title: Thrombin receptors activate G(o) proteins in endothelial cells to regulate intracellular calcium and cell shape changes.
abstract: Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but not G(i1/2) minigenes. We conclude that the G(o) proteins play a role in thrombin signaling distinct from G(i1/2) proteins, which are mediated through their Gbetagamma subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.
authors.affiliation: Institute for Neuroscience, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Jurgen F
authors.initials: JF
authors.lastname: Vanhauwe
authors.forename: Tarita O
authors.initials: TO
authors.lastname: Thomas
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: Anli
authors.initials: A
authors.lastname: Li
authors.forename: Annette
authors.initials: A
authors.lastname: Gilchrist
authors.forename: Eun-ja
authors.initials: EJ
authors.lastname: Yoon
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000502:physiology*; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D037342:Pertussis Toxin / Q000494:pharmacology; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000502:physiology*; D015398:Signal Transduction; D018411:Spodoptera; D022502:Stress Fibers / Q000502:physiology
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium