Gs protein-coupled receptor agonists induce transactivation of the epidermal growth factor receptor in T84 cells -: Implications for epithelial secretory responses

We have previously shown that G(q) protein-coupled receptor (G(q)PCR) 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 G(q)PCR 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 G(s)PCRs. All experiments were performed using monolayers of T-84 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 (DeltaI(sc)) across voltage-clamped T-84 cells. The G(s)PCR agonist, vasoactive intestinal polypeptide (VIP; 100 nm), rapidly stimulated EGFr phosphorylation in T-84 cells. This effect was mimicked by a cell-permeant analog of cAMP, Bt(2)cAMP/AM (3 muM), and was attenuated by the protein kinase A (PKA) inhibitor, H-89 (20 muM). The EGFr inhibitor, tyrphostin AG1478 (1 muM), inhibited both Bt(2)cAMP/ AM-stimulated EGFr phosphorylation and I., responses. VIP and Bt(2)cAMP/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 muM), attenuated Bt(2)cAMP/AM-stimulated secretory responses. We conclude that G(s)PCR agonists rapidly transactivate the EGFr in T-84 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.