2003/02/01 by Jeremiah Stitham, Aleksandar Stojanović, Aleksandar Stojanovic +4 · 8 citations
Biochemistry, Genetics and Molecular Biology · Neuroscience · #Receptor Mechanisms and Signaling #Eicosanoids and Hypertension Pharmacology #Neuropeptides and Animal Physiology
paper · pdf · doi:10.1074/jbc.m207420200
The human prostacyclin receptor is a seven-transmembrane α-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 agonist-binding 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. The human prostacyclin receptor is a seven-transmembrane α-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 agonist-binding 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. G-protein coupled receptor prostacyclin 5′-guanlyl-imidodiphosphate analysis of variance human prostacyclin receptor transmembrane 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 (1Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). 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 ligand-binding 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 (2Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar), the putative binding pocket for GPCRs with small nonpeptide ligands is believed to be located predominantly within the hydrophobic core of the transmembrane domain in close proximity to the extracellular boundary of the receptor. However, the crucial anchoring points that comprise the fundamental structure of the binding pocket, securing important receptor-ligand associations between prostacyclin and its receptor, have yet to be determined. As is the case with all prostanoids, prostacyclin (PGI2) is a derivative of the C20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid) (Fig. 1). The general structure of prostanoid molecules consists of a centralized cyclopentane ring (thromboxane has an oxane ring) flanked by two hydrocarbon chains, the α- and ω-chains, whose configuration and functional groups determine further classification (Fig. 1). In particular, the prostacyclin molecule contains an additional oxolane (cyclic ether) ring fused to the cyclopentane ring as well as two hydroxyl groups located at C11 and C15. A characteristic terminal carboxylate group is present at the C1 position as well as carbon-carbon double bonds linking C5 to C6 and C13 to C14. Similar molecular features can be seen in synthetic prostacyclin analogues such as iloprost, a stable high affinity agonist that substitutes a secondary cyclopentane ring in place of the PGI2 oxolane ring, carries an additional C16-methyl group and a ω-chain triple bond (Fig. 1). Side chains of certain amino acids have been shown in receptors to interact directly with substituents of ligands, conferring binding affinity (3Hwa J. Graham R.M. Perez D.M. J. Biol. Chem. 1995; 270: 23189-23195Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,4Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: 6322-6327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) through major forces such as hydrogen bonding, hydrophobic interactions, and ionic interactions. Thus, structural similarities and differences between both prostanoid receptors and prostanoid ligands play an important role in determining sites of interaction between receptor and ligand. For example, conserved serine residues found in TMV of the adrenergic receptors have been shown to interact with the conserved hydroxyl groups extending from the catechol ring of the biogenic amines (4Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: 6322-6327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Recent studies have begun to identify generalized regions within the prostacyclin receptor and other prostanoid receptors that appear crucial for ligand-binding specificity and affinity. Studies using chimeric combinations of mouse prostaglandin D and prostaglandin I 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, whereas TMI along with a portion of the first extracellular loop confers broader binding functions, incorporating recognition and interaction with the cyclopentane ring (5Kobayashi T. Kiriyama M. Hirata T. Hirata M. Ushikubi F. Narumiya S. J. Biol. Chem. 1997; 272: 15154-15160Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Kobayashi T. Ushikubi F. Narumiya S. J. Biol. Chem. 2000; 275: 24294-24303Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Additionally, glycosylation at Asn-7 and Asn-78 (7Zhang Z. Austin S.C. Smyth E.M. Mol. Pharmacol. 2001; 60: 480-487PubMed Google Scholar) and proline residues within the transmembrane domains (8Stitham J. Martin K.A. Hwa J. Mol. Pharmacol. 2002; 61: 1202-1210Crossref PubMed Scopus (36) Google Scholar) have also been shown to be essential for proper binding and activation. Although neither of the two recently identified naturally occurring polymorphisms (i.e. V25M and R212H) have revealed inherent effects on binding, R212H in the third intracellular loop has been shown to exclusively effect activation and exhibits defective binding only under acidic conditions (9Stitham J. Stojanovic A. Hwa J. J. Biol. Chem. 2002; 277: 15439-15444Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Using site-directed mutagenesis, prostanoid ligand and receptor comparisons, and a three-dimensional computer-generated homology model of the hIP receptor derived from the recently published crystal structure of bovine rhodopsin T. T. I. T. M. M. 2000; PubMed Scopus Google Scholar), four crucial points of interaction between prostacyclin and the of the transmembrane domain of the hIP were These crucial points Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), which interact with the ring and and of This agonist-binding pocket is distinct from that of the biogenic amine receptors and ligands, and iloprost, were from were from whereas the hIP a from were to identify important residues within the of the transmembrane domain (i.e. the of the putative whose side-chain functional groups may interact with prostacyclin interactions, hydrogen bonding, or hydrophobic Thus, candidate hIP residues with on ligand binding were for site-directed mutagenesis, to A of binding were using iloprost, a stable high affinity of prostacyclin. For a change in binding further to specific were to determine the specific of the amino acid with effects on ligand binding. The on binding affinity to be the direct molecular interactions residues and ligand side chains within the binding molecular interactions receptor residues involved in of the binding or interactions amino further the receptor amino acids that with sequence for all prostanoid receptor transmembrane domains were from prostanoid receptor including and from were with other as well as with Similar were in functional groups of prostacyclin well as its to other prostaglandin of amino acids at on receptors along with the of certain functional groups on other functional and identified within the putative binding pocket and on ligand binding affinity were on a computer-generated hIP model upon the crystal structure of A prostacyclin molecule the model receptor all of the a three-dimensional model of the pocket the and were using of were extending and from the mutation The of of and and of and and at for for and for for The were with for to of of to by from were using and all were were on as the of with by of in and were at with for which were subsequently for and through with were of were as were in and using for in by for and the a high for the in and by in and at A protein to for the receptors were through a of binding using the ligand analysis involved the of of and along with of 11 of from to of at were by the of and using a The were with and in the of of binding by the of a of The of from to for binding were using were to using the and were as a analysis of variance and were to determine differences The of protein with affinity to through analysis using the (8Stitham J. Martin K.A. Hwa J. Mol. Pharmacol. 2002; 61: 1202-1210Crossref PubMed Scopus (36) Google Scholar). of of protein to This to a using a and secondary and with of the of the native ligand a synthetic (Fig. in all However, that both are and the that binding specificity with the hIP ring, and in to binding that the prostacyclin molecule would be and within molecular of the prostacyclin ligand agonist to the hIP using 1997; PubMed Scopus Google Scholar). A three-dimensional PGI2 molecule upon the structure as well as a of a PGI2 J. Mol. PubMed Scopus Google Scholar). the of such that would to the of the ligand This the of all bonds to with and bond and double bond and bond and and double bond and of all for both were at with the for the bond of the oxane ring, which to The for all bonds and at and with the the two were to the configuration with the characteristic four with a of The structural of prostacyclin molecule is similar to that of in of PGI2 J. Mol. PubMed Scopus Google Scholar) where the is in a upon the two centralized whereas the hydrophobic ω-chain is in an This configuration using where the to all exhibited such a ring and with the major in position of the ω-chain A three-dimensional homology model of the seven-transmembrane of the hIP receptor using the 1997; PubMed Scopus Google Scholar). acid from all prostanoid receptors were from the in and with of the bovine rhodopsin receptor. Based upon this alignment, distinct segments amino to the seven-transmembrane domains of the hIP were and a homology model using the 2.8-Å x-ray crystallographic structure of the bovine rhodopsin receptor as the Receptor residues were by to rhodopsin transmembrane and by of of by the transmembrane domains were the to the of the model and and of the model as well as of the prostacyclin ligand using the amino acids were to the extracellular transmembrane domain and to an α-helical using the crystal structure of rhodopsin as a the crystal structure of the rhodopsin, of the transmembrane amino acids the binding of prostacyclin may the extracellular The were from model as are in rhodopsin to be with from the crystal In no homology with the of the Thus, exclusively on the putative binding pocket within the transmembrane domain of the The prostacyclin receptor important roles in vascular smooth muscle platelet and Pharmacol. 1996; PubMed Scopus Google Scholar, R.M. Pharmacol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, I. 1998; PubMed Scopus Google Scholar, M. Sugimoto Y. A. 1995; PubMed Scopus Google Scholar). are for the treatment of pulmonary hypertension Y. Pharmacol. Physiol. 1997; PubMed Scopus Google Scholar, M. Y. T. S. F. T. J. 1999; PubMed Scopus Google Scholar, T. J. J. 2000; PubMed Scopus Google Scholar, R.M. J. Pharmacol. 1998; Google Scholar). has also been that prostacyclin may also be as a in Y. 2002; Google Scholar) and M. 1996; PubMed Scopus Google Scholar, T. S. T. S. T. Ushikubi F. Narumiya S. T. 1999; Google Scholar). of the hIP receptor is with only a of studies the of this important receptor. there are no high affinity antagonists and only a stable high affinity understanding of the residues that the ligand-binding pocket would be in determining features this receptor may and assist in the development of specific hIP ligands, selective residues were first to to determine that an on binding affinity These amino acids were upon to interact with prostacyclin side with to the binding pocket position within the seven-transmembrane all binding pocket residues such as and side-chain amino acids and in the of the TM domain were The prostacyclin receptor well with a binding affinity for of The binding to a binding In the of a high affinity the agonist for binding. studies were on the receptor in the of a of to G-protein from the receptor. The of no effect on binding affinity for protein that this affinity the of G-protein Of the 29 residues 11 a effect on binding (Fig. These were located in and and TMV and and and In the of a high affinity for ligand binding, only receptors with differences of in binding affinity and with receptor were to a binding (Fig. For with binding affinity and binding of protein analysis (Fig. and binding yet of protein for Moreover, analysis also revealed the that are with receptors (8Stitham J. Martin K.A. Hwa J. Mol. Pharmacol. 2002; 61: 1202-1210Crossref PubMed Scopus (36) Google binding and binding for the 29 transmembrane to cyclopentane ring, are the from at of by in binding are the of at two of in a binding for and that exhibited a decrease in binding of are are the and that binding affinity as with of a and to the as with the analysis for and with binding affinity. analysis as under using the high affinity to the and in binding were in using of of protein with the of which only of are the from at of by in binding are the of at two of Receptor configuration and TM were upon sequence homology and with the crystal structure of the rhodopsin receptor. conserved ligand substituents on are to interact with conserved amino acids on the prostaglandin receptor. This has been and in the of GPCRs including the adrenergic receptors (4Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: 6322-6327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and rhodopsin J. PubMed Scopus Google Scholar). the computer-generated homology model of the hIP transmembrane domain and using the of the model with the crystal structure of rhodopsin structural further determine the of hIP a analysis Of the amino acids the transmembrane were within the for a whereas were within the This well with the seven-transmembrane α-helical secondary structure of the hIP receptor. also (via that of the residues involved in or with the protein or other side-chain were there of prostacyclin the model of the of distinct receptor-ligand interactions The major for the position of prostacyclin upon the for the to interact with The binding pocket amino acid is the highly conserved Arg-279 found within the transmembrane domain of the hIP receptor. Upon mutation to a decrease in agonist binding affinity as with the receptor In protein in with the protein studies on the M. A. J. 1997; PubMed Scopus Google Scholar, Y. D.M. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and Mol. Pharmacol. 1998; PubMed Scopus (36) Google Scholar) receptors have the of this in both ligand binding as well as receptor activation. Moreover, has been shown that this has the to only an ionic bond with the group of ligands as a hydrogen for groups as receptor studies have that the interaction between this and ligand is ionic hydrogen to in a affinity and S. M. Mol. Pharmacol. 2001; PubMed Scopus Google Scholar). Thus, the level of of Arg-279 all of the prostanoid marked effect on ligand binding to and of the all of the native prostanoid a direct ionic interaction between Arg-279 and the of prostacyclin (Fig. to the crucial Arg-279 is an found only in the receptor at this The of this to directly with unique structural features found on receptor ligands, in the additional oxolane (cyclic ether) ring found on the native prostacyclin ligand. synthetic ligands such as and secondary cyclopentane at this whereas other naturally occurring (Fig. 1). A in binding affinity in the mutation also as by the of the the Phe-278 contains a side Thus, the between this and prostacyclin a hydrophobic interaction between the oxolane ring of prostacyclin ring of and the Phe-278 As with model (Fig. there may be an additional interaction with the is that both of ring as unique yet that are crucial for proper ligand binding and to the bond at The of Phe-278 and Arg-279 key anchoring points at the and secondary oxolane ring of the such that in a (Fig. as with the of prostacyclin. The Tyr-75 on is conserved the of the prostanoid receptors with the of the which a a group is present on all native with the of In the this hydroxyl is to a of a cyclopentane in the is as of a oxane ring (Fig. 1). from the group on prostacyclin is in close proximity with the This is by the close proximity between Arg-279 and further for Thus, and a direct interaction between Tyr-75 and the group as binding of agonist with the revealed a decrease in affinity with specific binding as with the receptor determine the structural features involved in this interaction (i.e. the of a ring, a hydroxyl group, or further and no in binding were with of the additional with and to for all of and protein for and hydrogen to be the major for interaction (Fig. in the ligand or the receptor may for the of hydrophobic interactions between the centralized cyclopentane ring of prostacyclin and the Thus, interaction (i.e. hydrophobic or hydrogen is in receptor-ligand affinity. a high level of all prostanoid receptors from the structural groups with ligands from close proximity to the Arg-279 and affinity upon to Tyr-75 as an anchoring to the of prostacyclin. The Phe-95 is of a within TM domain to model and binding Phe-95 plays a crucial role in the hydrophobic ω-chain of prostacyclin. A of hydrophobic residues is present in on all prostanoid receptors in the of the putative binding pocket. The binding affinity for the mutation from the a binding The for The Phe-95 side a hydrophobic that to the ω-chain (Fig. studies have shown this TM to be between mouse prostaglandin I and mouse prostaglandin D receptors T. Ushikubi F. Narumiya S. J. Biol. Chem. 2000; 275: 24294-24303Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). is of two regions for activation 1996; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that this is also to be important in the hIP (8Stitham J. Martin K.A. Hwa J. Mol. Pharmacol. 2002; 61: 1202-1210Crossref PubMed Scopus (36) Google Scholar). Thus, with for the carboxylate group TMVII) and both centralized and Tyr-75 the hydrophobic of which the fundamental of between receptor and the effects on binding amino acid residues and are by model to be directly involved in receptor-ligand binding (TMII), highly conserved all prostanoid from ligand-binding pocket. in conserved is to be directly involved in binding. to the hydrophobic found in Although is close to its Phe-95 is from the prostacyclin molecule and has no direct on ligand binding. to a marked decrease in affinity suggests a role as a position through interactions with the is also an important as by the marked in binding affinity upon mutation to However, to ligand binding are to the from the bound ligand as by in TMV is found in only of all prostanoid receptors and has been shown to ligand-binding affinity upon mutation to to is in close proximity to both and The position is conserved only in the prostanoid with the of amino acids at this position the is to be directly involved in receptor-ligand interactions may as a structural in hydrophobic interactions with in is conserved in of all prostanoid no direct interaction the proximity to the ligand with a carboxylate group suggests that is an essential structural the binding through the of a residues are important in the fundamental binding pocket and anchoring points for the prostacyclin model (i.e. and model were the prostacyclin molecule in place and the structure the to for other receptor-ligand interactions using the as an additional and (TMIII), were identified as binding pocket receptor-ligand associations within the ω-chain of prostacyclin. additional as a mutation candidate for the binding by The and with model lend to both its and A binding pocket is found in only a small number of prostanoid receptors including the such a small and molecule would be as a direct to ligand binding as by an which exhibited affinity (Fig. However, to a amino acid (i.e. a decrease in binding affinity A between the side and the ω-chain of prostacyclin is by model (Fig. with and side-chain other hydrophobic residues in the ω-chain of prostacyclin as is bound to the hIP receptor. binding pocket with direct interaction with ligand (TMIII), which is highly conserved and present in of all prostanoid In model (Fig. that may to binding affinity through hydrogen bond with the group of which is a highly in all prostaglandin However, a change at this position exhibited no change in binding affinity as with This is with studies on the and receptor which that the conserved group may play an important role in agonist affinity S. M. Mol. Pharmacol. 2001; PubMed Scopus Google Scholar, M. M. Y. M. S. M. M. Y. M. 2000; PubMed Scopus Google Scholar). As both the to we that of the binding pocket the important Phe-95 that mutation to the hydrophobic interaction with the Upon of in to be a candidate for of the binding to (Fig. Thus, we both and in with the The mutation exhibited a binding affinity in with of this in binding, no in The of model by this should be that model is a of the binding of ligand to receptor. an interaction in is with in both receptor and ligand an prostacyclin and within the binding pocket would in conformational receptor activation. has been for rhodopsin as well as other GPCRs including hIP studies that receptor upon mutation of residues for ligand binding Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. A. 1997; PubMed Scopus Google Scholar). This suggests in to binding, residues may also as important structural in the Moreover, binding pocket residues may to the on receptors that by ligand to receptor activation Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). As a of prostacyclin or binding, such may be to both ligand and receptor conformational the position of prostacyclin in the binding pocket, we would that and would and upon agonist binding. is for such as and on rhodopsin upon of to have shown in and 1996; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar, J. Hwa J. 2001; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar). These major are by ligand and ω-chain Thus, the first of this has been further studies are for This specific residues that comprise the fundamental structure of the pocket, securing crucial receptor-ligand associations as well as amino acids in close proximity to the general binding findings a structural model of prostacyclin in which four distinct anchoring sites by TM amino ligand to receptor. These were the pocket at the level in an opposing to the ligands of rhodopsin and the biogenic amine both of which also have ligands of a carbon ring with a hydrocarbon and For rhodopsin, the ring of in TMV and and the carbon is a to T. T. I. T. M. M. 2000; PubMed Scopus Google Scholar). the biogenic amines hydroxyl groups from the catechol interact with on TMV and the amine group with an acidic in (4Hwa J. Perez D.M. J. Biol. Chem. 1996; 271: 6322-6327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). and the biogenic amines has important interactions with and the TMI and TMV and (Fig. that this may be a unique of the prostacyclin receptor that has the of high affinity selective This may assist in the development of unique and highly including additional selective antagonists for the treatment and of prostanoid-related disorders. to Martin Graham and for the of for and in the of prostacyclin.