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• W2040489780 abstract "Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation). Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation). Prostacyclin and its receptor play key roles in vascular smooth muscle relaxation and inhibition of platelet aggregation. A host of studies, including IP 2The abbreviations used are: IP, prostacyclin receptor; GPCR, G-protein-coupled receptor; h-, human; PGI2, prostacyclin; TP, thromboxane receptor; TMI-VII, transmembrane α-helices 1-7; β-ME, β-mercaptoethanol; DP, prostaglandin D receptor; EP2, prostaglandin E receptor type 2; FP, prostaglandin F receptor. 2The abbreviations used are: IP, prostacyclin receptor; GPCR, G-protein-coupled receptor; h-, human; PGI2, prostacyclin; TP, thromboxane receptor; TMI-VII, transmembrane α-helices 1-7; β-ME, β-mercaptoethanol; DP, prostaglandin D receptor; EP2, prostaglandin E receptor type 2; FP, prostaglandin F receptor. receptor knock-out (IP-/-) mice, has implicated dysfunctional IP activity in numerous cardiovascular abnormalities, including thrombosis, myocardial infarction, stroke, hypertension, and atherosclerosis (2Cheng Y. Austin S.C. Rocca B. Koller B.H. Coffman T.M. Grosser T. Lawson J.A. FitzGerald G.A. Science. 2002; 296: 539-541Crossref PubMed Scopus (719) Google Scholar, 5Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Despite significant progress in our understanding of G-protein coupled receptors (GPCRs) in general, the details of human prostacyclin receptor structure-function remains largely unknown.Cysteine chemistry is both fascinating and intriguing. The sulfhydryl or thiol (SH) reactive groups of this amino acid are very susceptible to oxidation and can readily form stable dimers (i.e. disulfide S-S bridges), which play important roles in the organization and maintenance of protein tertiary structure. Somewhat analogous to hydroxyl groups (OH) found on serines, sulfhydryl (SH) side chains are also polar and can participate in hydrogen bonding interactions and can additionally coordinate trace metals (e.g. zinc) (6Stojanovic A. Stitham J. Hwa J. J. Biol. Chem. 2004; 279: 35932-35941Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Sulfur atoms are also quite nucleophilic and react readily with electrophilic molecules to form a variety of thiol-linked derivatives (e.g. thioethers, thioesters, and thioacetals). Thus, cysteine side chains are common sites for various biological coupling and conjugation reactions, including palmitoylation, isoprenylation, disulfide cross-linking, and thiol-disulfide exchange (7Hayes J.S. Lawler O.A. Walsh M.T. Kinsella B.T. J. Biol. Chem. 1999; 274: 23707-23718Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9Perlman J.H. Wang W. Nussenzveig D.R. Gershengorn M.C. J. Biol. Chem. 1995; 270: 24682-24685Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In this study our goal was to assess the structural and functional contributions of cysteines within the human prostacyclin receptor (hIP) receptor. Early mutagenesis studies in bovine rhodopsin (using Cys→ Ser mutations) revealed the importance of amino acids Cys-110 and Cys-187, which are essential in the formation of normal functional rhodopsin protein (10Karnik S.S. Sakmar T.P. Chen H.B. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8459-8463Crossref PubMed Scopus (346) Google Scholar). It was later shown that these two highly conserved cysteine residues form a disulfide bond in the extracellular domain (11Karnik S.S. Khorana H.G. J. Biol. Chem. 1990; 265: 17520-17524Abstract Full Text PDF PubMed Google Scholar). Further mutagenesis and mass spectrometry confirmed the presence of this disulfide bond and its importance in stabilizing rhodopsin (12Davidson F.F. Loewen P.C. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4029-4033Crossref PubMed Scopus (129) Google Scholar, 13Hwa J. Klein-Seetharaman J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4872-4876Crossref PubMed Scopus (62) Google Scholar). Because of the initial discovery of this imperative disulfide bond within rhodopsin, many other investigations have gone on to demonstrate the presence and importance of similar S-S bonds in other GPCRs (14Fay J.F. Dunham T.D. Farrens D.L. Biochemistry. 2005; 44: 8757-8769Crossref PubMed Scopus (48) Google Scholar, 18Lisenbee C.S. Dong M. Miller L.J. J. Biol. Chem. 2005; 280: 12330-12338Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mutagenesis of the thromboxane receptor (TP) at the conserved extracellular S-S cysteine positions (Cys-105 and Cys-183) revealed decreased binding affinity and low amplitude calcium signaling (19D'Angelo D.D. Eubank J.J. Davis M.G. II Dorn G.W. J. Biol. Chem. 1996; 271: 6233-6240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 20Chiang N. Kan W.M. Tai H.H. Arch. Biochem. Biophys. 1996; 334: 9-17Crossref PubMed Scopus (56) Google Scholar). Interestingly, in addition to this conserved disulfide bridge, further studies on the β2-adrenergic receptor as well as the gonadotropin-releasing hormone receptor have suggested the presence of a second, non-conserved disulfide bond (8Cook J.V.F. Eidne K.A. Endocrinology. 1997; 138: 2800-2806Crossref PubMed Scopus (92) Google Scholar, 21Noda K. Saad Y. Graham R. Karnik S. J. Biol. Chem. 1994; 269: 6743-6752Abstract Full Text PDF PubMed Google Scholar).The C-terminal tail of the human prostacyclin receptor contains six resident cysteines, which have been extensively studied. Five of these cysteine residues are located within CAAX consensus motifs thought to comprise sites for lipid anchoring (e.g. palmitoylation or isoprenylation). Given that a good deal of evidence has already been presented for both palmitoylation as well as isoprenylation at these C-terminal cysteine sites on the human prostacyclin receptor (1Miggin S.M. Lawler O.A. Kinsella B.T. J. Biol. Chem. 2003; 278: 6947-6958Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 7Hayes J.S. Lawler O.A. Walsh M.T. Kinsella B.T. J. Biol. Chem. 1999; 274: 23707-23718Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we have opted to exclude this region from our current investigation. The remaining 12 cysteine residues within the extracellular and transmembrane domains of the hIP receptor were individually converted to alanine using site-directed PCR mutagenesis. We report that in addition to the highly conserved disulfide interaction between Cys-92 (top of transmembrane (TM) III) and Cys-170 (exoloop 2), an additional, putative non-conserved disulfide bridge may exist between Cys-5 (N terminus) and Cys-165 (exoloop 2), within the extracellular domain of the hIP, as well as other prostanoid receptors (hDP and hEP2). Moreover, this interaction seems to serve a critical, yet distinct purpose compared with its conserved counterpart. Furthermore, in the transmembrane domain Cys-118 (TMIII), Cys-202 (TMV), and Cys-251 (TMVI) were found to be necessary for preserving normal receptor binding affinity, activation capacity, and/or cell-surface expression through probable hydrogen bonding networks. Such observations provide further insights into the molecular functioning of the hIP receptor and contribute to the mechanistic understanding of how the hIP protein is stabilized during the continuum of conformational changes that occur upon agonist-induced activation.EXPERIMENTAL PROCEDURESMaterials—Radiolabeled [3H]iloprost (17.0 Ci/mmol) and nonradiolabeled iloprost were purchased from Amersham Biosciences. Oligonucleotide primers were purchased from Sigma-Genosys (The Woodlands, TX), whereas hIP cDNA was a generous gift from Dr. Mark Abramovitz (Merck Frosst, Quebec, Canada).Cysteine-to-Alanine Mutations—Twelve (within the extracellular and transmembrane domains) of a total of 18 cysteine residues were individually mutated to alanine (Fig. 1A). The choice of amino acid substitution was critical as cysteine-to-serine (Cys→ Ser) mutations in rhodopsin have been shown to have greater adverse effects on receptor conformation than cysteine-to-alanine (Cys→ Ala) substitutions (10Karnik S.S. Sakmar T.P. Chen H.B. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8459-8463Crossref PubMed Scopus (346) Google Scholar, 12Davidson F.F. Loewen P.C. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4029-4033Crossref PubMed Scopus (129) Google Scholar). Additionally, the C-terminal residues were not included in this study because these have been extensively characterized (1Miggin S.M. Lawler O.A. Kinsella B.T. J. Biol. Chem. 2003; 278: 6947-6958Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Human IP cDNA tagged at the C terminus with the 1D4 epitope tag (last 14 amino acids of rhodopsin) was cloned into the plasmid vector pMT4, and point mutations were generated using conventional methods of PCR mutagenesis as previously described (23Stitham J. Martin K.A. Hwa J. Mol. Pharmacol. 