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Center for Basic Research in Digestive Diseases (M.D., M.Z., D.I.P., L.J.M.), Departments of Internal Medicine and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905; and Vanderbilt University (Z.L., T.P.L.), Department of Chemistry and Center for Structural Biology, Nashville, Tennessee 37232-8725
Address all correspondence and requests for reprints to: Laurence J. Miller, M.D., Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, Minnesota 55905. E-mail: miller{at}mayo.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-p-benzoylbenzoyl)lysine in position 18 and a site for oxidative radioiodination [(tyrosine10,(benzoyl-benzoyl)lysine18)rat secretin-27]. This analog represented a full agonist, stimulating cAMP accumulation in Chinese hamster ovary-SecR cells in a concentration-dependent manner. It bound to the SecR specifically and saturably, and was able to efficiently label that molecule within its amino terminus. Sequential specific cleavage, purification, and sequencing demonstrated that this probe labeled receptor residue arginine14, in the same subdomain as that labeled by previous probes. Consistent with the importance of this residue, alanine replacement mutagenesis (R14A) resulted in substantial reductions in the potency (127-fold) and binding affinity (400-fold) of secretin relative to its action at the wild-type receptor. We have been able to accommodate all four extant pairs of residue-residue approximations between divergent regions of the secretin pharmacophore and the first forty residues of the SecR into a credible molecular model of this interaction. Additional experimentally derived constraints will be necessary to determine the spatial positioning of this complex with the remainder of the SecR. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The unique amino-terminal domain of the SecR has been shown to be critical for agonist binding and receptor activation using site-directed mutagenesis, receptor truncation and deletion studies, and chimeric receptor studies (5, 6). This represents a consistent theme for other class II family members (7, 8, 9, 10). Photoaffinity labeling is a powerful complementary approach for characterization of receptor ligand-binding domains. This technique can provide direct evidence for spatial approximation between residues within a ligand and within its receptor. Using this approach, we have demonstrated that a photolabile p-benzoyl-L-phenylalanine (Bpa) residue in the carboxyl-terminal half of secretin, in position 22, covalently labels leucine (Leu)17 within the amino-terminal tail of the SecR (11, 12). Another probe with a Bpa residue in the amino-terminal half of the ligand, in position 6, labeled another amino-terminal residue, valine (Val)4 (13). Still another probe, having a Bpa residue at a position closer to the carboxyl terminus of the ligand, in position 26, labeled another residue within the amino-terminal tail of the SecR (Leu36) (12). Of note, all three of these labeled residues reside within the same subdomain of the amino-terminal tail region, within the first 40 residues.
With three pairs of experimentally derived residue-residue approximations that can be used as constraints, we attempted to establish a fourth such constraint focused on a secretin residue in still another location within the peptide pharmacophore. This provided the opportunity to link the distal amino-terminal region of this receptor having the three previous peptide interactions either to another receptor region or to the same region, thereby refining the basis of the interaction. Indeed, the latter is what occurred. We designed and synthesized an additional probe having its photolabile residue [-p-benzoyl-benzoyl (BzBz) lysine (Lys)] at a site near the mid-region of the ligand, in position 18 {[Tyrosine (Tyr)10,(BzBz)Lys18]rat secretin-27, referred to as (BzBz)Lys18 analog or probe}. We used this to determine which receptor residue might reside adjacent to this position within secretin. This probe was a full agonist and bound specifically and saturably to the SecR. It was also able to efficiently label the SecR within the amino-terminal tail. Sequential specific cleavage reactions, purification, and characterization demonstrated that this probe labeled the arginine (Arg)14 residue that also resides within the same receptor subdomain as the previous probes.
The implication that receptor residue Arg14 is likely an important site of functional interaction between secretin and its receptor was further supported by receptor mutagenesis. In these studies, replacement of Arg14 with alanine (Ala) resulted in a 400-fold reduction in the affinity of binding natural secretin and in a 127-fold reduction in the potency of secretin to stimulate a biological response (intracellular cAMP). There were also similar reductions in the binding and agonist activity of the (BzBz)Lys18 analog of secretin. Replacement of an adjacent residue Arg15 with Ala had substantially smaller impact on secretin action (12-fold reduction in affinity and 13-fold reduction in potency).
