Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.
OBJECTIVE
Dysregulation of the thromboxane A₂ (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis.


METHODS
Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions.


RESULTS
The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response.


CONCLUSIONS
In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.
Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, MB, Canada.
K T
KT
Santhosh
A S
AS
Sikarwar
M
M
Hinton
P
P
Chelikani
S
S
Dakshinamurti
D019943:Amino Acid Substitution; D000818:Animals; D000831:Animals, Newborn; D020013:Calcium Signaling / Q000187:drug effects; D015687:Cell Hypoxia; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004195:Disease Models, Animal; D020536:Enzyme Activators / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010547:Persistent Fetal Circulation Syndrome / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013047:Specific Pathogen-Free Organisms; D034421:Sus scrofa; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D020536:Enzyme Activators; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D011994:Recombinant Proteins; D012694:Serine; D013928:Thromboxane A2; D017868:Cyclic AMP-Dependent Protein Kinases
PKA; hypoxia; pulmonary hypertension; thromboxane