2002; 61: 1202-1210Crossref PubMed Scopus (35) Google Scholar). Ten microliters of the PCR product was used to transform competent DH5α Escherichia coli cells (∼2 × 109 cells) followed by DNA extraction from selected clones. Large plasmid preparations were performed using Wizard® Plus Maxiprep kits (Promega, Madison, WI), and all mutant constructs were confirmed via PCR DNA dideoxynucleotide chain-termination sequencing (Molecular Biology Core Facility, Dartmouth Medical School, Hanover, NH).Transfection of COS-1 Cells and Membrane Preparations—Transient transfections were performed on COS-1 cells as previously described (24Stitham J. Stojanovic A. Hwa J. J. Biol. Chem. 2002; 277: 15439-15444Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In brief, cells were incubated in DNA (10 or 20 μg/plate) in diethylaminoethyldextran (DEAE-dextran; Sigma) (0.2 mg/ml Dulbecco's modified Eagle's medium) and harvested 72 h post-transfection. Transfected COS-1 cells were washed in phosphate-buffered saline and harvested. Vortexing (providing shear forces) for 3 min in sucrose (0.25 m) was followed by low speed spin (∼1260 × g) for 5 min, and the supernatant was collected. After a high speed centrifugation (∼30,000 × g for 15 min) the pellet was then washed twice in 1× HEM (20 mm Hepes, pH 7.4, 1.5 mm EGTA, and 12.5 mm MgCl2) followed by re-suspension in 1× HEM containing 10% glycerol and stored at -70 °C (25Stitham J. Stojanovic A. Merenick B.L. O'Hara K.A. Hwa J. J. Biol. Chem. 2003; 278: 4250-4257Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Either Bradford or BCA protein assays were performed to quantitate membrane proteins.Ligand Binding Affinity—Ligand binding characteristics for the expressed receptors were initially determined through a series of competition binding assays using the radiolabeled ligand [3H]iloprost. Analysis involved construction of reaction mixtures (in duplicate wells) containing 50 μg of membrane, HEM buffer, and 15 nm [3H]iloprost along with 1 of 11 different concentrations of cold (nonradiolabeled) iloprost ranging from 10 μm to 0.1 nm. After 1.5 h of incubation at 4 °C, reactions were stopped by the addition of ice-cold 10 mm Tris/HCl buffer, pH 7.4, and filtered onto Whatman® GF/C glass-fiber filters using a Brandel® cell harvester. The filters were washed 5 times with ice-cold Tris/HCl buffer, and radioactivity was measured in the presence of 5 ml of Ecoscint™ H scintillation fluid (National Diagnostics, Atlanta, GA). Nonspecific binding was determined by the addition of a 500-fold excess of nonradiolabeled iloprost. Data were analyzed using GraphPad Prism® software (GraphPad software, Inc., San Diego, CA). IC50 values were converted to Ki using the Cheng-Prusoff equation, and Ki values were expressed as a mean ± S.E. For saturation binding experiments to determine Bmax and KD, the concentration of [3H]iloprost was varied from 1 to 100 nm. Nonspecific binding was determined by the addition of a 500-fold excess of nonradiolabeled iloprost. Data were analyzed using GraphPad Prism® software (GraphPad Software). Analysis of variance and Student's t tests were used to determine significant differences (p < 0.05).Receptor Activation cAMP Determination—The wild-type and cysteine mutant constructs were analyzed for signal transduction capabilities. COS-1 cells were transiently transfected with 2 μg of receptor DNA in 25-mm plates as described above. [3H]cAMP was used in competition for a cAMP-binding protein against known concentrations of nonradiolabeled 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 Ecoscint™ H (National Diagnostics). Results were analyzed with GraphPad Prism® software. For the dose response, a non-linear, curve-fitting program (GraphPad Prism®) was used, and the EC50 (mean ± S.E.) was determined for wild-type hIP1D4 and mutant constructs. Analysis of variance and Student's t tests were used to determine statistically significant differences (p < 0.05).Confocal Immunofluorescence Microscopy—COS-1 cells were seeded into six-well tissue culture plates containing sterilized poly-l-lysine (Sigma)-treated glass coverslips and transiently transfected with 1.0 μg/ml wild-type or mutant (C5A, C92A, C165A, and C170A) hIP DNA according to the aforementioned transfection protocol. Cells were fixed and permeabilized 48 h post-transfection in ice-cold methanol. Prostacyclin receptors were labeled using anti-1D4 monoclonal antibody (C-terminal-tagged hIP), 1:1000 dilution, for 60-90 min followed by goat anti-mouse IgG F(ab′)2 AlexaFluor®568 fluorescent antibody (Molecular Probes, Inc.) 1:200 dilution, for 60-90 min. The endoplasmic reticulum was labeled using anti-calnexin polyclonal antibody (StressGen Biotechnologies), 1:400 dilution, for 60-90 min followed by goat anti-rabbit IgG AlexaFluor®488 fluorescent antibody (Molecular Probes), 1:200 dilution, for 60-90 min. Cells were post-fixed with 4% paraformaldehyde (Fluka) for 5 min, mounted with ProLong Gold with 4′, 6-diamidino-2-phenylindole, and examined via confocal microscopy using a Zeiss LSM 510 Meta Laser Scanning Confocal Microscope System (i.e. inverted Zeiss Axiovert 200 microscope with two conventional photomultipliers and one meta polychromatic multi-channel detector) (Carl Zeiss Microimaging, Inc., Thornwood, NY). Representative cells were selected, and images were captured at high resolution (63×) for cytoplasmic or plasma membrane localization.Molecular Modeling of hIP Receptor—A theoretical, three-dimensional homology model of the seven transmembrane α-helices of the hIP was constructed using the internet-based protein-modeling server, SWISS-MODEL (GlaxoSmithKline) (26Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9472) Google Scholar). The homology model was generated using the 2.8-Å-resolution x-ray crystallographic structure of bovine rhodopsin as the template (PDB code 1HZX). The transmembrane domains were energy-minimized, utilizing the Gromos96 force field to improve the stereochemistry of the model and remove unfavorable clashes (SWISS-MODEL). Polypeptide chains corresponding to the extracellular and cytoplasmic loop regions were manually constructed. The large C-terminal tail region of the hIP was excluded from this particular model. Initial torsion angles were derived from full polypeptide secondary structure prediction using the JPred multiple sequence alignment consensus server (27Cuff A.a.C.M.E.J. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (916) Google Scholar). The subsequent loop peptides were energy-minimized using the NAMD molecular dynamics simulator (28Phillips J.C. Braun R. Wang W. Gumbart J. Tajkhorshid E. Villa E. Chipot C. Skeel R.D. Kale L. Schulten K. J. Comput. Chem. 2005; 26: 1781-1802Crossref PubMed Scopus (12926) Google Scholar). Resulting structures were sequentially attached to the transmembrane homology model. Compiled structures were energy-minimized using NAMD, including constraints for either 1) no extracellular disulfide bonds, 2) a single, conserved extracellular disulfide bond (Cys-92-Cys-170), or 3) dual extracellular disulfide bonds (Cys-92-Cys-170 and Cys-5-Cys-165). This latter structure, with dual extracellular (disulfide) constraints, yielded the lowest energy conformation and was utilized as the preferred hIP receptor model for subsequent comparisons. Although our model was a useful first pass tool in providing preliminary insights and predictions of sulfhydryl hIP structure function, biochemical and molecular pharmacological techniques were necessary to confirm our hypothesis.RESULTSOur current knowledge of the hIP receptor is limited. Of particular interest has been the role of cysteine residues within the extracellular and TM domains of the protein. Cysteines contain a highly nucleophilic sulfhydryl or thiol (SH) side chain that is capable of acting as a nucleophilic catalyst. Moreover, with a pKa of ∼8, the chemical reactivity of cysteine sulfhydryls is easily modified by environmental conditions. Within a reducing environment, cysteine residues may be involved in bioconjugation reactions, whereas conversely, within an oxidizing environment, side chains may dimerize to form disulfide (S-S) linkages. These latter interactions play an invaluable role in intramolecular cross-linking of proteins that can increase molecular stability within harsh environments as well as confer resistance to proteolytic cleavage. We wished to elucidate the differential contributions of conserved and non-conserved cysteines within the transmembrane region of the hIP receptor as well as explore the potential existence of a second, putative non-conserved disulfide bond within the extracellular domain.Extracellular Cysteines Are Required for Receptor Trafficking, Binding, and Activation—Site-directed mutagenesis was performed targeting all cysteine residues present within the hIP receptor, excluding those located in the C-terminal tail. Thus, 12 of the total 18 cysteine residues (Table 1, Fig. 1A) were individually mutated to alanine (Cys→ Ala) and assessed for functional effects on agonist binding (affinity) and receptor activation (potency), as measured via cAMP production. Binding affinity (Ki) could not be detected using competition binding (15 nm [3H]iloprost) for individual mutations of all four extracellular cysteines (C5A, C92A, C165A, and C170A) (Table 1, Fig. 2A). Only with saturation binding using increasing concentrations of [3H]iloprost could binding affinities (Table 2, Fig. 2B) be determined. These binding affinities are estimates due an inability to saturate the expressed receptors (Fig. 2B). They nevertheless indicate severe defects in binding affinity. Related to the severe binding defects, potency (EC50) values could not be determined due to negligible efficacy (Table 1). These results demonstrate the severity of functional perturbation with mutation of any one of the four extracellular cysteine residues within the hIP and lend preliminary support to the notion that 1) the highly conserved Cys-92 and Cys-170 may form a disulfide bond (Fig. 1B), and 2) an alternative interaction may exist between the remaining extracellular cysteines Cys-5 and Cys-165 (perhaps a second disulfide bond), which appears to be equally important for hIP receptor binding and activation.TABLE 1Functional comparison of binding affinity (Ki from competition binding using 15 nm [3H]iloprost and 1 μg of DNA/ml transfection solution) and dose-response (EC50), characteristics for wild-type hIP and cysteine-to-alanine mutationsKi ± S.E. (n)EC50 ± S.E. (n)Expression pmol/mg of proteinnmnmWild type7.9 ± 1.7 (9)1.2 ± 0.1 (10)2.1 ± 0.5C5AND (3)ap < 0.001.ND (3)ap < 0.001.0.2 ± 0.1ap < 0.001.C92AND (4)ap < 0.001.ND (3)ap < 0.001.0.6 ± 0.2bp < 0.05.C118AND (4)ap < 0.001.ND (3)ap < 0.001.0.5 ± 0.2bp < 0.05.C135A6.4 ± 1.1 (3)0.9 ± 0.3 (3)1.5 ± 0.5C147A5.3 ± 2.1 (3)1.2 ± 0.3 (4)2.1 ± 0.9C151A8.9 ± 2.3 (3)1.9 ± 0.6 (4)1.4 ± 0.6C165AND (4)ap < 0.001.ND (3)ap < 0.001.0.5 ± 0.2bp < 0.05.C170AND (4)ap < 0.001.ND (3)ap < 0.001.0.8 ± 0.3bp < 0.05.C202A4.4 ± 1.7 (3)1.2 ± 0.4 (3)0.4 ± 0.1bp < 0.05.C211A9.4 ± 2.3 (3)0.9 ± 0.1 (3)1.4 ± 0.4C251AND (4)ap < 0.001.3.1 ± 0.2 (4)bp < 0.05.0.4 ± 0.2bp < 0.05.C259A11.3 ± 2.4 (3)0.8 ± 0.2 (3)3.2 ± 0.3a p < 0.001.b p < 0.05. Open table in a new tab FIGURE 2Determination of ligand binding affinity (Ki) for wild-type and extracellular cysteines. Competition binding curves using identical amounts of membrane protein are shown. Raw counts (cpm) are plotted against increasing concentrations of iloprost. Panel A, wild-type (WT, ▪) competition binding performed in parallel with the four extracellular cysteine mutants, C92A (gray diamond), C170A (gray square), C5A (black diamond), and C165A (black inverted triangle). Panel B, saturation binding studies on C92A (gray diamond), C170A (gray square), C5A (black diamond), and C165A (black inverted triangle) using increased DNA transfection and six different concentrations of [3H]iloprost in duplicate. Each graph is the composition of at least three separate experiments. Shown in Table 2 are the corresponding results for KD. Panel C, corresponding confocal microscopy (63× resolution) images for each mutation showing predominant membrane trafficking only for wild-type protein. The hIP receptors, both wild type and mutants, are in red (1D4 monoclonal antibody). The endoplasmic reticulum is shown in green (anti-calnexin antibody), and the overlay additionally has blue nuclear staining (4′, 6-diamidino-2-phenylindole) and a phase contrast microscopic image of the cell to localize the cells perimeter. Red arrows are used to localize areas of cell surface membrane.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Binding affinity (Kd) characteristics for wild-type hIP and the four extracellular cysteine-to-alanine mutations in which binding affinity was not able to be detected using standard competition binding (Table 1)Kd ± S.E. (n)nMWild type13.4 ± 2.6 (10)C5A121.5 ± 29.4ap < 0.001. (4)C92A113.9 ± 31.4ap < 0.001. (3)C165A142.4 ± 18.0ap < 0.001. (6)C170A140.0 ± 14.2ap < 0.001. (4)a p < 0.001. Open table in a new tab Estimates of cell surface trafficking using saturation binding on plasma membrane preparations confirmed decreased plasma membrane expression for all four extracellular Cys→ Ala substitutions compared with wild-type hIP (Table 1). The additional use of confocal microscopy corroborated reduced cell surface expression and demonstrated defective trafficking with endoplasmic reticulum co-localization (calnexin) and retention (Fig. 2C). Wild-type hIP predominantly trafficked to the cell surface as observed with the red fluorescence surrounding the cell in the phase contrast micrograph overlay (Fig. 2C). Because of the nature of overexpression systems, some wild-type hIP was also detectable in the endoplasmic reticulum as observed with co-localization with the green fluorescence. Both C5A and C165A showed a marked reticular pattern co-localized predominantly to the endoplasmic reticulum, with reduced cell surface. C92A and C170A were also found predominantly in an endoplasmic reticulum location (perinuclear), again with reduced cell surface expression. Thus, in conjunction with the findings from previous binding and activation studies, these results also show defects for the four extracellular cysteines, suggesting that there may be dual functional connections between the highly conserved Cys-92-Cys-170 and the less conserved Cys-5-Cys-165.Sequence Alignments and Treatment with Reducing Agents Suggest Formation of Dual Extracellular Disulfide Bonds (Cys-5 to Cys-165 and Cys-92 to Cys-170)—In addition to our initial experimental findings, further supportive evidence that a second disulfide bond exists between the non-conserved extracellular cysteine residues Cys-5 and Cys-165 can be found in human prostanoid receptor sequence alignments (Fig. 1B), which shows that both residues are either exclusively present or exclusively absent within the various prostanoid receptors. One of the cysteines (Cys-5 or Cys-165) is always found in the presence of the other. Thus, at equivalent positions within the human IP, EP2, and DP receptors, both the Cys-5 and Cys-165 residues are found, whereas conversely, at equivalent positions within the human EP1, EP3, EP4, TP, and FP receptors, both cysteines are absent (Fig. 1B). In contrast, Cys-92 and Cys-170 are conserved at equivalent positions among all the prostanoid receptors, suggesting that Cys-92 interacts with Cys-170, and (when present) Cys-5 interacts with Cys-165.If critical disulfide (S-S) bonds are present within the extracellular domain of the hIP receptor, then chemical reduction of the wild-type receptor should be associated with defects in structure (folding-conformation-stability) and function (binding and activation). Competition binding was performed in the presence of increasing concentrations of 1.4-140 mm β-mercaptoethanol (β-ME; Fig. 3A) and 1-100 mm 1,4-dithiothreitol (Fig. 3B), which break disulfide (S-S) bonds and maintain sulfhydryl (SH) groups in the reduced state. As can be viewed in Fig. 3, a sequential decrease in agonist binding was observed for wild-type hIP receptors treated with increasing concentrations of either 1,4-dithiothreitol or β-ME. The significant difference and decline in raw counts (specific binding) in response to increasingly higher concentrations of reducing agent is indicative of an increasing pool of defective IP (i.e. reduced S-S bonds to SH). Clear sigmoidal competition binding curves suggest a population of receptors in which the disulfide bond remained intact. As shown, the IC50 values, measured in log-fold molar concentrations of iloprost agonist, log[iloprost](m), remained unchanged for wild-type hIP -7.4 ± 0.2 log m (untreated), -7.4 ± 0.2 log m (1.4 mm β-ME), -7.6 ± 0.3 log m (14 mm β-ME), and -7.8 ± 0.4 log m (140 mm β-ME) (Fig. 3A). A corresponding decrease in cAMP signaling levels was also observed, demonstrating a parallel reduction in the levels of functioning receptor (Fig. 4B) with no significant change in EC50 -9.3 ± 0.4 log m (wild-type hIP-untreated), -9.4 ± 0.8 log m (140 mm β-ME), and -9.5 ± 0.8 log m (14 mm β-ME). Additional evidence, albeit indirect, for the presence of dual disulfide bonds can be gleaned from the observation that the low levels of specific binding for both the C92A and C170A mutations quickly dissipates upon the addition of β-mercaptoethanol (Fig. 5), implying further reduction of a" @default.
• W2040489780 created "2016-06-24" @default.
• W2040489780 creator A5003763243 @default.
• W2040489780 creator A5004307066 @default.
• W2040489780 creator A5053908198 @default.
• W2040489780 creator A5077834308 @default.
• W2040489780 creator A5012292085 @default.
• W2040489780 date "2006-12-01" @default.
• W2040489780 modified "2023-10-17" @default.
• W2040489780 title "Versatility and Differential Roles of Cysteine Residues in Human Prostacyclin Receptor Structure and Function" @default.
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