Visit our Expo - Redox and Inflammation signaling 2012

Session II : Receptor signaling and G proteins Poster II, 2
Galpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial
junctions and regulates barrier function
Alexandra V. Andreeva, Mikhail A. Kutuzov, Rita Vaiskunaite, Jasmina Profirovic,
Sanda Predescu, Asrar B. Malik and Tatyana A. Voyno-Yasenetskaya
Department of Pharmacology, College of Medicine, University of Illinois at Chicago,
Chicago, Illinois 60607, U.S.A. Email: aandreev@uic.edu
In vascular endothelium, the endothelial-specific cadherin (VE-cadherin) is essential for the
control of permeability and angiogenesis. Newly synthesized VE-cadherin is transported to
the plasma membrane and forms a homotypic interaction with VE-cadherin from other
endothelial cells, resulting in the assembly of adherens junctions. The involvement of
heterotrimeric G proteins in the regulation of adherens junction function is unclear. Galpha12
has recently been reported to bind directly to the C-terminal region of E-cadherin. In this
study, we identified alphaSNAP, an essential component of the membrane fusion machinery,
as an interactive partner of Galpha12 using yeast two hybrid screening. GST pull-down assays
showed the selective interaction of alphaSNAP with Galpha12 in COS-7 as well as human
umbilical vein endothelial cells (HUVECs). Using domain swapping experiments, we
demonstrated that the N-terminal region of Galpha12 (1-37 aa) was necessary and sufficient
for its interaction with alphaSNAP. Galpha13 with its N-terminal extension replaced by that
of Galpha13 acquired the ability to bind to alphaSNAP, whereas Galpha12 with its Nterminus
replaced by that of Galpha13 lost this ability. Using four point mutations of
alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the
convex rather than concave surface of alphaSNAP was involved in its interaction with
Galpha12. Co-transfection of HUVECs with Galpha12 and alphaSNAP stabilized VEcadherin
at the plasma membrane, whereas downregulation of alphaSNAP using siRNA
resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with
Galpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a
direct link between the alpha-subunit of G12 and alphaSNAP, and suggest a role for this
interaction in regulating the membrane localization of VE-cadherin and hence formation of
adherens junctions, control of cell confluence and endothelial barrier function.
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