Carbachol Stimulates Transactivation of Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase in T84Cells

Abstract

We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 μm) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 μm), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 μm). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nm), but was not altered by the PKC inhibitor GF 109203X (1 μm). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 μm), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated. We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 μm) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 μm), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 μm). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nm), but was not altered by the PKC inhibitor GF 109203X (1 μm). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 μm), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated. carbachol G-protein-coupled receptor epidermal growth factor EGF receptor: ERK, extracellular signal-regulated kinase mitogen-activated protein kinase short circuit current phorbol myristate acetate protein kinase C polyacrylamide gel electrophorsis 4-diphenylacetoxy-N-methylpiperidine. Chloride secretion across intestinal epithelial cells is under close regulation by hormonal, neural, paracrine, and endocrine factors. Such factors stimulate chloride secretion by binding to specific receptors on the surface of epithelial cells and consequently stimulating elevations in the levels of intracellular second messengers, such as cyclic nucleotides and calcium. In turn, increases in the levels of intracellular messengers activate the transport proteins that comprise the chloride secretory mechanism (1Barrett K.E. Am. J. Physiol. 1997; 272: C1069-C1076Crossref PubMed Google Scholar). However, even though both calcium and cyclic nucleotides are both capable of stimulating epithelial chloride secretion, it has been known for many years that these two classes of intracellular messengers stimulate temporally distinctive chloride secretory responses. While responses to cyclic nucleotide-mediated agonists, such as vasoactive intestinal peptide, are sustained, secretory responses to calcium-mediated agonists, such as the muscarinic agonist, carbachol (CCh),1 are transient, even though levels of intracellular calcium remain elevated after the secretory response has resolved (2Dharmsathaphorn K. Pandol S.J. J. Clin. Invest. 1985; 77: 348-354Crossref Scopus (238) Google Scholar, 3Mandel K.G. Dharmsathaphorn K. McRoberts J.A. J. Biol. Chem. 1986; 261: 704-712Abstract Full Text PDF PubMed Google Scholar, 4Vajanaphanich M. Schultz C. Tsien R.Y. Traynor-Kaplan A.E. Pandol S.J. Barrett K.E. J. Clin. Invest. 1995; 96: 386-393Crossref PubMed Scopus (53) Google Scholar). This implies that negative signals exist within intestinal epithelial cells that limit, or “switch off” epithelial secretory responses to calcium-mediated agonists. Since epithelial cells are continuously exposed, in vivo, to endogenously generated hormones and neurotransmitters that stimulate epithelial secretion via calcium-dependent mechanisms (5Kaunitz J.D. Barrett K.E. McRoberts J.A. Yamada T. Gastroenterology. 2nd Ed. Lippencott, Philadelphia, PA1995: 326-361Google Scholar), such inhibitory signals may represent an intrinsic epithelial “braking” mechanism by which excessive chloride, and consequently water, secretion may be prevented. Previous work from our laboratory has identified several intracellular messengers which appear to negatively influence calcium-mediated chloride secretion. Influx of extracellular calcium, generation of inositol (3,4,5,6)-tetrakisphosphate, and activation of protein kinase C (PKC) have all been shown to inhibit secretory responses to calcium-dependent agonists (1Barrett K.E. Am. J. Physiol. 1997; 272: C1069-C1076Crossref PubMed Google Scholar). More recently we have shown that receptor tyrosine kinase-dependent signaling pathways, such as those activated by epidermal growth factor (EGF) also activate intracellular pathways that are inhibitory to calcium-dependent secretion (6Uribe J.M. Gelbmann C.M. Traynor-Kaplan A.E. Barrett K.E. Am. J. Physiol. 1996; 271: C914-C922Crossref PubMed Google Scholar, 7Uribe J.M. Keely S.J. Traynor-Kaplan A.E. Barrett K.E. J. Biol. Chem. 1996; 271: 26588-26595Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Furthermore, since influx of extracellular calcium (8Bischof G. Beate I. Reenstra W.W. Machen T.E. Am. J. Physiol. 1995; 268: C154-C161Crossref PubMed Google Scholar) and stimulation of inositol (3,4,5,6)-tetrakisphosphate production (9Vajanaphanich M. Wasserman M. Buranawuti T. Traynor-Kaplan A.E. Barrett K.E. Gastroenterology. 1993; 104: A286Crossref PubMed Scopus (323) Google Scholar) in response to calcium-dependent secretagogues have both been demonstrated to be dependent upon stimulation of tyrosine kinase activity, tyrosine kinases appear to play important role(s) in negative regulation of epithelial secretory processes. Indeed, recent studies demonstrate that tyrosine kinases may function in the regulation of apical cystic fibrosis transmembrane conductance regulator Cl− channels (10Illek B. Fischer H. Santos G.F. Widdicombe J.H. Machen T.E. Reenstra W.W. Am. J. Physiol. 1995; 268: C886-C893Crossref PubMed Google Scholar, 11Lehrich R.W. Forrest J.N. Am. J. Physiol. 1995; 269: F594-F600PubMed Google Scholar, 12Sears C.L. Firoozmand F. Mellander A. Chambers F.G. Eromar I.G. Bot A.G.M. Scholte B. DeJonge H.R. Donowitz M. Am. J. Physiol. 1995; 269: C874-C882PubMed Google Scholar, 13Illek B. Fischer H. Machen T.E. Am. J. Physiol. 1996; 270: C265-C275Crossref PubMed Google Scholar) and basolateral K+ channels (13Illek B. Fischer H. Machen T.E. Am. J. Physiol. 1996; 270: C265-C275Crossref PubMed Google Scholar), both of which are transport proteins essential for epithelial chloride secretion. Although it has been demonstrated previously that calcium-dependent secretagogues stimulate intestinal epithelial tyrosine kinase activity (8Bischof G. Beate I. Reenstra W.W. Machen T.E. Am. J. Physiol. 1995; 268: C154-C161Crossref PubMed Google Scholar), there is little known of the signaling pathways involved in such processes. However, some insight into how G-protein-coupled receptors (GPCRs), such as the muscarinic receptor for CCh on colonic epithelia, might be linked to alterations in intracellular tyrosine kinase activity comes from studies in other models. In recent years, there has been a growing body of evidence to suggest that many GPCR agonists can activate the MAPK signaling cascade in a variety of cell types including epithelial cells (14Dabrowski A. Groblewski G.E. Schafer C. Guan K.-U. Williams J.A. Am. J. Physiol. 1997; 42: C1472-C1479Crossref Google Scholar, 15Wilkie N. Morton C.M. Ng L.L. Boarder M.R. J. Biol. Chem. 1996; 271: 32447-32453Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Gerthoffer W.T. Yamboliev I.A. Pohl J. Haynes R. Dang S. McHugh J. Am. J. Physiol. 1997; 272: L244-L252PubMed Google Scholar, 17Heasley L.E. Senkfor S.I. Winitz S. Strasheim A. Teitelbaum I. Berl T. Am. J. Physiol. 1994; 267: F366-F373PubMed Google Scholar, 18Nakamura K. Zhou C.-J. Parente J. Chew C.S. Am. J. Physiol. 1996; 271: G640-G649PubMed Google Scholar, 19Zou Y. Komuro I. Yamazaki T. Aikawa R. Sumiyo K. Shiojima I. Hiroi Y. Mizuno T. Yazaki Y. J. Biol. Chem. 1996; 271: 33592-33597Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The MAPK signaling cascade is classically involved in mediating the mitogenic actions of growth factors, such as EGF, and the mechanisms by which the EGF receptor (EGFr) is coupled to MAPK activation have been well elucidated (20Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3205) Google Scholar). Upon ligand binding the EGFr becomes autophosphorylated and recruits the adapter molecules, Shc and Grb2, which bind to tyrosine-phosphorylated residues on the receptor by virtue of their SH2 domains. Formation of the EGFr-Shc-Grb2 complex leads to activation of a guanine nucleotide exchange factor, mSOS, which in turn activates the low molecular weight G-protein, p21ras. This, in turn brings about activation of the upstream components of the MAPK cascade, Raf and MEK, ultimately leading to the stimulation of the ERK isoforms of MAPK. The mechanisms by which GPCRs stimulate ERK activation are less well understood but current evidence suggests the involvement of two predominant pathways which may be differentially activated depending on the nature of receptor coupling to heterotrimeric G-proteins (21van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 22Sugden P.H. Clerk A. Cell Signal. 1997; 9: 337-351Crossref PubMed Scopus (282) Google Scholar, 23Luttrell L.M. van Biesen T. Hawes B.E. Della-Rocca G.J. Luttrell D.K. Lefkowitz R.J. Adv. Pharmacol. 1998; 42: 466-470Crossref PubMed Scopus (6) Google Scholar). Gi protein-coupled receptors (GiPCR) appear to stimulate ERK activation predominantly by a mechanism involving increases in intracellular tyrosine kinase activity, and which may be mediated by transactivation of the EGF receptor. In contrast, Gq protein-coupled receptors (GqPCR) are believed to stimulate ERK predominantly via a tyrosine kinase-independent pathway mediated by However, recent studies that these two pathways for ERK activation may not be and that in some may into a pathway G.J. van Biesen T. Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the we to the involvement of tyrosine kinase-dependent signaling pathways in negative regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. We CCh as a calcium-mediated which via coupled muscarinic receptors on colonic epithelial cells to stimulate chloride secretion K.E. M. J. Pharmacol. Pharmacol. PubMed Scopus Google Scholar, K.E. K. J. Pharmacol. 1995; PubMed Scopus Google Scholar). In we to a role for the ERK isoforms of MAPK in regulation of CCh-stimulated secretion, and to mechanisms CCh stimulation of ERK activity in T84 cells. vasoactive intestinal and genistein were from Tyrphostin GF and were from growth factor was from EGF receptor and were from were from were from other were of and were for of T84cells in were as previously A. McRoberts J.A. Dharmsathaphorn K. J. Clin. Invest. 1985; PubMed Scopus Google Scholar). T84 cells were in with were by cells were as previously A. McRoberts J.A. Dharmsathaphorn K. J. Clin. Invest. 1985; PubMed Scopus Google Scholar). and MAPK cells were on T84 cells are known to the of colonic were for to of T84cells were in and in The of the was and were to by the of current these in in response to agonists are of chloride secretion K.G. McRoberts J.A. G. Dharmsathaphorn K. Am. J. Physiol. 1986; PubMed Google Scholar). T84 cell on were with to for and were stimulated with agonists for the The was by in and the cells were in 100 and in for were into for and the was were for protein protein and that an of studies were with EGFr for by with protein were for and the was The were in and in and were in gel were for and were a polyacrylamide gel and proteins were by proteins were a the was with a of for by a with the of in in with were for in or in This was by in were an of was were and with as in the and ERK activity was a were in and in MAPK were for with 100 and of in of were by the of of gel and were for to Phosphorylation of by MAPK was by with specific for the of are as for a of or of with the were to as were to represent We to the of CCh on protein tyrosine phosphorylation in T84 cells. blot of T84 cell revealed stimulated with CCh (100 μm) on the basolateral a tyrosine phosphorylation of several T84 cell proteins, with molecular masses ranging up to 180 the increases in protein tyrosine phosphorylation stimulated by CCh are involved in regulation of CCh-stimulated chloride secretion, we examined the of the tyrosine kinase inhibitor, responses to CCh across of T84 cells. of T84cells with genistein (5 μm), which reversed CCh-stimulated protein tyrosine phosphorylation significantly potentiated responses to CCh (100 a role for CCh-stimulated