The Unique Ligand-binding Pocket for the Human Prostacyclin Receptor

Abstract

The human prostacyclin receptor is a seven-transmembrane ␣-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-Å x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonistbinding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.Vascular smooth muscle relaxation and inhibition of platelet aggregation are two key physiological processes mediated by human prostacyclin. 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 (1). In contrast to other members of the rhodopsin-like G-protein coupled receptor (GPCR) 1 subfamily such as the adrenergic receptors or other members of the prostanoid family, there are currently no high affinity selective antagonists for the prostacyclin receptor. This finding suggests that the prostacyclin receptor may possess a unique ligandbinding pocket.Receptor activation is contingent upon ligand binding interactions, which initiate a conformational change in protein structure that is subsequently transmitted to the G-protein.Determining the exact nature and location of receptor-ligand binding interactions at the molecular level is essential for understanding the functions of prostanoid receptor physiology. Moreover, such insights would lend to the development of novel and highly specific modes of treatment for prostanoid-related disorders. Based upon the position of the chromophore (covalently bound 11-cis-retinal) within the binding pocket of rhodopsin along with the location of other ligands within similar rhodopsin-type GPCRs (2), the putative binding pocket for GPCRs with small nonpeptide ligands is beli...

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors

“…The improved homology model based on the hybrid template predicts that EL2 could play an important role in anchoring a free fatty acid at GPR84 by means of a positively charged arginine. Given that phylogenetically GPR84 belongs to the prostanoid receptor subfamily, for which the positively charged residue is predicted to coordinate the negatively charged ligands, as exemplified by the prostacyclin receptor (Stitham et al 2003) it is suggested that GPR84 also could attract the anionic part of the ligand via a counter-charged residue. Figure 7.…”

The Pharmacology and Function of Receptors for Short-Chain Fatty Acids

“…Previous studies had shown that positively charged amino acids within the TM regions are essential for the binding and function of other GPCRs whose ligands contain a carboxylic acid group (Stitham et al, 2003;He et al, 2004;Tunaru et al, 2005;Sabirsh et al, 2006). This, together with the observation that uncharged ester derivatives of SCFAs are inactive at FFA2 and FFA3 (Le Poul et al, 2003), led Milligan's group to hypothesize that basic residues might also play a crucial role in the binding of SCFAs to their receptors (Stoddart et al, 2008a).…”

Acceleration of Cardiovascular Disease by a Dysfunctional Prostacyclin Receptor Mutation

“…[15][16] Like other prostanoid receptors, the hIP has been categorized (based on sequence homology, ligand structure, and overall receptor functionality) as a class A rhodopsin-like GPCR, and shares many structural commonalities with rhodopsin, the class A representative and "prototypical" GPCR. These common traits can be divided into 3 major receptor domains: (1) the extracellular domain, consisting of a short amino N-terminal tail and 3 extracellular loops (exoloops); (2) a TM domain, comprised of 7-TM-spanning ␣-helices, whose upper third ␣-helices contain the putative binding pocket 17 ; and (3) the cytoplasmic or intracellular domain that is made up of 3 helix-joining intracellular loops (cytoloops), a fourth loop produced by lipid anchoring (palmitoylation) of intracellular cysteines, and a fairly lengthy carboxyl terminus ( Figure 1). Genetic variants may, therefore, have differential effects on binding, expression, and activation, dependent on localization within the protein structure, analogous to the retinitis pigmentosa rhodopsin mutations.…”