation of one receptor system by another and is usually mediated by broad spectrum serine/threonine kinase such as protein kinases C and A. A special form of heterologous desensitization may occur when RTKs desensitize GPCRs. RTKs can desensitize GPCRs by phosphorylating the GPCR (13Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar), by phosphorylating heterotrimeric G proteins (14Leibmann C. Graness A. Boehmer A. Kovalenko M. Adomeit A. Steinmetzer T. Nurnberg B. Wetzker R. Boehmer F. J. Biol. Chem. 1996; 271: 31098-31105Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), or by other mechanisms (15Lamm M. Rajagopalan-Gupta R. Hunzicker-Dunn M. Endocrinology. 1999; 140: 29-36Crossref PubMed Scopus (10) Google Scholar, 16Arimura S. Saito Y. Nakata H. Fukishima K. Nishio E. Watanabe Y. Life Sci. 1998; 63: 1563-1570Crossref PubMed Scopus (3) Google Scholar). It is also possible that GPCRs could desensitize RTKs, but little is known about this phenomenon. Renal mesangial cells possess many mitogenic GPCRs, including angiotensin II AT1A (17Schlondorff D. DeCandido S. Satriano J.A. Am. J. Physiol. 1987; 253: C113-C120Crossref PubMed Google Scholar), bradykinin B2 (18El-Dahr S.S. Dipp S. Baricos W.H. Am. J. Physiol. 1998; 275: F343-F352Crossref PubMed Google Scholar,19Jaffa A.A. Miller B.S. Rosenzweig S.A. Naidu P.S. Velarde V. Mayfield R.K. Am. J. Physiol. 1997; 273: F916-F924PubMed Google Scholar), lysophosphatidic acid (20Goppelt-Struebe M. Fickel S. Reiser C.O. Biochem. J. 2000; 345: 217-224Crossref PubMed Scopus (42) Google Scholar, 21Gaits F. Salles J.P. Chap H. Kidney Int. 1997; 51: 1022-1027Abstract Full Text PDF PubMed Scopus (34) Google Scholar), and 5-hydroxytryptamine (5-HT2A) receptors (22Nebigil C.G. Garnovskaya M.N. Spurney R.F. Raymond J.R. Am. J. Physiol. 1995; 268: F122-F127PubMed Google Scholar). Mesangial cells also express RTKs, which may participate in the proliferative phase of chronic renal failure (23Floege J. Eng E. Young B.A. Johnson R.J. Kidney Int. Suppl. 1993; 39: S47-S54PubMed Google Scholar) or in the recovery from renal failure (24Ranieri E. Gesualdo L. Petrarulo F. Schena F.P. Kidney Int. 1996; 50: 1990-2001Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Mesangial cells possess an epidermal growth factor (EGF) receptor (25Takemura T. Murata Y. Hino S. Okada M. Yanagida H. Ikeda M. Yoshioka K. J. Pathol. 1999; 189: 431-438Crossref PubMed Scopus (31) Google Scholar) that stimulates proliferative cascades in those cells (26Ghosh Choudhury G. Jin D.C. Kim Y. Celeste A. Ghosh-Choudhury N. Abboud H.E. Biochem. Biophys. Res. Commun. 1999; 258: 490-496Crossref PubMed Scopus (28) Google Scholar). It is somewhat paradoxical that mesangial cells should express so many mitogenic receptors in that, under normal circumstances, proliferation is highly restrained within the confines of the glomerulus. This suggests that the responsiveness of mitogenic receptors must be rigidly controlled in mesangial cells. One mechanism through which rigid control of mitogenic signaling in mesangial cells might be exercised is desensitization. In this study, we report that pretreatment of kidney mesangial cells with GPCR ligands (5-HT, bradykinin, lysophosphatidic acid) results in a PKC-dependent transactivation of EGFR followed by a profound decrease in the ability of EGF to initiate multiple signals including autophosphorylation of the EGF receptor (EGFR), phosphorylation of ERK, and regulation of transcription factor activities (NF-κB, E2F, CREB). Furthermore, the desensitization pathway involves PKC and results in a dramatic internalization of native EGF receptors and transfected EGFR-GFP fusion proteins. Thus, preconditioning of cells by GPCR ligands may be a novel method to abrogate deleterious signals initiated by EGFR and other RTK. Drugs and reagents were obtained from the following sources. 5-HT, bradykinin, lysophosphatidic acid, epidermal growth factor, and phorbol 12-myristate 13-acetate were from Sigma. Phospho-ERK antibodies were obtained from New England Biolabs (Beverly, MA). GF109203X (bisindolylmaleimide I) and protease inhibitors (4-(2-aminoethyl)-benzenesulfonyl fluoride, EDTA, E-64, leupeptin, and aprotinin) were from Calbiochem. Anti-phosphotyrosine antibody (PY99), protein A-agarose, and E2F oligonucleotides were from Santa Cruz Biotechnology (Santa Cruz, CA). NF-κB and CREB oligonucleotides were from Promega (Madison, WI). Rat renal mesangial cells were obtained from cortical sections of kidneys from young 100–150-gram Harlan Sprague-Dawley rats using standard sieving techniques (27Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar). The kidneys were harvested in accordance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. Cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 and were subcultured every 1–2 weeks by trypsinization until a pure culture of mesangial cells was obtained. These cells were plated at a density of 2–5 × 104cells/ml in RPMI medium supplemented with 20% heat-inactivated fetal bovine serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Cells used were from passages 6–16. HEK293 cells were maintained in F12 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 50 µg/ml gentamicin (Life Technologies, Inc.) at 37 °C in a humidified 5% CO2 atmosphere. Transfections were performed on 50–70% confluent monolayers in 100-mm dishes, using LipofectAMINE, Lipofectin (Life Technologies), or FuGene™ 6 (Roche Molecular Biochemicals). Empty vectors were added to transfections to keep the total mass of DNA added per dish constant within experiments. 48 h prior to studies, cells were placed in serum-free medium supplemented with antibiotics and 0.1% bovine serum albumin. Cells (∼1 × 107) were grown in 100-mm culture dishes, washed twice with phosphate-free buffer (10 mm HEPES, pH 7.4, 137 mm NaCl, 3 mm KCl), and incubated in phosphate-free RPMI medium supplemented with 20 mmdextrose, 20 mm HEPES, pH 7.4, and 100 µCi of [32P]phosphoric acid for 4 h at 37 °C. Cells were then treated with mitogens for 3 min with or without pretreatment with inhibitors 30 min before stimulation. Cells were placed on ice, washed three times with ice-cold phosphate-buffered saline, and lysed in a modified radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, 500 µm 4-(2-aminoethyl)-benzenesulfonyl fluoride, 500 µm EDTA, 1 µm E-64, 1 µmleupeptin, and 1 µg/ml aprotinin). The cell lysates were rocked at 4 °C for 1 h and then centrifuged to remove insoluble debris. The lysates were then diluted to a protein concentration of 1–2 mg/ml, and 500 µl was used for immunoprecipitation using 5 µg of anti-EGFR monoclonal antibody. The mixture was rocked for 1 h at 4 °C, then 20 µl of protein A- or protein G/A-agarose beads were added, and the mixture was incubated on a rocker for another 20 min at 4 °C. The immune complexes were isolated by centrifugation, washed three times with radioimmune precipitation buffer, and then dissociated from the agarose beads by adding Laemmli buffer. The samples were heated to 90 °C for 2 min and then loaded onto precast 4–20% polyacrylamide gels (Novex, San Diego) and resolved under nonreducing conditions. The gels were dried and analyzed with a PhosphorImager. ERK immunoblots were performed essentially as described previously (28Garnovskaya M.N. Mukhin Y. Raymond J.R. Biochem. J. 1998; 330: 489-495Crossref PubMed Scopus (41) Google Scholar). The phospho-ERK antibody was used at 1:1000 dilution, whereas the control antibody, which recognizes equally well the phosphorylated and nonphosphorylated mitogen-activated protein kinase, was used at a 1:500 dilution as per the manufacturers recommendations. After treatment, cells were scraped into Laemmli buffer, boiled for 3 min, and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions with 4–20% pre-cast gels (Novex). After semi-dry transfer to polyvinylidene difluoride membranes, the membranes were blocked with a BLOTTO buffer (5% defatted dried milk in 10 mm Tris, 150 mm NaCl, 1% Tween 20, pH 8.0). The membranes were incubated overnight with the BLOTTO containing the phospho-ERK antibody. The membranes were washed, then exposed to goat anti-rabbit alkaline phosphatase-conjugated IgG (1:1000) in BLOTTO for 1 h, and then washed again. Immunoreactive bands were visualized by a chemiluminescent method (CDP Star™, New England Biolabs) using pre-flashed Kodak X-AR film. For other immunoblots, cell extracts were incubated with 5 µg/ml anti-EGFR or anti-phosphotyrosine monoclonal antibodies and visualized as described above, except the secondary antibody was a rabbit anti-mouse IgG alkaline phosphatase conjugate. A bright green mutant of GFP, enhanced GFP (CLONTECH, Palo Alto, CA), was attached to the carboxyl terminus of human EGFR as previously described (29Carter R.E. Sorkin A. J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This