Palmitoylation of G alpha q Determines Its Association with the Thromboxane Receptor in Hypoxic Pulmonary Hypertension

Pulmonary arterial vasoconstriction is a hallmark of persistent pulmonary hypertension of the newborn (PPHN). We reported increased calcium responses to thromboxane and selectively increased thromboxane prostanoid (TP) association with G alpha q in hypoxic pulmonary artery. Palmitoylation of G alpha q is important for efficient receptor-G alpha q-phospholipase-C interactions. TP alpha receptor is not itself amenable to palmitoylation. We studied the role of G alpha q palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition, the effects of hypoxia on palmitoylation, and the effects of site-specific cysteine substitution mutations of G alpha q on G alpha q membrane targeting, TP alpha association, and calcium dose-response curve to a TP agonist. PPHN pulmonary artery and HEK293T cells expressing TP alpha were exposed to irreversible palmitoylation inhibitor 2-bromopalmitate before challenge with TP agonist U46619. Palmitate uptake was studied in hypoxic and normoxic myocytes. Wild-type G alpha q and G alpha q cysteine-to-alanine mutants C9A, C10A, and C9A/C10A were transiently coexpressed in HEK293T cells stably expressing TP alpha. We examined membrane localization of G alpha q, TP receptor-G alpha q association by coimmunoprecipitation, and Ca2+ responses to U46619 in hypoxic and normoxic cells. G alpha q palmitoylation is essential for the Ca2+ response to TP alpha stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not in control pulmonary artery. Hypoxia increases palmitoylation of G alpha q; the hypoxic. but not the normoxic, response to thromboxane is palmitoylation sensitive. Palmitoylation of one N-terminal cysteine is required for physical association of G alpha q with the TP alpha receptor. Palmitoylation of both cysteines is required for G alpha q membrane localization and Ca2+ mobilization. Depalmitoylation of any one G alpha q cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.