tyrosine kinase activity in the of CCh-stimulated secretory responses. of the proteins increased tyrosine phosphorylation in response to CCh molecular masses of and to the molecular masses of the and isoforms of We to a role for ERK in negative regulation of CCh-stimulated secretory responses. blot were which that ERK is in T84 epithelial cells in response to CCh stimulation Furthermore, kinase in which we the of to the ERK demonstrated that CCh-stimulated increases in ERK phosphorylation were by increases in ERK activity In to CCh stimulation of ERK phosphorylation is a specific mediated by activation of we examined the of the muscarinic receptor on CCh-stimulated responses. (100 responses to CCh across T84 cells. responses to CCh were with cells (100 also ERK activation in response to CCh. ERK phosphorylation in cells was that in cells with a stimulation in cells In to the that CCh-stimulated ERK activation may be involved in regulation of CCh-stimulated chloride secretion, we examined the of a specific inhibitor of ERK on both ERK phosphorylation and responses to CCh. of T84 cell with a in phosphorylation of ERK in response to CCh Furthermore, (20 μm), which CCh-stimulated ERK phosphorylation by potentiated responses to CCh across of T84 cells responses to CCh were in cells with in cells also responses that in or those in up to after of the two mechanisms have been by which GPCRs may about ERK is mediated by a tyrosine kinase-dependent which may transactivation of the EGFr, the other is a tyrosine kinase-independent pathway (21van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 22Sugden P.H. Clerk A. Cell Signal. 1997; 9: 337-351Crossref PubMed Scopus (282) Google Scholar, 23Luttrell L.M. van Biesen T. Hawes B.E. Della-Rocca G.J. Luttrell D.K. Lefkowitz R.J. Adv. Pharmacol. 1998; 42: 466-470Crossref PubMed Scopus (6) Google Scholar). pathways for ERK activation appear to be and in T84 cells since both EGF (100 and the phorbol (100 nm), stimulate T84 cell ERK phosphorylation, with and to CCh with In the of we to the to which of these pathways is involved in mediating the of CCh on ERK Since of the proteins increased phosphorylation in response to CCh was a protein to the molecular of the EGFr we examined a role for the EGFr in mediating the of CCh on ERK studies were which that CCh (100 μm) stimulated a time-dependent increase in tyrosine phosphorylation of EGFr The of the response was to that for CCh stimulation of ERK phosphorylation CCh also stimulated an increase in co-immunoprecipitation of the adapter proteins, Shc and with EGFr and EGF (100 also stimulated phosphorylation of the EGFr and increased co-immunoprecipitation of both Shc and with the EGFr these that CCh transactivation of EGFr in T84 in a to that of EGF, the of Formation of such is a for ERK activation in many cell EGFr phosphorylation and increases co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. were stimulated with basolateral CCh (100 μm) for the or with basolateral EGF (100 and cell were with by and to were or proteins were by blot is of We examined the of a specific inhibitor of EGFr tyrphostin on CCh-stimulated ERK of T84 cells with tyrphostin AG1478 (1 μm) the of CCh on EGFr phosphorylation and significantly CCh-stimulated ERK activation In the of tyrphostin AG1478 CCh-stimulated ERK phosphorylation was by transactivation of the EGFr to a of the of CCh on ERK activation in Furthermore, we to with the ERK inhibitor tyrphostin AG1478 also responses to CCh across of T84 cells a role for transactivation of the EGFr, with activation of ERK, in negative regulation of CCh-stimulated epithelial secretion. In to a role for PKC in the of CCh on ERK we examined the of a inhibitor of PKC activity, on CCh-stimulated ERK of T84cells with (1 μm) significantly ERK phosphorylation responses to in cells with in the of were of those in cells stimulated with In contrast, not CCh stimulation of ERK phosphorylation it have that induced by the EGFr inhibitor, tyrphostin AG1478 both were in that isoforms of PKC other of are involved in mediating the of CCh on ERK activity in T84 epithelial cells. In the we have a role for tyrosine kinase-dependent signaling pathways in negative regulation of calcium-mediated epithelial chloride secretion. In with studies in colonic epithelial cell (8Bischof G. Beate I. Reenstra W.W. Machen T.E. Am. J. Physiol. 