construct behaves like wild-type EGFR in assays of phosphorylation, protein-protein interactions, signal transduction, internalization, and degradation (29Carter R.E. Sorkin A. J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Cells grown in 6-well plates were incubated with vehicle, EGF, or 5-HT prior to incubation with various concentrations of EGF (1–100 ng/ml) for various times at 37 °C. Monolayers were then washed twice with ice-cold Hanks' balanced salt solution. Cells were then washed with cold acid wash buffer (50 mm glycine, 100 mm NaCl, pH 3.0) to dissociate bound EGF followed by three cold Hanks' balanced salt solution washings. Cells were then incubated with 50 pm125I-EGF for 90 min at 4 °C in HEPES binding medium (RPMI 1640 with 40 mm HEPES, pH 7.4, 0.1% bovine serum albumin) in the continuing presence of vehicle or 5-HT. Cells were then washed three times with Hanks' balanced salt solution and dissolved in 1 ml of 1 m NaOH. The solubilized material was collected in scintillation vials and counted in a γ-counter. Nonspecific binding was determined in quadruplicate wells containing 100 ng/ml unlabeled EGF and was subtracted from total binding to yield specific 125I-EGF binding at each time point. Data were analyzed using Prism 2.0 software (GraphPad Software, San Diego, CA). Oligonucleotides (E2F1, CREB, or NF-κB transcription factor consensus binding sites) were end-labeled using T4 polynucleotide kinase and [γ32P]CTP. Nuclear extracts were prepared exactly as described (30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar), and the electrophoretic mobility shift assay was modified from a previously published protocol (31Grewal J.S. Bag J. FEBS Lett. 1996; 383: 267-272Crossref PubMed Scopus (5) Google Scholar). The reaction mixture comprised 10 µg of nuclear extracts, 1–2 µg of poly(dI-dC), 5 µl of 5× binding buffer (50 mm HEPES, pH 7.8, 5 mm spermidine, 15 mm MgCl2, 36% glycerol, 3 mg/ml bovine serum albumin, and 15 mmdithiothreitol). This mixture was incubated on ice for 15 min, and then 40,000–70,000 cpm of 32P-labeled oligonucleotide were added. The reaction mixture was incubated further for 15 min at room temperature. DNA-protein complexes were then resolved on 5% native polyacrylamide gels and quantified with a PhosphorImager after drying. For the CREB assays, cells were pretreated with Ro20–1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a selective cAMP-specific phosphodiesterase inhibitor. For competition assays, nuclear extracts were preincubated with unlabeled oligonucleotides at room temperature for 15 min before adding 32P to the labeled oligonucleotide. When possible, reactions were also carried out by using a 32P-labeled oligonucleotide carrying mutations in the consensus regions to check the specificity of the binding reaction. Assays for the inhibition of endocytosis were carried out as described previously by Jockerset al. (32Jockers R. Angers S. Da Silva A. Benaroch P. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1999; 274: 28900-28908Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Cells that were serum-starved for 24 h in 6-well culture plates or 100-mm dishes were treated with various conditions that inhibit endocytosis, including potassium depletion (33Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar), hypertonic medium (34Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar, 35Moss A.L. Ward W.F. J. Cell. Physiol. 1991; 149: 319-323Crossref PubMed Scopus (22) Google Scholar), concanavalin A (36Henis Y.I. Elson E.L. Exp. Cell Res. 1981; 136: 189-201Crossref PubMed Scopus (18) Google Scholar), and monodansylcadaverine (37Davies P.J. Davies D.R. Levitzki A. Maxfield F.R. Milhaud P. Willingham M.C. Pastan I.H. Nature. 1980; 283: 162-167Crossref PubMed Scopus (412) Google Scholar, 38Phonphok Y. Rosenthal K.S. FEBS Lett. 1991; 281: 188-190Crossref PubMed Scopus (64) Google Scholar). Cells were incubated with cycloheximide (5 µg/ml) for 30 min prior to treatment with mitogens to prevent the confounding effects of protein synthesis. Procedures to inhibit endocytosis were also performed for 30 min prior to experimentation coincident with cycloheximide. The buffers used were as follow: potassium depletion buffer (20 mm HEPES, pH 7.4, 140 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 4.5 mg/liter dextrose); hypertonic medium (RPMI 1640, 0.5% bovine serum albumin, 4.5 g/liter dextrose, and 500 mm sucrose); and medium for chemical inhibition by concanavalin A (250 µg/ml) or 500 µmmonodansylcadaverine (RPMI 1640, 0.5% bovine serum albumin, 4.5 g/liter dextrose). Cells were then shifted to 4 °C, and cell-surface EGFRs were measured by radioligand binding. HEK293 or mesangial cells were grown on round coverslips by placing coverslips at the bottom of the wells in 6- or 12-well culture plates. After rinsing in PBS, adherent cells transfected with the EGFR-GFP fusion protein were identified by incubating with rhodamine-concanavalin A (10 µg/ml in PBS) for 2 min at 4 °C. Cells were rinsed with PBS several times and then fixed with 4% paraformaldehyde in PBS for 15 min followed by quenching the fixative with three 5-min washes with 50 mm NH4Cl at room temperature. For single labeling of EGFR, cells on coverslips were fixed as described above and were then inverted onto 20 µl of fluorescein isothiocyanate-conjugated anti-EGFR antibody raised against an extracellular epitope of the receptor (1:100) dilution in PBS with 1% goat serum and incubated in the dark for 2 h at room temperature. Cells were rinsed four times with PBS supplemented with 1% goat serum for 10 min. Coverslips were then mounted on a slide with Slow-Fade medium (Molecular Probes, Eugene, OR) and sealed with Cytoseal (Electron Microscopy Sciences, Fort Washington, PA) solution before scanning under a confocal microscope (Olympus Merlin™ IX70, Melville, NY). Cells in 100 mm-culture dishes were treated with 30 µg/ml cycloheximide or puromycin dihydrochloride for 1 h before treatment with 5-HT for different time periods, after which cells were washed and scraped into a modified radioimmune precipitation buffer as described above. Total EGFR protein was visualized by immunoprecipitation and immunoblotting as described above using anti-EGFR polyclonal antibody for immunoprecipitation and an anti-EGFR monoclonal antibody for immunoblotting. Fig. 1 shows that when rat renal mesangial cells were treated with 1 µM 5-HT for 3 min, EGFRs became phosphorylated as detected by metabolic labeling and immunoprecipitation of EGFRs. The increase was dependent upon both the concentration of 5-HT (EC50 = 160 nm) and time of incubation, peaking at 3–10 min. EGFR phosphorylation was blocked almost completely when cells were pretreated with the specific EGFR tyrosine kinase inhibitor AG1478 for 30 min prior to exposure to 5-HT. Thus, the 5-HT2A receptor transactivates the EGFR through the intrinsic kinase activity of the EGFR in a manner already shown to occur with other GPCRs such as those for angiotensin II (6Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar), carbachol, lysophosphatidic acid, and thrombin (12Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 39Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar). Because both ERK and PKC can induce phosphorylation of the EGFR (40Morrison P. Takishima K. Rosner M.R. J. Biol. Chem. 1993; 268: 15536-15543Abstract Full Text PDF PubMed Google Scholar), and because the 5-HT2A receptor has been shown to activate both ERK and PKC (27Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar, 41Ganz M.B. Boron W.F. Am. J. Physiol. 1994; 266: F576-F585Crossref PubMed Google Scholar), we used inhibitors of ERK kinase (MEK1) and PKC to determine which of those intermediates might be involved in 5-HT-induced phosphorylation of the EGFR. Fig. 1 c shows that a PKC inhibitor (5 µm GF109203X) greatly attenuated 5-HT-induced phosphorylation of the EGFR, whereas a MEK inhibitor (100 µm PD98059) did not. This concentration of PD98059 nearly completely attenuates ERK activation by the 5-HT2A receptor in these cells as previously determined by us (27Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar). Thus, PKC (and not MEK/ERK) seems to be involved in the transphosphorylation of the EGFR by the 5-HT2A receptor in mesangial cells. Next, we examined the effects of 5-HT on the activation of three EGFR-stimulated transcription factors (E2F, CREB, and NF-κB). Fig. 2 shows that acute treatment with either 5-HT or EGF induced activation of all three transcription factors as assessed by electrophoretic mobility shift assay. Moreover, the stimulation of all three transcription factors by 5-HT could be attenuated by preincubation with AG1478. Similarly, AG1478 blocked 5-HT-induced phosphorylation of ERK in mesangial cells (not shown). Those results suggest that the 5-HT2A receptor in mesangial cells activates transcription factors through the intermediary actions of the EGFR. To further explore the similarities between the effects of 5-HT and EGF on EGFR function, we next studied the effects of prior treatment with 5-HT on the activation of downstream signals by EGF. Our rationale for those studies is that EGF treatment has been shown to desensitize the EGFR to subsequent activation by EGF. Thus, we hypothesized that 5-HT pretreatment might also desensitize the EGFR. Fig. 3 shows the results of studies in which EGF-induced ERK phosphorylation was assessed after pretreatment with vehicle or 5-HT. Those results clearly demonstrate that pretreatment of mesangial cells with 5-HT results in a marked attenuation of the ability of EGF to induce phosphorylation of ERK. We used a similar paradigm to assess the effects of prior treatment with 5-HT on the ability of EGF to activate the three transcription factors shown in Fig. 2. Those results are shown in Fig.4, a–c. Pretreatment with 5-HT greatly reduced transcription factor activation as reflected by their binding with respective labeled consensuscis-elements. The specificity of the interactions of the transcription factors with their consensus oligonucleotides was confirmed by competition with unlabeled oligonucleotides and mutant (nonbinding) oligonucleotides. Figs. 3 and 4 show that multiple signals residing downstream from the EGFR can be attenuated by pretreatment with 5-HT, which suggested to us that desensitization of the EGFR most likely occurs at the level of the receptor itself. Therefore, we tested the effects of pretreatment with 5-HT on the ability of EGF to induce autophosphorylation of the EGFR. Fig. 5shows that pretreatment of mesangial cells with 5-HT leads to a marked decrease in the ability of multiple concentrations of EGF to induce the phosphorylation of its receptor. The attenuation was consistent over a broad range of concentrations of EGF, suggesting that that this effect may be relevant under physiological conditions. If the effect of pretreatment of cells with 5-HT is truly important, we would expect that other GPCRs might also desensitize the EGFR. Indeed, attenuation of EGF-induced phosphorylation of the EGFR was observed when cells were pretreated with other mitogenic GPCR ligands such as bradykinin and lysophosphatidic acid (Fig.6, a and b) as well as angiotensin (not shown). Thus, the ability of GPCR to desensitize EGF-induced phosphorylation of EGFR is not limited to the 5-HT2A receptor. One of the major pathways that links Gi and Gq-coupled receptors to mitogenic signals in mesangial and other cells involves PKC (3van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 27Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar, 39Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (608) Google Scholar). We therefore examined the effects of direct stimulation of PKC on the ability of EGF to induce phosphorylation of the EGFR. Fig.6 c shows that when cells were pretreated with 1 µm phorbol 12-myristate 13-acetate (PMA), the ability of EGF to induce tyrosine phosphorylation of the EGFR was reduced by at least 60%. These results suggest that PKC is involved in both transactivation and desensitization of the EGFR by GPCRs. One potential mechanism through which the EGFR could be desensitized is by internalization such that cell-surface EGFRs available for binding by EGF would be diminished. Thus, we measured cell-surface EGFRs by ligand binding and by confocal microscopy. For ligand binding, cells were treated for 1 h with vehicle, EGF, or 5-HT and subjected to acid wash, and then cell-surface125I-EGF binding was measured. Fig.7 shows that preincubation with either 5-HT (300 nm or 3 µm) or EGF (20 ng/ml) resulted in a marked down-regulation of cell-surface125I-EGF binding, which was nearly complete after 10 min of pre-incubation. This down-regulation of binding could be due either to decreased numbers of cell-" @default.
• W2031835913 created "2016-06-24" @default.
• W2031835913 creator A5033763744 @default.
• W2031835913 creator A5037713958 @default.
• W2031835913 creator A5054100392 @default.
• W2031835913 date "2001-07-01" @default.
• W2031835913 modified "2023-09-27" @default.
• W2031835913 title "G Protein-coupled Receptors Desensitize and Down-regulate Epidermal Growth Factor Receptors in Renal Mesangial Cells" @default.
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