Subproteome Of Thromboxane A2 Receptor In Transfected Hek293t Cells

The thromboxane A2 receptor (TP) densities are significantly increased in atherosclerotic coronary arteries and aortae. A potential reason for the elevated TP protein levels in the diseased vasculature could be the rise in TP protein synthesis/traffic. To uncover TP synthesis/traffic mechanisms, we analyzed its subproteome in transfected HEK293T cells. TP immunoprecipitates were separated by 4-20% SDS-PAGE and bands at 37, 65 and 110 kDa were excised and trypsin digested. LC/MS/MS identified a group of endoplasmic reticulum (ER)-membrane spanning proteins in complex with TP in transfected cells (n=3) but not in non-transfected cells (n=3). As positive control, TP peptides were found in the 37 and 65 kDa bands that correspond to the size of its monomeric and dimeric forms. TP partners were: sarcoplasmic/ER calcium ATPase 2 (ATP2A2), phosphatidylinositide phosphatase SAC1 (SAC1), Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3A (STT3A) and Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 2 (RPN2). Twenty peptides of ATP2A2, 6 peptides of SAC1, 4 peptides of STT3A and 6 peptides of RPN2 were identified. STT3A and RPN2 are components of the oligosaccharyltransferase complex, which catalyzes co-translational N-glycosylation and mediates protein translocation across the ER membrane. N-glycosylation of either Asn4 or Asn16 is required for TP expression predicting that STT3A and RPN2 might be involved in TP N-glycosylation. The dynamic localization of SAC1 in ER and the Golgi apparatus regulates protein secretion from the Golgi apparatus in response to proliferating signals. SAC1 might aid TP secretion and trafficking to the plasma membrane of cells undergoing proliferation. Since intracellular Ca2+ can regulate protein traffic it is possible that TP-ATP2A2 association serves as a feedback mechanism for TP expression. In summary, LC/MS/MS analysis identified ER membrane-spanning proteins that form macromolecular complexes with TP, which may be involved in the receptor increased synthesis/traffic in diseased/proliferating vasculature. Supported by NIH.