Arginine (CGC) codon targeting in the human prostacyclin receptor gene (PTGIR) and G-protein coupled receptors (GPCR).

The human prostacyclin receptor (hIP) has recently been recognized as an important seven transmembrane G-protein coupled receptor that plays critical roles in atheroprevention and cardioprotection. To date, four non-synonymous genetic variants have been identified, two of which occur at the same Arg amino acid position (R212H, R212C). This observation instigated further genetic screening for prostacyclin receptor variants on 1455 human genomic samples. A total of 31 distinct genetic variants were detected, with 6 (19%) involving Arg residues. Distinct differences in location and frequencies of genetic variants were noted between Caucasian, Asian, Hispanic and African Americans, with the most changes noted in the Asian cohort. From the sequencing results, three Arg-targeted changes at the same 212 position within the third cytoplasmic loop of the human prostacyclin (hIP) receptor were detected: 1) R212C (CGC-->TGC), 2) R212H (CGC-->CAC), and 3) R212R (CGC-->CGT). Three additional Arg codon variants (all exhibiting the same CGC to TGC change) were also detected, R77C, R215C, and R279C. Analysis (GPCR and SNP databases) of 200 other GPCRs, with recorded non-synonymous mutations, confirmed a high frequency of Arg-targeted missense mutations, particularly within the important cytoplasmic domain. Preferential nucleotide changes (at Arg codons), were observed involving cytosine (C) to thymine (T) (pyrimidine to pyrimidine), as well as guanine (G) to adenine (A) (purine to purine) (p<0.001, Pearson's goodness-of-fit test). Such targeting of Arg residues, leading to significant changes in coding amino acid size and/or charge, may have potentially-important structural and evolutionary implications on the hIP and GPCRs in general. In the case of the human prostacyclin receptor, such alterations may reduce the cardio-, vasculo-, and cytoprotective effects of prostacyclin.

Human prostacyclin receptor structure and function from naturally-occurring and synthetic mutations.

Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation (anti-thrombotic), and moderator of vascular smooth muscle cell proliferation-migration-differentiation (anti-atherosclerotic). These actions are mediated via a seven transmembrane-spanning G-protein coupled receptor (GPCR), known as the human prostacyclin receptor or hIP. Animal studies using prostacyclin receptor knock-out (IP-/-) mice have revealed increased propensities towards thrombosis, intimal hyperplasia, atherosclerosis, restenosis, as well as reperfusion injury. Of further importance has been the world-wide withdrawal of selective COX-2 inhibitors, due to their discriminating suppression of COX-2-derived PGI2 and its cardioprotective effects, leading to increased cardiovascular events, including myocardial infarction and thrombotic stroke. Over the last decade, mutagenesis studies of the IP receptor, in conjunction with in vitro functional assays and molecular modeling, have provided critical insights into the molecular mechanisms of both agonist binding and receptor activation. Most recently, the discovery of naturally-occurring and dysfunctional mutations within the hIP has provided additional insights into the proposed cardioprotective role of prostacyclin. The aim of this review is to summarize the most recent findings regarding hIP receptor structure-function that have developed through the study of both synthetic and naturally-occurring mutations.