Spatial Approximation between the Amino Terminus of a Peptide Agonist and the Top of the Sixth Transmembrane Segment of the Secretin Receptor
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310407200
ISSN1083-351X
AutoresMaoqing Dong, Zhijun Li, Delia I. Pinon, Terry P. Lybrand, Laurence J. Miller,
Tópico(s)Photochromic and Fluorescence Chemistry
ResumoDistinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr10]secretin-27) and -2 (rat [Bpa-2,Gly-1,Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation. Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr10]secretin-27) and -2 (rat [Bpa-2,Gly-1,Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation. The secretin receptor is prototypic of the Class B family of guanine nucleotide-binding protein (G protein)-coupled receptors which includes many important drug targets. Understanding of the molecular basis of ligand binding of receptors is important for the rational design of receptor-active drugs. However, the molecular basis of ligand binding of Class B receptors is less well understood than that of members of the Class A family, such as rhodopsin and the adrenergic receptor. This likely reflects the facts that natural ligands for Class B G protein-coupled receptors are relatively large peptides with diffuse phamacophoric domains and that these receptors have long and complex amino-terminal domains that are important for binding. Both of these interacting domains are flexible, with active conformations that have not been clearly defined. One of the distinct characteristics of the Class B receptor family is the long amino terminus that exceeds 120 residues in length. It contains 6 conserved Cys residues that have been demonstrated to form intradomain disulfide bonds (1.Qi L.J. Leung A.T. Xiong Y. Marx K.A. Abou-Samra A.B. Biochemistry. 1997; 36: 12442-12448Crossref PubMed Scopus (69) Google Scholar, 2.Perrin M.H. Fischer W.H. Kunitake K.S. Craig A.G. Koerber S.C. Cervini L.A. Rivier J.E. Groppe J.C. Greenwald J. Moller N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 3.Grauschopf U. Lilie H. Honold K. Wozny M. Reusch D. Esswein A. Schafer W. Rucknagel K.P. Rudolph R. Biochemistry. 2000; 39: 8878-8887Crossref PubMed Scopus (106) Google Scholar, 4.Bazarsuren A. Grauschopf U. Wozny M. Reusch D. Hoffmann E. Schaefer W. Panzner S. Rudolph R. Biophys. Chem. 2002; 96: 305-318Crossref PubMed Scopus (74) Google Scholar, 5.Asmann Y.W. Dong M. Ganguli S. Hadac E.M. Miller L.J. Mol. Pharmacol. 2000; 58: 911-919Crossref PubMed Scopus (39) Google Scholar) and to be critical for ligand binding. These disulfide bonds could provide key constraints for building a model of the secretin receptor, but definitive mapping of these bonds in an active receptor has not yet been achieved. A bonding pattern has been proposed for three other Class B G protein-coupled receptors, binding parathyroid hormone (PTH) 1The abbrevations used are: PTH, parathyroid hormone; Bpa, p-benzoyl-l-phenylalanine; Bpa1 probe, rat lsqb]Bpa1,Tyr10]secretin-27; Bpa-1 probe, rat [Bpa-1,Tyr10]secretin-27; Bpa-2 probe, rat [Bpa-2,Gly-1,Tyr10]secretin-27; (BzBz)Lys, p-benzoylbenzoyl-l-lysine; CHO-SecR, secretin receptor-bearing Chinese hamster ovary cell line; ECL, extracellular loop domain; HPLC, high performance liquid chromatography; ICL, intracellular loop domain; Lys-C, endoproteinase Lys-C; MES, 4-morpholineethanesulfonic acid; TM, transmembrane domain. (3.Grauschopf U. Lilie H. Honold K. Wozny M. Reusch D. Esswein A. Schafer W. Rucknagel K.P. Rudolph R. Biochemistry. 2000; 39: 8878-8887Crossref PubMed Scopus (106) Google Scholar), glucagon-like peptide (4.Bazarsuren A. Grauschopf U. Wozny M. Reusch D. Hoffmann E. Schaefer W. Panzner S. Rudolph R. Biophys. Chem. 2002; 96: 305-318Crossref PubMed Scopus (74) Google Scholar), and corticotropin-releasing factor (2.Perrin M.H. Fischer W.H. Kunitake K.S. Craig A.G. Koerber S.C. Cervini L.A. Rivier J.E. Groppe J.C. Greenwald J. Moller N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), based on analysis of the refolded nonglycosylated amino-terminal peptides of these receptors that had been produced in Escherichia coli. It should be noted that the pattern of disulfide bonds obtained from the refolded amino-terminal extracellular domain of the corticotropin-releasing factor is different from that predicted by mutagenesis of that entire receptor (1.Qi L.J. Leung A.T. Xiong Y. Marx K.A. Abou-Samra A.B. Biochemistry. 1997; 36: 12442-12448Crossref PubMed Scopus (69) Google Scholar). Like that of other members of the Class B family, the complex amino terminus of the secretin receptor has been demonstrated to play a critical role in ligand binding by mutagenesis and chimeric receptor analysis (6.Holtmann M.H. Hadac E.M. Miller L.J. J. Biol. Chem. 1995; 270: 14394-14398Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 7.Holtmann M.H. Ganguli S. Hadac E.M. Dolu V. Miller L.J. J. Biol. Chem. 1996; 271: 14944-14949Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We have taken a more direct approach to understanding the molecular basis of secretin binding to its receptor, utilizing intrinsic photoaffinity labeling. This approach can provide information regarding spatial approximations between photolabile residues situated within a ligand and residues adjacent to these when docked at the receptor. We have used a series of probes with a photolabile residue (p-benzoyl-l-phenylalanine (Bpa) or p-benzoylbenzoyl-l-lysine (BzBz)Lys)) in different regions of the peptide, in positions 6, 12, 13, 14, 18, 22, and 26 (8.Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9.Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10.Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11.Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar, 12.Zang M. Dong M. Pinon D.I. Ding X.Q. Hadac E.M. Li Z. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar, 13.Dong M. Li Z. Zang M. Pinon D.I. Lybrand T.P. Miller L.J. J. Biol. Chem. 2003; 278: 48300-48312Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Interestingly, all of these probes have been demonstrated to label residues within the amino-terminal domain of the secretin receptor, with six probes labeling the first 40 residues of the amino terminus (8.Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9.Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10.Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11.Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35) Google Scholar, 13.Dong M. Li Z. Zang M. Pinon D.I. Lybrand T.P. Miller L.J. J. Biol. Chem. 2003; 278: 48300-48312Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and one labeling a residue close to the first transmembrane segment (12.Zang M. Dong M. Pinon D.I. Ding X.Q. Hadac E.M. Li Z. Lybrand T.P. Miller L.J. Mol. Pharmacol. 2003; 63: 993-1001Crossref PubMed Scopus (36) Google Scholar). This strongly supports a critical role for the amino terminus of the secretin receptor in ligand binding. However, mutagenesis data have shown that, in addition to the amino-terminal region of the secretin receptor, the extracellular loop regions play complementary roles that affect ligand binding. It is not known whether such effects are direct, interacting with the ligand, or indirect, interacting with other portions of the receptor itself. Because the distal amino-terminal region of secretin has been shown to be functionally important and has not yet been studied directly by photoaffinity labeling, in the current work we focused on this region as a possible site of interaction with the body of the receptor. The amino terminus of PTH, the natural ligand of another Class B G protein-coupled receptor, has been shown to interact with the top of the sixth transmembrane segment (TM6) of that receptor (14.Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: 22498-22505Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 15.Behar V. Bisello A. Bitan G. Rosenblatt M. Chorev M. J. Biol. Chem. 2000; 275: 9-17Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, incorporation of a photolabile residue at the amino terminus of secretin may be challenging because of the critical importance of His1 in structure-activity studies (16.Hefford M.A. Kaplan H. Biochim. Biophys. Acta. 1989; 998: 267-270Crossref PubMed Scopus (21) Google Scholar). Therefore, in addition to replacing His1 with a photolabile Bpa (rat [Bpa1,Tyr10]secretin-27), we also placed a Bpa at the amino-terminal extension of secretin, in positions -1 (rat [Bpa-1,Tyr10]secretin-27) and -2 (rat [Bpa-2,Gly-1,Tyr10]secretin-27), to minimize possible negative functional impact. In this work, we have successfully synthesized three photolabile analogs and have shown that they are full agonists that bind to the secretin receptor specifically and saturably and that they are able to label this receptor efficiently. All three probes consistently labeled receptor residues in a distinct region that included the top of the secretin receptor TM6. Together with other photoaffinity labeling data we reported previously, these constraints were built into a three-dimensional model of the agonist-bound secretin receptor. These findings add important constraints to the docking of secretin to its receptor and may suggest a mechanism for ligand binding and activation which may be shared by other members of the Class B family of G protein-coupled receptors. Materials—Cyanogen bromide (CNBr), solid phase oxidant, N-chloro-benzenesulfonamide (IODO-BEADS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS), and acetic anhydride were purchased from Pierce. Endoproteinase Lys-C was from Calbiochem. 3-Isobutyl-1-methylxanthine and N-(2-aminoethyl-1)-3-aminopropyl glass beads were from Sigma. Endoglycosidase F was produced in our laboratory (17.Pearson R.K. Miller L.J. Hadac E.M. Powers S.P. J. Biol. Chem. 1987; 262: 13850-13856Abstract Full Text PDF PubMed Google Scholar). All other reagents were analytical grade. Peptides—The probes, rat [Bpa1,Tyr10]secretin-27 (Bpa1 probe), rat [Bpa-1,Tyr10]secretin-27 (Bpa-1 probe), and rat [Bpa-2,Gly-1,Tyr10]secretin-27 (Bpa-2 probe), were designed to contain a Tyr residue at position 10 for radioiodination and a photolabile residue, Bpa, in positions near the amino terminus, for covalent labeling of the secretin receptor. These three probes and other peptides used in this study, i.e. rat secretin-27 and the peptide used for radiolabeling, rat [Tyr10]secretin-27, were synthesized by manual solid phase techniques and purified to homogeneity by reversed-phase HPLC, as described previously (18.Powers S.P. Pinon D.I. Miller L.J. Int. J. Pept. Protein Res. 1988; 31: 429-434Crossref PubMed Scopus (99) Google Scholar). Their chemical identities were established by mass spectrometry. The above three photolabile probes and the radioligand used in receptor binding studies were radioiodinated oxidatively with Na 125I upon exposure to an IODO-BEAD for 15 s and purified by reversed-phase HPLC to yield specific radioactivities of 2,000 Ci/mmol (18.Powers S.P. Pinon D.I. Miller L.J. Int. J. Pept. Protein Res. 1988; 31: 429-434Crossref PubMed Scopus (99) Google Scholar). Receptor Preparations—Secretin receptor-bearing Chinese hamster ovary cell line (CHO-SecR), which has been previously established and characterized (19.Ulrich C.D. Pinon D.I. Hadac E.M. Holicky E.L. Chang-Miller A. Gates L.K. Miller L.J. Gastroenterology. 1993; 105: 1534-1543Abstract Full Text PDF PubMed Scopus (68) Google Scholar), was used as source of wild type receptors for the current study. In this work, it was necessary to develop new secretin receptor mutants that incorporated additional sites for CNBr cleavage in the first extracellular loop (ECL1) and TM6. These represented Ala175 to Met (A175M), Leu324 to Met (L324M), Ile331 to Met (I331M), and Ile334 to Met (I334M) receptor constructs. In addition, two dual mutants were developed to introduce a new CNBr cleavage site while simultaneously eliminating a naturally occurring Met residue, one representing Ile331 to Met, Met344 to Ile (I331M/M344I), with another one representing Ile334 to Met, Met344 to Ile (I334M/M344I). These mutants were prepared using an oligonucleotide-directed approach with the QuikChange™ site-directed mutagenesis kit from Stratagene, with the products verified by direct DNA sequencing (20.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). A CHO cell line stably expressing the I334M/M344I construct was produced using the same methodology utilized previously (21.Hadac E.M. Ghanekar D.V. Holicky E.L. Pinon D.I. Dougherty R.W. Miller L.J. Pancreas. 