title: Gs protein-coupled receptor agonists induce transactivation of the epidermal growth factor receptor in T84 cells: implications for epithelial secretory responses.
abstract: We have previously shown that Gq protein-coupled receptor (GqPCR) agonists stimulate epidermal growth factor receptor (EGFr) transactivation and activation of mitogen-activated protein kinases (MAPK) in colonic epithelial cells. This constitutes a mechanism by which Cl- secretory responses to GqPCR agonists are limited. In the present study we examined a possible role for the EGFr in regulating Cl- secretion stimulated by agonists that act through GsPCRs. All experiments were performed using monolayers of T84 colonic epithelial cells grown on permeable supports. Protein phosphorylation and protein-protein interactions were analyzed by immunoprecipitation and Western blotting. Cl- secretion was measured as changes in short-circuit current (DeltaIsc) across voltage-clamped T84 cells. The GsPCR agonist, vasoactive intestinal polypeptide (VIP; 100 nM), rapidly stimulated EGFr phosphorylation in T84 cells. This effect was mimicked by a cell-permeant analog of cAMP, Bt2cAMP/AM (3 microM), and was attenuated by the protein kinase A (PKA) inhibitor, H-89 (20 microM). The EGFr inhibitor, tyrphostin AG1478 (1 microM), inhibited both Bt2cAMP/AM-stimulated EGFr phosphorylation and Isc responses. VIP and Bt2cAMP/AM both stimulated ERK MAPK phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to the EGFr in a tyrphostin AG1478-sensitive manner. The PI3K inhibitor, wortmannin (50 nM), but not the ERK inhibitor, PD 98059 (20 microM), attenuated Bt2cAMP/AM-stimulated secretory responses. We conclude that GsPCR agonists rapidly transactivate the EGFr in T84 cells by a signaling pathway involving cAMP and PKA. Through a mechanism that likely involves PI3K, transactivation of the EGFr is required for the full expression of cAMP-dependent Cl- secretory responses.
authors.affiliation: Division of Gastroenterology, Department of Medicine, University of California, Medical Center, San Diego, California 92103-8414, USA.
authors.forename: Lone S
authors.initials: LS
authors.lastname: Bertelsen
authors.forename: Kim E
authors.initials: KE
authors.lastname: Barrett
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Keely
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D002713:Chlorine / Q000378:metabolism; D003106:Colon / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007477:Ions; D007546:Isoquinolines / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011799:Quinazolines; D013449:Sulfonamides; D013997:Time Factors; D015533:Transcriptional Activation; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002712:Chlorides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007477:Ions; D007546:Isoquinolines; D011799:Quinazolines; D013449:Sulfonamides; D020032:Tyrphostins; C101044:RTKI cpd; D014660:Vasoactive Intestinal Peptide; D014443:Tyrosine; D002713:Chlorine; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D017868:Cyclic AMP-Dependent Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin