RT Journal Article
SR Electronic
T1 Characterization of the Role of N-Linked Glycosylation on the Cell Signaling and Expression of the Human Thromboxane A<sub>2</sub>Receptor <em>Alpha</em> and <em>Beta</em>Isoforms
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 1026
OP 1036
VO 286
IS 2
A1 Marie T. Walsh
A1 John F. Foley
A1 B. Therese Kinsella
YR 1998
UL http://jpet.aspetjournals.org/content/286/2/1026.abstract
AB 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 TPα revealed that radioligand binding also was reduced greatly for both the single (TPαN4–Q4, TPαN16–Q16) and double (TPαN4,N16–Q4,Q16) mutants, yielding levels of 8% binding relative to the wild-type TPα 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 TPαN4–Q4 or TPαN16–Q16 but was reduced to levels of 55% of total expression in TPαN4,Q4–N16,Q16. Inhibition of glycosylation reduced, but did not abolish, agonist (U46619) mediated intracellular Ca++ mobilization by TPα or TPβ and cAMP production by TPα. Thus, N-linked glycosylation of the human TP isoforms is important for ligand binding, efficient second messenger signaling and efficient membrane expression. The American Society for Pharmacology and Experimental Therapeutics