Identification of proteins associating with glycosylphosphatidylinositol- anchored T-cadherin on the surface of vascular endothelial cells: role for Grp78/BiP in T-cadherin-dependent cell survival.
There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor alpha1 subunit, integrin beta3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin beta3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.
Philippova
Maria
M
Department of Research, Cardiovascular Laboratories, Basel University Hospital, Hebelstrasse 20, CH 4031 Basel, Switzerland. maria.filippova@unibas.ch
Ivanov
Danila
D
Joshi
Manjunath B
MB
Kyriakakis
Emmanouil
E
Rupp
Katharina
K
Afonyushkin
Taras
T
Bochkov
Valery
V
Erne
Paul
P
Resink
Therese J
TJ
D019921:Biotinylation; D015820:Cadherins / Q000378:metabolism*; D002462:Cell Membrane; D002470:Cell Survival; D000091342:Endoplasmic Reticulum Chaperone BiP; D042783:Endothelial Cells / Q000166:cytology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D015398:Signal Transduction
D015820:Cadherins; D000091342:Endoplasmic Reticulum Chaperone BiP; D017261:Glycosylphosphatidylinositols; C099972:H-cadherin; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D039661:Integrin beta3; D018832:Molecular Chaperones; D016271:Proto-Oncogene Proteins c-myc