Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote ...(hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-alpha variants were selected (C.sup.68 S, V.sup.80 E, E.sup.94 V, A.sup.160 T, V.sup.176 E, and V.sup.217 I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C.sup.68 S) to normal (V.sup.217 I), with most variants demonstrating functional alteration in binding, expression or activation (V.sup.80 E, E.sup.94 V, A.sup.160 T, and V.sup.176 E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.
Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered ...studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.
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.
Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote ...(hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP- alpha variants were selected (C68S, V80E, E94V, A160T, V176E, and V217I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C68S) to normal (V217I), with most variants demonstrating functional alteration in binding, expression or activation (V80E, E94V, A160T, and V176E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.
Single-nucleotide polymorphisms (SNPs), particularly non-synonymous variants, provide opportunities for deciphering pathophysiological protein influence. An ideal group for realizing these ...opportunities is the GPCR-superfamily. Principles from rhodopsin are applied to receptors for thromboxane (hTP) and prostacyclin (hIP), which play opposing cardiovascular roles. We report the identification and characterization of naturally-occurring genetic variants in these receptors. Initially, we analyzed zinc interactions with rhodopsin and Retinitis pigmentosa (RP) mutations. These studies identified two conserved zinc-binding motifs mediating receptor destabilization. Site-directed mutagenesis confirmed disruption of native histidine interactions by zinc-binding. Chelation reduced the destabilizing influence of these sites in both wild-type and a common RP mutation. Based on rhodopsin zinc-coordination, we identified similar hTP sites, but not hIP. Saturation binding demonstrates disruption of hTP, but not hIP, with a clear dose-response effect. We sequenced variants from approximately 1000 cardiovascular patients. From 33 variants, we focused on six within the ligand-binding pocket, the ligand-recognition domain, and the G-protein coupling region. Transmembrane variants (V80E and A160T) altered binding, with V80E decreasing affinity, and A160T increasing affinity. Other variants (C 68S, E94V, V176E, and V217I) produced non-significant dysfunctions. Altered activation was observed, highlighting agonist and antagonist differences. Finally, mutational “hot spots” observed within these receptors, where mutations preferentially occur, were investigated. We searched for nucleotide patterns in regions flanking the variants. From twelve million human SNPs, significant flanking motifs included CpG-island transition contributions, similar R- and K-type motifs (A/G and G/T, respectively), and an absence of immediately preceding nucleotide influence on S-type variants (C/G). Extending this to other species we find some of these patterns are conserved across species (human, chimpanzee, opossum, mouse, honey bee, bovine, canine, zebrafish, chicken, and rice). This analysis offers the potential to prospectively define sites prone to specific nucleotide alterations. Our studies identified and characterized naturally-occurring genetic variants in counterbalancing prostanoid receptors associated with cardiovascular disease. We developed a semi-automated system for predicting and testing the structural influence of genetic variants in GPCRs. Collectively, our findings provide useful experimental tools to streamline the interpretation of novel GPCR variants which may aid in treatments for cardiovascular disease and a plethora of other human disorders.
Thirty years have passed since Vane and colleagues first described a substance, prostanoid X, from microsomal fractions (later called prostacyclin) that relaxed rather than contracted mesenteric ...arteries. The critical role of prostacyclin in many pathophysiological conditions, such as atherothrombosis, has only recently become appreciated (through receptor knockout mice studies, selective cyclooxygenase-2 inhibition clinical trials, and the discovery of dysfunctional prostacyclin receptor genetic variants). Additionally, important roles in such diverse areas as pain and inflammation, and parturition are being uncovered. Prostacyclin-based therapies, currently used for pulmonary hypertension, are accordingly emerging as possible treatments for such diseases, fueling interests in structure function studies for the receptor and signal transduction pathways in native cells. The coming decade is likely to yield many further exciting advances.
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