G alpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial junctions and regulates barrier function.
The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 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 alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, 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.
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Alexandra V
AV
Andreeva
Mikhail A
MA
Kutuzov
Rita
R
Vaiskunaite
Jasmina
J
Profirovic
Thomas E
TE
Meigs
Sanda
S
Predescu
Asrar B
AB
Malik
Tatyana
T
Voyno-Yasenetskaya
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D019556:COS Cells; D015820:Cadherins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014471:Umbilical Veins / Q000166:cytology; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D005982:Glutathione Transferase; D005990:Glycerol; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000099152:Cadherin 5