GLYCOPROTEIN (GP)-IB-BETA IS THE CRITICAL SUBUNIT LINKING GP-IB-ALPHAAND GP-IX IN THE GP-IB-IX COMPLEX - ANALYSIS OF PARTIAL COMPLEXES

The glycoprotein (GP) Ib-IX complex is the receptor on platelet surfac es that mediates their adhesion to subendothelium. It comprises three polypeptides (GP Ib alpha, GP Ib beta, GP IX), each of which belongs t o a superfamily of proteins containing conserved leucine-rich motifs. In this study, we used Chinese hamster ovary (CHO) cells expressing ev ery combination of two GP Ib-IX complex subunits to demonstrate that G P Ib beta plays an essential role in the synthesis of the heterotrimer by associating with both of the other two subunits. Confocal microsco py demonstrated that GP Ib beta was present in the same cellular locat ions as GP Ib alpha in CHO alpha beta cells (cells expressing only GP Ib alpha and GP Ib beta) and as GP IX in CHO beta IX cells. The two po lypeptides expressed in CHO alpha IX cells did not co-localize. Associ ation between GP Ib alpha and GP Ib beta was demonstrated biochemicall y on immunoblots of detergent lysates of CHO alpha beta cells; electro phoresis under nonreducing conditions revealed the two subunits to be covalently linked through a disulfide bond. Association of GP Ib alpha and GP Ib beta was further demonstrated by the finding that immunopre cipitations with antibodies against either polypeptide precipitated bo th. Similarly immunoprecipitations of lysates of CHO beta IX cells wit h antibodies against GP Ib beta or GP IX precipitated both polypeptide s. In contrast, co-immunoprecipitation of the two polypeptides express ed in CHO alpha IX cells could not be demonstrated. Transient expressi on in CHO cells of GP Ib beta with GP IX yielded higher GP IX levels o n the cell membrane than did expression of GP IX alone; supertransfect ion of CHO alpha IX cells with GP Ib beta also increased GP IX levels on the cell surface.