Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells.
We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton.
Calandrella
Sean O
SO
Department of Medicine, University of California, San Diego, San Diego, California, USA.
Barrett
Kim E
KE
Keely
Stephen J
SJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D018721:Muscarinic Agonists; D014443:Tyrosine; D002217:Carbachol; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium