Thrombin receptors activate G_o proteins in endothelial cells to regulate intracellular calcium and cell shape changes

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, G₁₂, or G₁₃ 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 (1). 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 [³⁵S]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 [Ca²⁺]_i from intracellular stores. Transfection of the β-adrenergic kinase C terminus blocked the [Ca²⁺]_i response to the same extent as with G_o1 minigene peptide, suggesting that this G_o-mediated [Ca²⁺]_i transient was caused by Gβγ stimulation of PLCβ. Transfection of a G_i1/2 minigene had no effect on thrombin-stimulated [Ca²⁺]_i signaling in HMEC, suggesting that Gβγ derived from G_o but not G_i could activate PLCβ. 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 Gβγ subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.