Expression of platelet glycoprotein (GP) V in heterologous cells and evidence for its association with GP Ib alpha in forming a GP Ib-IX-V complex on the cell surface.
The glycoprotein (GP) Ib-IX-V complex comprises four polypeptides: the subunits of the GP Ib-IX complex (GP Ib alpha, GP Ib beta, GP IX) and GP V. To determine the requirements for cell-surface expression of GPV, we transiently expressed the recombinant polypeptide in wild-type Chinese hamster ovary (CHO) cells by cotransfection with plasmids for the subunits of the GP Ib-IX complex and in CHO cells that stably express different combinations of the GP Ib-IX complex subunits. Glycoprotein V expressed alone was detectable on the cell surface, and the level was not augmented by cotransfection with any one of the subunits of the GP Ib-IX complex. However, when GP V was expressed in cells that stably express combinations of GP Ib-IX complex subunits, its expression on the cell surface was greater in all the cell lines that contained GP Ib alpha than in wild-type CHO cells. That GP V associates with GP Ib alpha was also suggested by confocal microscopy studies: GP V colocalized with GP Ib alpha in CHO alpha beta IX (cells that express GP Ib alpha, GP Ib beta, and GP IX), CHO alpha beta, and CHO alpha IX cells, but did not colocalize with GP Ib beta in CHO beta IX cells. Similarly, immunoprecipitation of GP V from cells expressing GP Ib alpha led to coprecipitation of the latter polypeptide; neither GP Ib beta nor GP IX coprecipitated with GP V from CHO beta IX cells. Taken together, these data indicate that GP V associates with the GP Ib-IX complex through a direct interaction with GP Ib alpha and establish the topology of the GP Ib-IX-V subunits on the cell surface.
Li
C Q
CQ
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94110, USA.
Dong
J F
JF
Lanza
F
F
Sanan
D A
DA
Sae-Tung
G
G
López
J A
JA
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D018613:Microscopy, Confocal; D008958:Models, Molecular; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins