Impaired Receptor Binding and Activation Associated with a Human Prostacyclin Receptor Polymorphism

The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structure-function characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4,Ki = 2.2 ± 1.2 nm; pH 6.8Ki = 45.6 ± 12.0 nm). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC50 = 2.8 ± 0.5 nmversus wild-type hIP EC50 = 0.5 ± 0.1 nm; pH 6.8, R212H EC50 = 3.2 ± 1.6 nmversus wild-type hIP EC50 = 0.5 ± 0.2 nm). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP. The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structure-function characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4,Ki = 2.2 ± 1.2 nm; pH 6.8Ki = 45.6 ± 12.0 nm). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC50 = 2.8 ± 0.5 nmversus wild-type hIP EC50 = 0.5 ± 0.1 nm; pH 6.8, R212H EC50 = 3.2 ± 1.6 nmversus wild-type hIP EC50 = 0.5 ± 0.2 nm). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP. human prostacyclin receptor transmembrane domain single nucleotide polymorphism Similar to other prostanoids, prostacyclin is a derivative of the C-20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid), and its cellular action is conveyed through cell surface G-protein-coupled receptors that predominantly couple to the heterotrimeric G-protein Gs stimulating the production of cAMP (1Kobayashi T. Ushikubi F. Narumiya S. J. Biol. Chem. 2000; 275: 24294-24303Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The human prostacyclin receptor (hIP)1 is expressed on platelets, where it mediates inhibition of platelet aggregation and on vascular smooth muscle cells, where it mediates vascular smooth muscle relaxation. 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 (2Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Accordingly, IP receptor knock-out mice exhibit increased thrombosis and reduced inflammatory and pain responses (3Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Ueno A. Oh-ishi S. Narumiya S. Nature. 1997; 388: 678-682Crossref PubMed Scopus (690) Google Scholar). Limited studies have begun to identify generalized regions within the IP and other prostanoid receptors that appear crucial for ligand-binding specificity and affinity. Studies using chimeric combinations of mouse prostaglandin D (mDP) and prostaglandin I (mIP) 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. In addition, TMI (along with a portion of the first extracellular loop) confers broader binding functions, incorporating recognition and interaction with the cyclopentane ring of prostacyclin (1Kobayashi T. Ushikubi F. Narumiya S. J. Biol. Chem. 2000; 275: 24294-24303Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 4Kobayashi 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). Glycosylation at Asn-17 and Asn-78 in the extracellular domain (see Fig. 1), has also been demonstrated to be essential for proper binding and G-protein activation (5Zhang Z. Austin S.C. Smyth E.M. Mol. Pharmacol. 2001; 60: 480-487PubMed Google Scholar). As observed with other G-protein-coupled receptors, genetic variants of the hIP receptor may act as predisposing and/or modifying factors for disease states or therapeutic response. In this investigation, we have undertaken a functional analysis of the first polymorphisms identified in the coding region of the hIP receptor, recently identified in the SNP database (6Sherry S.T. Ward M. Sirotkin K. Genome Res. 1999; 9: 677-679PubMed Google Scholar). The goal of this study is to determine the effects of these polymorphisms on agonist binding and G-protein activation at physiologic and pathological pH levels. Our results indicate that the V25M polymorph had no significant effects on agonist binding or Gs activation, functioning in a manner consistent with the wild-type hIP. In contrast, the R212H polymorph showed a significant decrease in signal transduction activation, requiring a 6-fold increase of agonist to elicit a wild-type-like response at both pH 7.4 and 6.8. Furthermore, under acidotic conditions (pH 6.8), a defect in binding was also observed for R212H. Iloprost ligands, radiolabeled [3H]iloprost (17.0 Ci/mmol), and non-radiolabeled iloprost as well as the cAMP radioimmunoassay system were purchased from Amersham Biosciences. Oligonucleotides were purchased from Sigma-Genosys (The Woodlands, TX). The hIP cDNA was a generous gift from Dr. Mark Abramovitz (Merck Frosst, Quebec, Canada). Human IP cDNA was cloned along with a C-terminal 1D4 epitope tag (native nine C-terminal amino acids from rhodopsin) into the pMT4 expression vector. Point mutations were generated using conventional methods of PCR mutagenesis as previously described (7Hwa J. Garriga P. Liu X. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10571-10576Crossref PubMed Scopus (82) Google Scholar). Complementary oligonucleotide primers were designed extending 10–12 nucleotides 3′ and 5′ from the desired mutation sites (V25 or R212). All mutant constructs were confirmed via PCR DNA dideoxynucleotide chain termination sequencing (Dartmouth Medical School Molecular Biology Core Facility). Transient transfections of COS-1 cells were performed initially at a DNA concentration of 2.0 μg/ml followed by decreasing concentrations of 1.0, 0.5, 0.25, 0.05, and 0.025 μg/ml using diethylaminoethyl-dextran (DEAE-Dextran; Sigma) as previously described (7Hwa J. Garriga P. Liu X. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10571-10576Crossref PubMed Scopus (82) Google Scholar). Preparations of COS-1 cell membranes were carried out as follows. Cells were washed in phosphate-buffered saline and harvested by scraping. Subsequent washes in 0.25m sucrose solution were followed by vigorous vortexing (providing shear forces) for 3 min. A low speed spin (∼1,260 ×g) was performed for 5 min, and the supernatant was collected. After a high speed centrifugation (∼30,000 ×g for 15 min) the pellet was washed twice in 1× HEM (20 mm Hepes pH 7.4, 1.5 mm EGTA, and 12.5 mm MgCl2) followed by resuspension in 1× HEM containing 10% glycerol and was stored at −70 °C. A Bradford protein assay was performed to quantitate membrane proteins. Ligand-binding characteristics for the expressed receptors were determined through a series of competition binding assays using radiolabeled [3H]iloprost (fixed concentration), an IP receptor-specific agonist, versusnon-radiolabeled iloprost (varied concentrations). Mock transfected COS-1 cell membranes revealed no specific binding to iloprost. 2J. Stitham, A. Stojanovic, and J. Hwa, unpublished data. Reaction mixtures (performed in duplicate) contained 50 μg of membrane, 1× HEM buffer (pH 7.4, 6.8, and 5.9), 15 nm [3H]iloprost, and one of 12 different concentrations (10 μm to 0.1 nm) of cold (non-radiolabeled) iloprost. After a 1.5-h incubation at 4 °C, reactions were stopped by the addition of ice-cold 10 mm Tris/HCl buffer (pH 7.4), and the reaction mixture was filtered onto Whatman® GF/C glass fiber filters using a Brandel® cell harvester. The filters were washed five times with ice-cold Tris/HCl buffer, and radioactivity remaining on the filter paper (trapped membranes) was measured in the presence of 5 ml of Liquiscint™ scintillation fluid (National Diagnostics, Atlanta, GA). Nonspecific binding was determined by the addition of a 500-fold excess of non-radiolabeled iloprost, whereas the concentration of [3H]iloprost was varied from 1 to 100 nm for saturation binding studies. Data were analyzed using GraphPad Prism® software. IC50values were converted to Ki using the Cheng-Prusoff equation, and Ki values were expressed as means ± S.E. An analysis of variance (post-test Newman-Keuls) and Student’st tests were used to determine statistically significant differences (p < 0.05). The wild-type hIP with the epitope tag hIP1D4 and mutant constructs were analyzed for signal transduction capabilities. COS-1 cells were transiently transfected with 2.0 μg/ml receptor DNA in 25-mm plates as described above. After 72 h, cells were washed twice with phosphate-buffered saline plus 4 mm EDTA and 2 mm IBMX (Sigma) (pH 7.4, 6.8, or 5.9) and incubated at 20 °C for 10 min. This was followed by addition of defined concentrations of iloprost to selected plates. Dose-response curves were determined by the addition of six different concentrations (1 μm to 10 pm) in duplicate. After 20 min, the cells were harvested and boiled for 3 min, followed by high speed (10,000 rpm) centrifugation. Fifty microliters of the resultant supernatant (a total of 300 μl) was used to determine cAMP production in the competition assay. cAMP levels were measured using the radio-receptor competition assay (Amersham Biosciences). In brief, [3H]cAMP was used in competition for a cAMP-binding protein against known concentrations of non-radiolabeled cAMP, followed by determination of the unknowns. The reaction was allowed to proceed for 2 h at 4 °C. Charcoal was used to remove excess unbound cAMP. Samples were counted in 5 ml of Liquiscint™(National Diagnostics). Results were analyzed with GraphPad Prism® software. Mean ± S.E. was calculated for basal and maximal cAMP production. For the dose response, a non-linear, curve-fitting program (GraphPad Prism®) was used, and the EC50 was determined for wild-type hIP1D4 and mutant constructs. An analysis of variance (post test Newman-Keuls) and Student’s t tests were used to determine statistically significant differences (p < 0.05). Two polymorphisms in the coding region of the hIP were recently identified and appeared on the SNP database (6Sherry S.T. Ward M. Sirotkin K. Genome Res. 1999; 9: 677-679PubMed Google Scholar). Using PCR mutagenesis we have reproduced these polymorphisms, V25M and R212H (Fig.1). The overall goal of our study was to determine whether these naturally occurring mutations would modify hIP receptor function. In particular, we analyzed binding of the high-affinity agonist iloprost (a stable derivative of the native hIP ligand prostacyclin), activation of the native Gs pathway, and cell surface expression of the receptor. Receptor binding was initially evaluated at physiological pH 7.4 with iloprost, a stable high-affinity analogue of prostacyclin. No significant difference was detected in theKi values for wild-type hIP1D4, V25M, or R212H (Table I). All binding curves were best fit by a one-site model. Thus, iloprost binding for both polymorphism mutants remained unaffected as compared with the wild-type hIP1D4 receptor. Saturation binding performed on the constructs showed expression levels of ± membrane protein for the hIP1D4 = and 1.5 ± membrane protein for the V25M = However, the R212H expressed (p < lower ± 0.2 membrane = the binding studies for hIP1D4, V25M, and R212H under different pH ± in is the number of ± 3.2 ± ± ± ± ± 1.2 ± 12.0 < ± < are Ki ± S.E. from at as under of 12 different concentrations Student’s t tests were used to determine significant from wild-type in is the number of < in a are Ki ± S.E. from at as under of 12 different concentrations Student’s t tests were used to determine significant from wild-type Receptor activation, as measured by in the production of cAMP, revealed a significant defect associated with the R212H which exhibited an EC50 ± 0.5 nm; < 6-fold that of the wild-type hIP1D4 receptor = 0.5 ± 0.1 nm) (Table the V25M mutant significant difference from the wild-type hIP1D4 in to cAMP (Table Thus, with to both ligand binding and activation the V25M variant exhibited wild-type-like In contrast, at pH 7.4 the R212H mutant had effects receptor activation with no significant on agonist studies under different pH ± 0.1 in is the number of ± 0.2 ± < ± ± 0.2 ± < ± 0.5 < ± 1.6 < ± 1.6 < response EC50 ± S.E. for hIP1D4, V25M, and R212H from at of six different are differences were determined using Student’s t tests and the results to wild-type in is the number of < < in a cAMP response EC50 ± S.E. for hIP1D4, V25M, and R212H from at of six different are differences were determined using Student’s t tests and the results to wild-type conditions of stress such as observed with renal, or acidosis and in pH levels The hIP receptor on the is to such pH Our results showed in ligand binding at pH 6.8 with the R212H polymorph = 45.6 ± 12.0 < which at a lower pH of However, the wild-type hIP1D4 the V25M variant showed effects in binding from the in pH I). The of pH on receptor activation was were transfected and in was no significant in activation from pH 7.4 to 6.8 for constructs (Table Fig. The R212H from wild-type hIP1D4 by pH was a significant decrease in EC50 for both wild-type hIP1D4 = ± < and V25M = ± < (Table Fig. The R212H remained abnormal at ± 1.6 For both the wild-type hIP1D4 and the two activation was at lower However, at pH was a defect observed with wild-type hIP1D4 and (Table the in affinity at lower pH for we a further of the abnormal activation at pH 7.4, 6.8, and ± S.E. of cAMP production from at in which was performed in with wild-type hIP1D4 is The by iloprost from 1 μm to EC50 were determined from the with (GraphPad production of cAMP performed at pH production of cAMP performed at pH 6.8. production of cAMP at pH Our cAMP activity assays showed maximal levels for constructs using 2.0 μg/ml hIP1D4 DNA for However, using the concentration of DNA our saturation binding that R212H expressed at the levels of V25M and that this difference from our the DNA used for our μg of using concentrations for hIP1D4 wild-type and R212H DNA concentrations of 1.0, and 0.5 μg of no significant differences in maximal cAMP However, lower DNA concentrations 0.05, and 0.025 showed a significant difference that with receptor μg of the was membrane protein for hIP1D4 and 0.2 membrane protein for R212H. 0.025 μg of expression was 0.2 for hIP1D4 and 0.1 for R212H. were performed to whether EC50 was by the in expression membrane protein the maximal cAMP values were the for both hIP1D4 and R212H. The EC50 for wild-type hIP1D4 was nm in to nm for R212H. lower expression levels for the R212H membrane the EC50 was and at 0.1 membrane protein the EC50 was nm a was in maximal cAMP were no significant differences in EC50 for both hIP1D4 and R212H at lower cell surface at the lower levels of responses described in Fig. were determined for different levels of expression using DNA concentrations of 1.0, 0.5, 0.05, and 0.025 the hIP1D4 expression of membrane 0.5 μg/ml DNA was For R212H expression μg/ml DNA was are the curves for hIP1D4 membrane and R212H and 0.1 membrane Receptor polymorphisms are as important to the of both disease and J. 1997; PubMed Scopus Google Scholar, K. M. U. J. 2001; PubMed Scopus Google Scholar, M. M. Mol. 2001; PubMed Scopus Google Scholar). naturally occurring variants have been in domains of G-protein-coupled receptors, ligand binding and to G-protein T. Rev. Pharmacol. 2001; PubMed Scopus Google Scholar). domain variants in (7Hwa J. Garriga P. Liu X. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10571-10576Crossref PubMed Scopus (82) Google the receptor P. S. J. PubMed Scopus Google and the receptor A. J. Mol. PubMed Scopus Google on ligand variants detected in the in the receptor A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the receptor K. J. N. S. M. M. T. M. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the receptor A. A. S. M. M. Mol. Biol. Google exhibit in G-protein studies have important T. Rev. Pharmacol. 2001; PubMed Scopus Google Scholar). polymorphisms that may be under normal physiological conditions with the functional in the J. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). in hIP receptor and by such naturally occurring mutations may to the differences observed in the of cardiovascular disease and responses to responses to iloprost in the of pulmonary T. J. J. 2000; PubMed Scopus Google Scholar, N. M. Y. T. S. F. N. K. T. J. 1999; PubMed Scopus Google Scholar). In this we the effects of the V25M and R212H polymorphisms on hIP function. Two hIP polymorphisms in the coding region of the were recently identified and appeared on the SNP database (6Sherry S.T. Ward M. Sirotkin K. Genome Res. 1999; 9: 677-679PubMed Google Scholar). The V25M was to from a single mutation at 1 to the amino acid number and PCR reactions from a of were used to this were an of mutant and further This polymorphism is in transmembrane I in the region of the agonist binding The R212H mutation from a single at 2 to the amino acid with a of 20 were also detected for this which is in the important The goal of our study was to determine the effects of these naturally occurring mutations on hIP receptor function. In particular, we analyzed agonist activation of the native Gs pathway, and cell surface expression of the receptor. Receptor binding was initially evaluated at physiological pH 7.4 with iloprost as described under No significant difference was observed in agonist binding wild-type hIP1D4 and the V25M or R212H Receptor activation, as measured by in the production of cAMP, revealed that was a significant defect associated with the R212H the V25M mutant significant difference from the wild-type hIP1D4 in to cAMP Thus, with to both ligand binding and activation, the V25M variant exhibited wild-type-like that the mutation was well a significant in amino acid In contrast, at pH 7.4 the R212H mutant in the important loop) had effects receptor activation with no significant effects on agonist A of conditions in in pH levels. The hIP receptor on the membrane is to such pH in the an important role in receptor activation, to the Gs of the heterotrimeric normal physiological conditions (pH 7.4), the amino acid is as compared with the native amino acid the of these amino acids are by the of the protein we that decreasing the pH of the to a may of the This may the activation defect at physiological pH by the and normal as with the native at of the wild-type hIP1D4 receptor. our results showed in ligand binding at pH 6.8 with the R212H which at a lower pH of However, the wild-type hIP1D4 the V25M variant showed effects in binding by the in pH I). the binding defect in R212H was no significant in activation from pH 7.4 to 6.8 for of the constructs Fig. and the R212H polymorphism from wild-type hIP1D4 by was at lower pH levels for as well as both at pH was a defect observed for hIP1D4 and V25M (Table the in affinity at lower pH for we a significant of the abnormal This may be to the of our assay system in significant in However, mutations have been in the prostacyclin receptor that decrease agonist binding affinity an on activation K. and Hwa, J. Mol. Pharmacol. Scholar). that this from amino interactions binding that to affinity to receptor required for pH (6.8) may such such The defect in receptor activation for R212H abnormal at acidic Our cAMP activity assays revealed maximal activation for constructs using 2.0 μg/ml DNA for However, using the concentration of DNA saturation binding that R212H at the levels of V25M and This difference may be to our system in which the of in G-protein-coupled receptor plays an important role J. Pharmacol. 2000; Google Scholar). has been shown that is the that maximal response to the receptor J. Pharmacol. 2000; Google Scholar). Thus, of the receptor in cells Y. J. PubMed Scopus Google or PubMed Scopus Google results in in the DNA used for our μg of using concentrations for hIP1D4 and R212H DNA concentrations 0.05, and 0.025 showed a significant difference in maximal that with receptor a was in maximal cAMP were no significant differences in EC50 for both hIP1D4 and R212H at lower cell surface The amino acid at in mouse and IP and receptors is a the for human and mouse IP are amino acid in the of the the the in an pH As and the low of human and mouse IP receptors has been signal transduction Recent studies of cloned in or revealed EC50 levels for cAMP of nm J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. 2001; PubMed Scopus Google as compared with nm for cloned hIP in cells and COS-1 (5Zhang Z. Austin S.C. Smyth E.M. Mol. Pharmacol. 2001; 60: 480-487PubMed Google Scholar, K. and Hwa, J. Mol. Pharmacol. Scholar, E.M. Austin S.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). that the hIP R212H mutation Thus, this may at in for these In this study the resultant and functional associated with the first known naturally occurring human prostacyclin receptor polymorphisms V25M and R212H in with a in may during of acidosis in hIP polymorphisms such as R212H. In these an urgent correction of bodily pH may be required to restore normal hemostasis and as well as to therapeutic This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of human prostacyclin Dr. (Dartmouth Medical for the

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