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 GP V, 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 expr ess different combinations of the GP Ib IX complex subunits. Glycoprot ein V expressed alone was detectable on the cell surface, and the leve l 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 s tably express combinations of GP Ib-IX complex subunits, its expressio n on the cell surface was greater in an 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 coloc alized 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 PIX cells, Similarly, immunoprecipitation of GP V from cells expressing GP Ib alpha led to c oprecipitation of the latter polypeptide; neither GP Ib beta nor GP IX coprecipitated with GP V from CHO beta IX cells. Taken together, thes e data indicate that G;P V associates with the GP Ib-IX complex throug h a direct interaction with GP Ib alpha and establish the topology of the GP Ib-IX-V subunits on the cell surface.