Characterization of the role of N-linked glycosylation on the cell signaling and expression of the human thromboxane A2 receptor alpha and beta isoforms.
The alpha and beta isoforms of the thromboxane A2 receptor (TP) mediate the actions of the prostanoid thromboxane A2 and its mimetics in humans. The amino terminal region of the TPs contains two consensus N-linked glycosylation sites at asparagine (N) residues N4 and N16. In this study, we explored the significance of N-linked glycosylation on the signaling and surface expression of the human TP isoforms. Inhibition of N-linked glycosylation reduced selective radioligand ([3H]SQ29,548) binding by either TP in both human erythroleukemia cells and in transfected human embryonic kidney 293 cells. Moreover, site-directed mutagenesis of the putative glycosylation sites of TPalpha revealed that radioligand binding also was reduced greatly for both the single (TPalphaN4-Q4, TPalphaN16-Q16) and double (TPalphaN4,N16-Q4,Q16) mutants, yielding levels of 8% binding relative to the wild-type TPalpha for the double mutants. Reductions in ligand binding were caused by decreased maximal binding and not by changes in affinity (Kd) or in specificity of the receptors for [3H]SQ29,548 or other ligands. Subcellular fractionation confirmed that, in relation to total TP expression, membrane expression was not altered in TPalphaN4-Q4 or TPalphaN16-Q16 but was reduced to levels of 55% of total expression in TPalphaN4,Q4-N16,Q16. Inhibition of glycosylation reduced, but did not abolish, agonist (U46619) mediated intracellular Ca++ mobilization by TPalpha or TPbeta and cAMP production by TPalpha. Thus, N-linked glycosylation of the human TP isoforms is important for ligand binding, efficient second messenger signaling and efficient membrane expression.
Walsh
M T
MT
Department of Biochemistry, Merville House, University College Dublin, Belfield, Dublin, Ireland.
Foley
J F
JF
Kinsella
B T
BT
D001667:Binding, Competitive; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011869:Radioligand Assay; D017482:Receptors, Thromboxane / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013928:Thromboxane A2 / Q000378:metabolism*; D014162:Transfection
D017482:Receptors, Thromboxane; D013928:Thromboxane A2; D000242:Cyclic AMP; D006596:Hexosaminidases; D019204:GTP-Binding Proteins; D002118:Calcium