1995; 268: C154-C161Crossref PubMed Google Scholar), we that the calcium-mediated CCh, stimulated tyrosine phosphorylation of several proteins in T84 cells. tyrosine phosphorylation was in within with tyrosine phosphorylation after a to that which CCh-stimulated secretory responses to This increase in tyrosine kinase activity to be involved in regulation of CCh-stimulated secretion since the tyrosine kinase inhibitor, significantly potentiated both the and of responses. suggest to stimulation of chloride secretion, CCh epithelial tyrosine kinase activity which to function in the of CCh-stimulated secretory responses. Since two of the proteins increased tyrosine phosphorylation in response to CCh molecular to those of the ERK and isoforms of we our studies to a role for these in regulation of epithelial secretion. Although MAPK activation is classically with regulation of cell growth and our studies also an role for ERK in negative regulation of calcium-mediated chloride secretion across intestinal epithelial cells. This from the that not CCh stimulate increases in ERK phosphorylation and activity in T84 but of T84 cells with leads to increases in both the and of CCh-stimulated secretory responses. is to be a specific inhibitor of the upstream of the MAPK cascade, MEK, and has been shown to inhibit ERK activation in a variety of N. Morton C.M. Ng L.L. Boarder M.R. J. Biol. Chem. 1996; 271: 32447-32453Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. S. A. 1995; PubMed Scopus Google Scholar). The that CCh activates both and signaling pathways is by the that the muscarinic receptor not CCh-stimulated but also the of CCh on ERK it that CCh-stimulated secretory responses are not by the of stimulation of pathways, but also by the to which negative signaling such as ERK, are The mechanisms by which GPCRs stimulate ERK activation are complex and appear to be dependent not on the nature of receptor coupling to heterotrimeric but also upon cell (21van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 22Sugden P.H. Clerk A. Cell Signal. 1997; 9: 337-351Crossref PubMed Scopus (282) Google Scholar, 23Luttrell L.M. van Biesen T. Hawes B.E. Della-Rocca G.J. Luttrell D.K. Lefkowitz R.J. Adv. Pharmacol. 1998; 42: 466-470Crossref PubMed Scopus (6) Google Scholar, G.J. van Biesen T. Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). However, in cell types to ERK activation in response to stimulation (14Dabrowski A. Groblewski G.E. Schafer C. Guan K.-U. Williams J.A. Am. J. Physiol. 1997; 42: C1472-C1479Crossref Google Scholar, B. N. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, B. K.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), including activation in cells S. Schultz G. J. 1993; PubMed Scopus Google Scholar), is a though our with that stimulation of PKC can be linked to ERK activation in T84 this pathway not appear to represent a mechanism by which CCh ERK activity in these cells. GF which T84 cell ERK and which has been shown to inhibit ERK activation in other cells (14Dabrowski A. Groblewski G.E. Schafer C. Guan K.-U. Williams J.A. Am. J. Physiol. 1997; 42: C1472-C1479Crossref Google Scholar), on ERK phosphorylation in response to CCh. our a role for transactivation of the EGFr, a pathway by (21van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 22Sugden P.H. Clerk A. Cell Signal. 1997; 9: 337-351Crossref PubMed Scopus (282) Google Scholar, 23Luttrell L.M. van Biesen T. Hawes B.E. Della-Rocca G.J. Luttrell D.K. Lefkowitz R.J. Adv. Pharmacol. 