New insights into human prostacyclin receptor structure and function through natural and synthetic mutations of transmembrane charged residues

Abstract

Background and purpose: The human prostacyclin receptor (hIP), a G-protein coupled receptor (GPCR) expressed mainly on platelets and vascular smooth muscle cells, plays important protective roles in the cardiovascular system. We hypothesized that significant insights could be gained into the structure and function of the hIP through mutagenesis of its energetically unfavourably located transmembrane charged residues. Experimental approach: Within its putative transmembrane helices fourteen hydrophilic residues, both unique and conserved across GPCRs, were systematically mutated to assess for effects on receptor structure and function. Key results: Mutations of ten of the fourteen charged residues to alanine exhibited defective binding and/or activation. Key potential interactions were identified between 6 core residues; E116 3.49 -R117 3.50 (salt bridge TMIII), D274 7.35 -R279 7.40 (salt bridge TMVII), and D60 2.50 -D288 7.49 (H-bond network TMII-TMVII). Further detailed investigation of E116 3.49 (TMIII) with mutation to a glutamine showed a 2.6-fold increase in agonist-independent basal activity. This increase in activity accounts for a proportion (B13%) of full agonist induced activation. We further characterized two novel naturally occurring human mutations, R77 2.33 C and R279 7.40 C recently identified in a 1455 human genomic DNA sample screen. The R77 2.33 C variant appeared to exclusively affect expression, while the R279 7.40 C variant, exhibited considerable deficiencies in both agonist binding and activation. Conclusions and implications: Transmembrane charged residues play important roles in maintaining the hIP binding pocket and ensuring normal activation. The critical nature of these charged residues and the presence of naturally occurring mutations have important implications in the rational design of prostacyclin agonists for treating cardiovascular disease.

Selexipag: an oral, selective prostacyclin receptor agonist for the treatment of pulmonary arterial hypertension

“…These potent vasodilators and inhibitors of platelet aggregation [7,8] also counteract the vasoconstrictor and prothrombotic activity of endothelin [9]. They bind to the prostacyclin receptor (IP receptor), a G-protein coupled receptor on the surface of platelets and vascular smooth muscle cells [10]. Activation of the receptor leads to production of cyclic adenosine monophosphate, which induces relaxation of vascular smooth muscle.…”

A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling

“…Likewise, the naturally occurring R279C mutation in the prostacyclin receptor dramatically reduces protein expression (38).…”

Comprehensive Biochemical Analysis of Rare Prostacyclin Receptor Variants

“…It was therefore not surprising that alterations at each of these positions produced severe defects, particularly in ligand binding as well as in activation and protein expression (Table 1). We have demonstrated previously that Arg-279 is an important counter-ion for the C1 carboxylate residue of prostacyclin (29) and that an SNP with a cysteine in this position has deleterious effects on binding and activation (22). The novel L104R and M113T mutations, located in transmembrane helix 3, each exhibited a lack of specific counts upon competition binding (Table 1), indicating severely compromised ligand binding.…”

“…Receptor Activation cAMP Determination-The wild-type and genetic variant constructs were analyzed for activation using COS-1 cells transfected with 2 g of receptor DNA (25-mm plates) (22) and iloprost as the agonist. [ 3 H]cAMP was used in competition for a cAMP-binding protein against known concentrations of non-radiolabeled cAMP followed by determination of the unknowns.…”

“…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). We recently initiated a large scale sequencing study to discover novel prostacyclin receptor genetic variants (22,23). Here we report complete characterization of 18 rare non-synonymous mutations in a COS-1 overexpression system.…”