Glycoprotein (GP) Ib beta is the critical subunit linking GP Ib alpha and GP IX in the GP Ib-IX complex. Analysis of partial complexes.
The glycoprotein (GP) Ib-IX complex is the receptor on platelet surfaces that mediates their adhesion to subendothelium. It comprises three polypeptides (GP Ib alpha, GP Ib beta, GP IX), each of which belongs to a superfamily of proteins containing conserved leucine-rich motifs. In this study, we used Chinese hamster ovary (CHO) cells expressing every combination of two GP Ib-IX complex subunits to demonstrate that GP Ib beta plays an essential role in the synthesis of the heterotrimer by associating with both of the other two subunits. Confocal microscopy demonstrated that GP Ib beta was present in the same cellular locations 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 polypeptides expressed in CHO alpha IX cells did not co-localize. Association between GP Ib alpha and GP Ib beta was demonstrated biochemically on immunoblots of detergent lysates of CHO alpha beta cells; electrophoresis 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 immunoprecipitations with antibodies against either polypeptide precipitated both. Similarly, immunoprecipitations of lysates of CHO beta IX cells with antibodies against GP Ib beta or GP IX precipitated both polypeptides. In contrast, co-immunoprecipitation of the two polypeptides expressed in CHO alpha IX cells could not be demonstrated. Transient expression in CHO cells of GP Ib beta with GP IX yielded higher GP IX levels on the cell membrane than did expression of GP IX alone; supertransfection of CHO alpha IX cells with GP Ib beta also increased GP IX levels on the cell surface.
López
J A
JA
Cardiovascular Research Institute, University of California, San Francisco 94141-9100.
Weisman
S
S
Sanan
D A
DA
Sih
T
T
Chambers
M
M
Li
C Q
CQ
D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D046911:Macromolecular Substances; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014162:Transfection
D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase