Gα12 Interaction with αSNAP Induces VE-cadherin Localization at Endothelial Junctions and Regulates Barrier Function

Abstract

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified ␣SNAP as an interactive partner of G␣ 12 using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of ␣SNAP with G␣ 12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G␣ 12 (1-37 amino acids) was necessary and sufficient for its interaction with ␣SNAP. G␣ 13 with its N-terminal extension replaced by that of G␣ 12 acquired the ability to bind to ␣SNAP, whereas G␣ 12 with its N terminus replaced by that of G␣ 13 lost this ability. Using four point mutants of ␣SNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of ␣SNAP was involved in its interaction with G␣ 12 . Co-transfection of human umbilical vein endothelial cells with G␣ 12 and ␣SNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of ␣SNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G␣ 12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the ␣ subunit of G 12 and ␣SNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.

Novel Mechanisms of G Protein-Dependent Regulation of Endothelial Nitric-Oxide Synthase

“…Although a traditional general paradigm for G␣ proteins has been that they transmit signals to their targets while in the GTP-bound (i.e., activated) state, the G␣ 12 -eNOS interaction was found to occur independently of the activation state of G␣ 12 . Similar observations have been reported for G␣ 12 interaction with Hsp90 (Niu et al, 2001), PP2A (Zhu et al, 2004), and ␣SNAP (Andreeva et al, 2005).…”

“…Several important signaling molecules have been shown to interact with G␣ 12 , including several RhoGEFs (Kozasa et al, 1998;Fukuhara et al, 1999Fukuhara et al, , 2000 that act between the actin cytoskeleton and the plasma membrane and regulate organization of cortical actin (Vaiskunaite et al, 2000); cadherin, a protein that mediates cell-cell interactions, and upon G␣ 12 binding, releases a transcriptional activator ␤-catenin (Meigs et al, 2001); ␣SNAP, a protein involved in membrane trafficking that in complex with G␣ 12 , increases cadherin presence at endothelial junctions (Andreeva et al, 2005); and zonula occludens proteins 1 and 2 that probably regulate properties of the tight junctions (Meyer et al, 2002). It was also shown that G␣ 12 interacts with a molecular chaperone Hsp90 and that this interaction is required for G␣ 12 function , possibly via Hsp90-dependent targeting of G␣ 12 to lipid rafts (Jones and Gutkind, 1998).…”

“…Inhibition of G␣ 12 expression was performed using G␣ 12 siGENOME SMARTpool reagent and individual siRNA duplexes (Dharmacon Research, Inc., Lafayette, CO). Assay was performed as described previously (Andreeva et al, 2005).…”

“…Another G␣ 12 (as well as G␣ 13 )-interacting protein is cadherin, which is involved in cell-cell adhesion (Meigs et al, 2001). We have previously determined that G␣ 12 (but not G␣ 13 ) binds to ␣SNAP, a protein involved in membrane trafficking (Andreeva et al, 2005) and Hsp90 ), a molecular chaperone that interacts with multiple signal transduction molecules and is essential to a variety of signaling pathways. It is noteworthy that Hsp90 is required for G␣ 12 -induced serum response element activation, cytoskeletal changes, mitogenic response , and probably G␣ 12 delivery to lipid rafts (Waheed and Jones, 2002).…”

Endothelial Nitric-Oxide Synthase Reveals a New Face in G Protein Signaling: Fig. 1.

“…Although the involvement of G 12 in cellular growth (Fukuhara et al, 2001;Radhika and Dhanasekaran, 2001), development (Dettlaff-Swiercz et al, 2005;Lin et al, 2005;Ruppel et al, 2005), apoptosis (Althoefer et al, 1997;Berestetskaya et al, 1998), and migration (Parks and Wieschaus, 1991;Offermanns et al, 1997;Gu et al, 2002;Xu et al, 2003) is well established, the mechanisms by which G 12 functions in these processes, including the identities of endogenous receptors coupled to G 12 activation (Riobo and Manning, 2005) and the direct effectors of G 12 signaling (Kurose, 2003;Zhu et al, 2004;Andreeva et al, 2005), are only just beginning to be discovered.…”