1998; 42: 466-470Crossref PubMed Scopus (6) Google Scholar), in mediating the of CCh on ERK of evidence this CCh tyrosine phosphorylation of the EGFr with a to that for ERK CCh of the adapter proteins, Shc and Grb2, to the EGFr. of these proteins with receptor tyrosine kinases is well as an in growth activation of the MAPK cascade (20Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3205) Google Scholar). which have been demonstrated to of the EGFr, but not that of other EGFr G. I. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. 1995; 267: PubMed Scopus Google Scholar), we that tyrphostin AG1478 not CCh-stimulated EGFr phosphorylation in T84 but also significantly CCh-stimulated ERK Furthermore, to the ERK inhibitor, tyrphostin AG1478 responses to CCh across T84 cell our a role for a CCh-stimulated signaling involving transactivation of the EGFr and ERK which negatively CCh-stimulated chloride secretory responses. our studies were on cells the EGFr and receptor are known to be to the basolateral This may that are to the receptor transactivation is also of that even though tyrphostin AG1478 CCh-stimulated transactivation of the EGFr, ERK phosphorylation in the of the pathway may exist by which CCh ERK This ERK phosphorylation not appear to be mediated by PKC since GF was not in CCh-stimulated ERK phosphorylation but was also in with tyrphostin Further studies are in to this and of T84 cell ERK activation in response to CCh. is not known how CCh-stimulated transactivation of the EGFr and ERK activation might with epithelial transport pathways to about of chloride secretion. studies cystic fibrosis transmembrane conductance regulator chloride which are believed to be an important pathway for chloride in response to calcium-dependent as a for inhibitory tyrosine kinase-dependent signals W.W. K. A. S. S. Am. J. Physiol. 1996; 271: PubMed Google Scholar, I. F. Am. J. Physiol. 1996; 272: Google Scholar). recent studies demonstrate for muscarinic transactivation of the EGFr in regulation of K+ channels in cells E.G. J. 1997; PubMed Scopus Google Scholar), and for ERK in regulation of K+ function in cells S. R. J.M. B. J. 1995; PubMed Scopus Google Scholar). Further studies are to are involved in of CCh-stimulated secretion in T84 cells. studies are to CCh-stimulated ERK activity and other signals believed to be involved in negative regulation of calcium-dependent chloride secretion, such as influx of extracellular calcium M. Schultz C. Tsien R.Y. Traynor-Kaplan A.E. Pandol S.J. Barrett K.E. J. Clin. Invest. 1995; 96: 386-393Crossref PubMed Scopus (53) Google Scholar). in this are the recent of and S. A. 1996; PubMed Scopus Google Scholar) have demonstrated that influx of calcium calcium channels EGFr phosphorylation, of and Shc with the EGFr, and ERK activation in cells. it has been demonstrated that calcium influx and a calcium-dependent tyrosine are important in mediating the of GPCR receptor activation on ERK activity and K+ in cells G.J. van Biesen T. Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. J.M. B. J. 1995; PubMed Scopus Google Scholar, I. G. S. J. 1996; PubMed Scopus Google Scholar). Although a role for CCh-stimulated calcium influx in mediating the of the agonist on ERK activation in T84 cells has to be studies from our laboratory that T84 cells not but that the becomes with the EGFr upon stimulation with CCh. J. S. and K. CCh-stimulated influx of extracellular calcium may represent an in a signaling cascade which ultimately in stimulation of ERK activity via transactivation of the EGFr. In we have shown that CCh activation of the ERK isoforms of MAPK in colonic epithelial and that this to be involved in the of CCh-stimulated chloride secretory responses. of ERK activity by CCh via a pathway of but which transactivation of the EGFr. We that since chloride is the predominant secretion in the (5Kaunitz J.D. Barrett K.E. McRoberts J.A. Yamada T. Gastroenterology. 2nd Ed. Lippencott, Philadelphia, PA1995: 326-361Google Scholar), transactivation of the EGFr and activation of ERK may represent a mechanism to excessive and the levels of calcium-dependent secretagogues are elevated within the intestinal We for with