The role of N-linked glycosylation in determining the surface expression, G protein interaction and effector coupling of the alpha (alpha) isoform of the human thromboxane A(2) receptor.
In humans, thromboxane (TX) A(2) signals through two TXA(2) receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within the carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn(4) and Asn(16). While it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn(4) or Asn(16) is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalpha(N4,N16-Q4,Q16) is almost completely retained within the endoplasmic reticulum (ER) and remains functionally inactive, failing to associate with its coupling G protein Galpha(q) and, in turn, failing to mediate phospholipase (PL) Cbeta activation.
Kelley
Leanne P
LP
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.
Kinsella
B Therese
BT
D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D011233:Precipitin Tests; D020033:Protein Isoforms; D017482:Receptors, Thromboxane / Q000032:analysis / Q000737:chemistry* / Q000502:physiology
D020033:Protein Isoforms; D017482:Receptors, Thromboxane; D019204:GTP-Binding Proteins