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Vol. 286, Issue 2, 1026-1036, August 1998
Department of Biochemistry, Merville House, University College
Dublin, Belfield, Dublin, Ireland
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 TP
revealed that radioligand binding also was
reduced greatly for both the single (TPN4-Q4,
TPN16-Q16) and double (TPN4,N16-Q4,Q16)
mutants, yielding levels of 8% binding relative to the wild-type TP
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 TPN4-Q4 or
TPN16-Q16 but was reduced to levels of 55% of total
expression in TPN4,Q4-N16,Q16. Inhibition of
glycosylation reduced, but did not abolish, agonist (U46619) mediated
intracellular Ca++ mobilization by TP or TP
and cAMP
production by TP. Thus, N-linked glycosylation of the human TP
isoforms is important for ligand binding, efficient second messenger
signaling and efficient membrane expression.
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