1996; 13: 130-139Crossref PubMed Scopus (101) Google Scholar). This cell line and the CHO-SecR line were cultured at 37 °C in a 5% CO2 environment on Falcon tissue culture plastic ware in Ham's F-12 medium supplemented with 5% fetal clone-2 (Hyclone Laboratories, Logan, UT). Cells were passaged twice a week and lifted mechanically before use. The A175M, L324M, I331M, I334M, and I331M/M344I mutant receptor constructs were expressed transiently on COS cells (American Type Cell Collection, Manassas, VA) after transfection using a modification of the DEAE-dextran method (7.Holtmann M.H. Ganguli S. Hadac E.M. Dolu V. Miller L.J. J. Biol. Chem. 1996; 271: 14944-14949Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These cells were harvested mechanically 72 h after transfection. Receptor-enriched plasma membranes were prepared from these cell lines using methods that we reported previously (21.Hadac E.M. Ghanekar D.V. Holicky E.L. Pinon D.I. Dougherty R.W. Miller L.J. Pancreas. 1996; 13: 130-139Crossref PubMed Scopus (101) Google Scholar). Biological Activity Assay—The agonist activities of the Bpa1, Bpa-1, and Bpa-2 probes were studied for stimulation of cAMP activity in the CHO-SecR cells using a competitive binding assay (Diagnostic Products Corporation, Los Angeles, CA). Cells were stimulated with the secretin analogs at 37 °C for 30 min, and the reaction was stopped by adding ice-cold perchloric acid. After adjusting the pH to 6 with KHCO3, cell lysates were cleared by centrifugation at 3,000 rpm for 10 min, and the supernatants were used in the assay, as described previously (22.Ganguli S.C. Park C.G. Holtmann M.H. Hadac E.M. Kenakin T.P. Miller L.J. J. Pharmacol. Exp. Ther. 1998; 286: 593-598PubMed Google Scholar). Radioactivity was quantified by scintillation counting in a Beckman LS6000. This assay was also used to characterize functionally the new receptor mutants expressed transiently in COS cells. Ligand Binding—Binding to the secretin receptor was characterized in a standard assay using membranes from the CHO-SecR cell line as source of receptor. Membranes (5 μg) were incubated with a constant amount of radioligand, rat 125I-[Tyr10]secretin-27 (5 pm), in the presence of increasing concentrations of rat [Bpa1,Tyr10]secretin-27, rat [Bpa-1,Tyr10]secretin-27, or rat [Bpa-2,Gly-1,Tyr10]secretin-27 (0–1 μm) for 1 h at room temperature in Krebs-Ringer-HEPES (KRH) medium (25 mm HEPES, pH 7.4, 104 mm NaCl, 5 mm KCl, 1 mm KH2PO4, 1.2 mm MgSO4,2mm CaCl2,1mm phenylmethylsulfonyl fluoride, 0.01% soybean trypsin inhibitor) containing 0.2% bovine serum albumin. Bound and free radioligand were separated using a Skatron cell harvester (Molecular Devices, Sunnyvale, CA) with glass fiber filtermats that had been soaked in 0.3% Polybrene for 1 h, and bound radioactivity was quantified in a γ-spectrometer. Nonspecific binding was determined in the presence of 1 μm secretin and represented <20% of total binding. The same assay was also utilized to characterize the binding activity of the new receptor mutants transiently expressed in COS cells. Photoaffinity Labeling of the Secretin Receptor—For covalent labeling studies, membranes from receptor-bearing CHO cells containing ∼50 μg of protein were incubated with 0.1 nm rat 125I-[Bpa1,Tyr10]secretin-27 or rat 125I-[Bpa-1,Tyr10]secretin-27 or rat 125I-[Bpa-2,Gly-1,Tyr10]secretin-27 in the presence of increasing concentrations of secretin (0–1 μm)for1hat room temperature prior to photolysis for 30 min at 4 °C in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) equipped with 3,500-Å lamps. To scale up receptor for purification, a larger amount of membranes (150–200 μg) was incubated with 0.5 nm rat 125I-[Bpa1,Tyr10]secretin-27 or rat 125I-[Bpa-1,Tyr10]secretin-27 or rat 125I-[Bpa-2,Gly-1,Tyr10]secretin-27 in the absence of competing secretin. After photolysis, membranes were washed, pelleted, solubilized in SDS sample buffer, and applied to a 10% SDS-polyacrylamide gel for electrophoresis (23.