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We previously reported that hypoxia inhibits<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptor phosphorylation, causing desensitization. Hence, we examined the role of<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>\u2010accessible serine residues in determining<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptor affinity, using site\u2010directed mutational analysis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Approach<\/jats:title><jats:p>Vasoconstriction to a thromboxane mimetic and phosphorylation of<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>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<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptors. Human TP\u03b1 serines at positions 324, 329 and 331 (<jats:styled-content style=\"fixed-case\">C<\/jats:styled-content>\u2010terminal tail) were mutated to alanine and transiently expressed in<jats:styled-content style=\"fixed-case\">HEK293T<\/jats:styled-content>cells. Saturation binding and displacement kinetics of a<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>antagonist and agonist were determined in porcine<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>, wild\u2010type human<jats:styled-content style=\"fixed-case\">TP\u03b1<\/jats:styled-content>and all<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>mutants. Agonist\u2010elicited calcium mobilization was determined for each<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>mutant, in the presence of a<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>activator or inhibitor, and in hypoxic and normoxic conditions.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key Results<\/jats:title><jats:p>The<jats:styled-content style=\"fixed-case\">Ser324A<\/jats:styled-content>mutant was insensitive to<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>activation and hypoxia, had a high affinity for agonist and increased agonist\u2010induced calcium mobilization.<jats:styled-content style=\"fixed-case\">Ser329A<\/jats:styled-content>was no different from wild\u2010type<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptors.<jats:styled-content style=\"fixed-case\">Ser331A<\/jats:styled-content>was insensitive to hypoxia and<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>with a decreased agonist\u2010mediated response.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions and Implications<\/jats:title><jats:p>In hypoxic pulmonary hypertension, loss of site\u2010specific phosphorylation of the<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptor causes agonist hyper\u2010responsiveness.<jats:styled-content style=\"fixed-case\">Ser324<\/jats:styled-content>is the primary residue phosphorylated by<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>, which regulates<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>receptor\u2010agonist interactions.<jats:styled-content style=\"fixed-case\">Ser331<\/jats:styled-content>mutation confers loss of<jats:styled-content style=\"fixed-case\">TP receptor<\/jats:styled-content>\u2010agonist interaction, regardless of<jats:styled-content style=\"fixed-case\">PKA<\/jats:styled-content>activity.<\/jats:p><\/jats:sec>","DOI":"10.1111\/bph.12487","type":"journal-article","created":{"date-parts":[[2013,10,30]],"date-time":"2013-10-30T09:45:21Z","timestamp":1383126321000},"page":"676-687","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Thromboxane receptor hyper\u2010responsiveness in hypoxic pulmonary hypertension requires serine 324"],"prefix":"10.1111","volume":"171","author":[{"given":"K T","family":"Santhosh","sequence":"first","affiliation":[{"name":"Biology of Breathing Group Manitoba Institute of Child Health Winnipeg MB Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"A S","family":"Sikarwar","sequence":"additional","affiliation":[{"name":"Biology of Breathing Group Manitoba Institute of Child Health Winnipeg MB Canada"},{"name":"Department of Physiology University of Manitoba Winnipeg MB 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