Homologous desensitization of signalling by the alpha (alpha) isoform of the human thromboxane A2 receptor: a specific role for nitric oxide signalling.
Thromboxane (TX) A(2) plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TP beta isoform of the human TXA(2) receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TP alpha isoform. TP alpha undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA(2) mimetic U46619 but, unlike that of TP beta, this is independent of GRKs. Similar to TP beta, TP alpha undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser(145) within intracellular domain (IC)(2) represents the key phospho-target. TP alpha also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr(337) and Ser(331), respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors L-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TP alpha involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser(145) partially and transiently impairs TP alpha signalling while PKG- and PKC-phosphorylation at both Ser(331) and Thr(337), respectively, within its C-tail domain profoundly desensitizes TP alpha, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TP alpha.
Kelley-Hickie
Leanne P
LP
School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
O'Keeffe
Martina B
MB
Reid
Helen M
HM
Kinsella
B Therese
BT
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054769:G-Protein-Coupled Receptor Kinase 2; D054770:G-Protein-Coupled Receptor Kinase 3; D006487:Hemostasis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D013928:Thromboxane A2 / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D014662:Vasoconstrictor Agents; D009569:Nitric Oxide; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D015544:Inositol 1,4,5-Trisphosphate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054770:G-Protein-Coupled Receptor Kinase 3; C496567:GRK2 protein, human; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; C070515:bisindolylmaleimide I