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Radiolabeled bands were detected by autoradiography. Identification of the Labeled Receptor Domains—Radioactive receptor bands were cut out from the gel and homogenized in water in a Dounce homogenizer, followed by elution, lyophilization, and ethanol precipitation. This material was used for chemical or enzymatic cleavage experiments. Aliquots of affinity labeled secretin receptor were deglycosylated with endoglycosidase F, as described previously (21.Hadac E.M. Ghanekar D.V. Holicky E.L. Pinon D.I. Dougherty R.W. Miller L.J. Pancreas. 1996; 13: 130-139Crossref PubMed Scopus (101) Google Scholar). CNBr and endoproteinase Lys-C were used separately or in sequence to cleave the labeled secretin receptor or its mutants using procedures described previously (8.Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The products of cleavage were resolved on 10% NuPAGE gels using MES running buffer (Invitrogen). After electrophoresis, labeled bands were identified by exposure to x-ray film with intensifying screens at -80 °C. In addition to peptide mapping by chemical and enzymatic cleavage, as described above, identification of the secretin receptor fragments affinity-labeled with the Bpa-1 and Bpa-2 probes was also achieved by Edman degradation sequencing of the purified CNBr fragments of the labeled receptor. For this, plasma membranes (10 mg) were photoaffinity labeled with 0.1 μm rat [Bpa-1,Tyr10]secretin-27 or rat [Bpa-2,Gly-1,Tyr10] secretin-27. These materials were mixed with 1-mg plasma membranes labeled with 1 nm rat 125I-[Bpa-1,Tyr10] secretin-27 or rat 125I-[Bpa-2,Gly-1,Tyr10] secretin-27 and were gel purified by the procedure described above. Labeled receptors were cleaved by CNBr and the resulting fragments were purified by gel electrophoresis followed by elution, lyophilization, and acetone precipitation. Purified fragments were then injected onto a PerkinElmer Life Sciences/Brownlee microbore C-18 HPLC column, utilizing a flow rate of 5 μl/min and 10 min of 1% solution B, followed by a linear gradient up to 80% solution B over 190 min (A, 0.1% trifluoroacetic acid, and B, 0.085% trifluoroacetic acid and acetonitrile). The separation was monitored at 280 nm with a UV absorbance detector. The eluant from this microbore column was collected directly onto a polyvinylidene difluoride membrane strip that was subsequently exposed to x-ray film for detection of the position of the radioactive peak. The peak area was excised from the membrane and subjected to Edman degradation sequencing using an Applied Biosystems automated instrument. Identification of Labeled Receptor Residues—Considering that the photolabile residue Bpa was located in the first position of the secretin analogs used in this study, caution was required to prevent cleavage of the Bpa in the first cycle of radiochemical Edman degradation sequencing of the attached receptor fragment. This was achieved by acetylation of the free amino group of the photolabile probe using acetic anhydride after photoaffinity labeling of the receptor, but prior to CNBr cleavage. For this, gel-purified and ethanol-precipitated labeled secretin receptor was dissolved in 200 μl of 0.1 m phosphate buffer (pH 7.8) containing 0.1% SDS. 20 μl of acetic anhydride was added in 5-μl aliquots over the course of 1 h, and the reaction was incubated for an additional hour with stirring at room temperature in the dark. Acetylated materials were ethanol precipitated prior to CNBr cleavage. After gel purification, the CNBr fragment was used for Edman degradation to localize the receptor residues labeled by these probes. Radiochemical sequencing was performed on cysteine-containing CNBr fragments of the receptor which were immobilized through sulfhydryl groups using the bifunctional cross-linker, Sulfo-MBS, and N-(2-aminoethyl-1)-3-aminopropyl glass beads. Edman degradation was manually repeated in a manner that has been reported previously in detail (24.Ji Z. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 25.Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R.M. Lybrand T.P. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and the radioactivity released in each cycle was quantified in a γ-spectrometer. Statistical Analysis—All observations were repeated at least three times in independent experiments and are expressed as the means ± S.E. Binding curves were analyzed and plotted using the nonlinear regression analysis routine for radioligand binding in the Prism software package (GraphPad Software, San Diego). Binding kinetics was determined by analysis with the LIGAND program of Munson and Rodbard (26.Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Molecular Modeling—Homology modeling techniques were employed to model both the receptor amino-terminal region and transmembrane region. The two regions were then connected guided by experimental data reported previously (27.Pellegrini M. Bisello A. Rosenblatt M. Chorev M. Mierke D.F. Biochemistry. 1998; 37: 12737-12743Crossref PubMed Scopus (65) Google Scholar). A detailed description of the receptor modeling has been reported elsewhere (13.Dong M. Li Z. Zang M. Pinon D.I. Lybrand T.P. Miller L.J. J. Biol. Chem. 2003; 278: 48300-48312Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Here we give a brief summary. For modeling of the amino-terminal region, sequence data bases were searched for homologs of each of the closely related Class B family amino-terminal domains using FASTA3 (28.Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar). Homologs with experimentally determined three-dimensional structures were assessed for suitability as templates in a homology modeling exercise, using structure-based sequence alignment of the secretin receptor amino terminus incorporated in MOE (29.Chemical Computing Group, Inc.MOE 2001.01. Chemical Computing Group, Inc., Montreal, Canada1997–2001Google Scholar). Three-dimensional homology models for the rat secretin receptor amino terminus were then built using Modeler (30.Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). Final models were assessed for stereochemical quality and side chain packing profiles using the PROCHECK (31.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and QPACK programs (32.Gregoret L.M. Cohen F.E. J. Mol. Biol. 1990; 211: 959-974Crossref PubMed Scopus (119) Google Scholar). The first eight residues of the amino-terminal region were then built into the homology model as an extended conformation, and the rest of carboxyl-terminal residues (109–122) were modeled as two α-helices approximately perpendicular to each other as suggested by experimental data (27.Pellegrini M. Bisello A. Rosenblatt M. Chorev M. Mierke D.F. Biochemistry. 1998; 37: 12737-12743Crossref PubMed Scopus (65) Google Scholar). To model the receptor transmembrane domain, the transmembrane helix sequences of the rat secretin receptor were aligned with those of bovine rhodopsin (PDB code: 1F88) based on the previously reported alignment derived using the “cold spot” method (33.Frimurer T.M. Bywater R.P. Proteins. 1999; 35: 375-386Crossref PubMed Scopus (64) Google Scholar). A homology model for the transmembrane domain was built using Sybyl 6.9 (34.Tripos Inc Sybyl 6.9. Tripos, Inc., St. Louis, MO2003Google Scholar). Extracellular and cytosolic loops were built by first defining anchoring residues located at each helix terminus, then searching a data base of protein loops for a proper template to model them. The last carboxyl-terminal helix of the amino-terminal domain model described above was treated as the beginning of transmembrane helix 1, these fragments were superimposed, and the two domains connected to create an intact secretin receptor model. The NMR-derived solution structure of secretin (35.Clore G.M. Nilges M. Brunger A. Gronenborn A.M. Eur. J. Biochem. 1988; 171: 479-484Crossref PubMed Scopus (39) Google Scholar) was manually docked close to its putative binding site in the three-dimensional model, guided by our previous photoaffinity labeling data (8.Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9.Dong M. Wang Y. Hadac E.M. Pinon D.I. Holicky E. Miller L.J. J. Biol. Chem. 1999; 274: 19161-19167Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10.Dong M. Asmann Y.W. Zang M. Pinon D.I. Miller L.J. J. Biol. Chem. 2000; 275: 26032-26039Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11.Dong M. Zang M.W. Pinon D.I. Li Z. Lybrand T.P. Miller L.J. Mol. Endocrinol. 2002; 16: 2490-2501Crossref PubMed Scopus (35
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