Low expression of cell-surface thromboxane A2 receptor beta-isoform through the negative regulation of its membrane traffic by proteasomes.
Human thromboxane A(2) receptor (TP) consists of two alternatively spliced isoforms, TP alpha and TP beta, which differ in their cytoplasmic tails. To examine the functional difference between TP alpha and TP beta, we searched proteins bound to C termini of TP isoforms by a yeast two-hybrid system, and found that proteasome subunit alpha 7 and proteasome activator PA28 gamma interacted potently with the C terminus of TP beta. The binding of TP beta with alpha 7 and PA28 gamma was confirmed by co-immunoprecipitation and pull-down assays. MG-132 and lactacystin, proteasome inhibitors, increased cell-surface expression of TP beta, but not TP alpha. Scatchard analysis of [(3)H]SQ29548 binding revealed that the B(max) was higher in transiently TP alpha-expressing cells than TP alpha-expressing cells. In addition, TP-mediated phosphoinositide hydrolysis was clearly observed in TP alpha-, but not TP beta-expressing cells. These results suggest that TP beta binds to alpha 7 and PA28 gamma, and the cell-surface expression of TP beta is lower than that of TP alpha through the negative regulation of its membrane traffic by proteasomes.
Sasaki
Masako
M
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Sukegawa
Jun
J
Miyosawa
Katsutoshi
K
Yanagisawa
Teruyuki
T
Ohkubo
Satoko
S
Nakahata
Norimichi
N
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D009124:Muscle Proteins; C085047:PSME1 protein, human; D010716:Phosphatidylinositols; D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C488662:PSMA7 protein, human; D046988:Proteasome Endopeptidase Complex