Association of Thromboxane A2 Receptor with Endoplasmic Reticulum-Membrane Spanning Proteins

Abstract

Human thromboxane A2 receptor (TP) density is significantly increased in atherosclerotic coronary arteries. The elevated TP protein levels in the diseased vasculature could be due to the increment of the TP protein synthesis/traffic. To reveal the mechanisms that regulate TP synthesis/traffic, we first analyzed its subproteome in TP-transfected HEK293T cells. TP immunoprecipitates were separated by SDS-PAGE and bands at 37, 65 and 110 kDa were excised for trypsin digestion and LC/MS/MS analysis. A group of endoplasmic reticulum (ER)-membrane spanning proteins were identified in complex with TP in transfected cells (n=3) but not in non-transfected cells (n=3). These proteins include phosphatidylinositide phosphatase SAC1 (SAC1), Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3A (STT3A) and Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 2 (RPN2). Six peptides of SAC1, 4 peptides of STT3A and 6 peptides of RPN2 were detected. Next, co-immunoprecipitation experiments showed that TP was able to pull down c-Myc tagged SAC1 (c-Myc-SAC1) in HEK293T cells coexpressing TP and c-Myc-SAC1 but not when TP or c-Myc-SAC1 were transfected alone (negative controls). The dynamic localization of SAC1 in the ER and Golgi apparatus regulates protein secretion from the Golgi apparatus in response to proliferating signals, while STT3A and RPN2 are components of the oligosaccharyltransferase complex, which catalyzes co-translational N-glycosylation and mediates protein translocation across the ER membrane. Thus, SAC1 likely aids TP secretion and trafficking to the plasma membrane of cells undergoing proliferation, and STT3A and RPN2 may be involved in TP Asn4- or Asn16-glycosylation which are required for TP expression. In summary, proteomic analysis identified ER membrane-spanning proteins that form macromolecular complexes with TP. Co-immunoprecipitation experiments confirmed TP and SAC1 association. Association of TP with ER-membrane spanning proteins may be involved in the regulation of TP synthesis, modification and trafficking in the proliferating vasculature. Supported by NIH.