Impaired Receptor Binding and Activation Associated with a Human Prostacyclin Receptor Polymorphism

Abstract

The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structurefunction characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4, K i ‫؍‬ 2.2 ؎ 1.2 nM; pH 6.8 K i ‫؍‬ 45.6 ؎ 12.0 nM). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC 50 ‫؍‬ 2.8 ؎ 0.5 nM versus wild-type hIP EC 50 ‫؍‬ 0.5 ؎ 0.1 nM; pH 6.8, R212H EC 50 ‫؍‬ 3.2 ؎ 1.6 nM versus wild-type hIP EC 50 ‫؍‬ 0.5 ؎ 0.2 nM). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP.Similar to other prostanoids, prostacyclin is a derivative of the C-20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid), and its cellular action is conveyed through cell surface G-protein-coupled receptors that predominantly couple to the heterotrimeric G-protein G s stimulating the production of cAMP (1). The human prostacyclin receptor (hIP) 1 is expressed on platelets, where it mediates inhibition of platelet aggregation and on vascular smooth muscle cells, where it mediates vascular smooth muscle relaxation. Dysfunctional prostacyclin activity has been implicated in the development of a number of cardiovascular diseases including thrombosis, myocardial infarction, stroke, myocardial ischemia, atherosclerosis, and systemic and pulmonary hypertension (2). Accordingly, IP receptor knock-out mice exhibit increased thrombosis and reduced inflammatory and pain responses (3).Limited studies have begun to identify generalized regions within the IP and other prostanoid receptors that appear crucial for ligand-binding specificity and affinity. Studies using chimeric combinations of mouse prostaglandin D (mDP) and prostaglandin I (mIP) receptors have shown that protein segments within transmembrane domains VI and VII (TMVI and TMVII) are involved in distinct binding interactions with prostacyclin side chains. In addition, TMI (along with a portion of the first extracellular loop) confers broader binding functions, incorporating recognition and interaction w...

Comprehensive Biochemical Analysis of Rare Prostacyclin Receptor Variants

“…1). Our initial goal for the current study was to analyze receptor variants for ligand binding (K D ) and activation (cAMP EC 50 ) in parallel using an in vitro COS-1 overexpression system previously used to successfully characterize five mutations (V25M, R77C, R212C, R212H, and R279C) (20,23,27,28). We now report the characterization of the 13 newly identified non-synonymous mutations, including two variants (L104R and M113T) at highly conserved positions (100%) across all prostanoid receptors.…”

“…We have determined previously that an R212H mutation (expressed in COS-1 cells) leads to defective ligand binding and activation, particularly when exposed to stress conditions such as acidosis, e.g. pH 6.8 (20). During pathological stress such as respiratory or cardiac failure (where severe acidosis may provoke defective prostacyclin binding), the added receptor defect may promote vasoconstriction and/or thrombosis, warranting urgent correction of pH to restore proper receptor function and prevent catastrophic ischemia.…”

“…Intracellular loop variants in the dopamine D2 receptor (43), the endothelin ETB receptor (44), and the vasopressin V2 receptor (45) exhibit impairment in G-protein coupling. We have previously described and characterized SNPs in the hIP (20). This was followed by extensive sequencing of patient samples to discover novel genetic variants (23).…”

“…3B). The remaining two mutations (R212C and R212H), which have been included to complete the set of 18, have previously been characterized (20,27,31) and exhibit clear activation defects (Table 1 and Fig. 3).…”

“…Our recent discovery and characterization of the first known naturally occurring hIP variants (20) provide further insight into the molecular mechanism and cardioprotective function of this receptor. Since these initial observations, there has only been one similar publication describing two additional genetic variants in the coding region of PTGIR within a Japanese cohort (21).…”