This further strengthened the key role of the distal amino terminus of the SecR for ligand binding and activation and provided an important additional constraint for the modeling of the agonist-bound receptor. We were able to demonstrate that all four extant residue-residue approximation constraints could be accommodated in a credible model of secretin bound to the first 40 residues of the receptor amino-terminal tail. Additional experimentally derived constraints will be necessary to position this complex relative to the remainder of the amino-terminal tail domain and relative to the extracellular loop domains of this receptor. This should help elucidate the molecular mechanism for activation of this receptor by peptide binding.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
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). The labeled protein band migrated on a 10% sodium dodecylsulfate (SDS)-polyacrylamide gel at approximate molecular weight (Mr) = 70,000, shifting to Mr = 42,000 after deglycosylation with endoglycosidase F. These migrations are consistent with previous labeling of the SecR with other secretin analogs (11, 12, 13). The labeling was inhibited in a concentration-dependent manner by unlabeled secretin (IC50 = 5.3 ± 1.8 nM). Bands of this size were absent when analogous affinity labeling procedures were performed with non-receptor-bearing CHO cell membranes.
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, CNBr cleavage of the SecR labeled with this probe resulted in a band that migrated on a 10% NuPAGE gel (Invitrogen, Carlsbad, CA) at approximate Mr = 19,000 and shifted to Mr = 10,000 after deglycosylation with endoglycosidase F. Given the mass of the radioiodinated probe (3,383 Da) and clear evidence of glycosylation, there were two candidate fragments that best fit these data. Both fragments are within the amino-terminal tail domain of the SecR, with one at the amino-terminal end of the receptor (fragment 1) and the other (fragment 3) adjacent to the first transmembrane domain.
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shows that both intact mutant receptors were labeled with the (BzBz)Lys18 probe, with both well recognized by the anti-HA antibody. However, after CNBr cleavage, only immunoprecipitated fragment 1 from the labeled SecR-HA37 mutant receptor was radioactive. This provides definitive identification of the domain of covalent attachment with the (BzBz)Lys18 probe as CNBr fragment 1.
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, Lys-C cleavage of labeled CNBr fragment 1 yielded a labeled fragment migrating on a 10% NuPAGE gel at approximate Mr = 6000 that did not shift after deglycosylation. Taking into account the mass of the probe (3383 Da), the first 30 residues at the distal amino terminus of the SecR was the most likely domain of labeling with this probe. Consistent with this, Lys-C cleavage of the labeled intact and deglycosylated SecR resulted in bands with the same electrophoretic migration (Fig. 5).
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).
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). CNBr cleavage of the V16M-HA37 mutant yielded a fragment migrating at approximate Mr = 4,500 that did not shift after deglycosylation, representing the fragment between Ala1 and Met16 (Fig. 7). Taken together, these data indicate that the site of labeling with the (BzBz)Lys18 probe was within the focused region between Arg14 and Val16.
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, a radioactive peak eluted consistently in cycle 1. This corresponds with the labeling of Arg14 of the SecR.
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shows that the HA-tagged constructs were demonstrated by immunohistochemistry to be normally synthesized and to traffic normally to the cell surface (inset). These site mutants were studied in this cell system for impact on the binding (left column) and biological activity (right column) of secretin (top row) and of the (BzBz)Lys18 analog of secretin (bottom row). Mutation of the Arg residue that was covalently labeled in the photoaffinity labeling studies (Arg14) had a very substantial negative effect on the binding and biological effects of these peptides, whereas mutation of the adjacent receptor residue having similar charge (Arg15) had less impact on these parameters (Table 1). Figure 10 shows a representative photoaffinity labeling experiment in which the R14A mutation interfered with the ability to covalently label the receptor, whereas the R15A mutation was still able to be covalently labeled with the (BzBz)Lys18 analog of secretin.
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). After structural refinement with a molecular dynamics simulation protocol, the resultant structures are stable in subsequent unconstrained energy minimization calculations. Because there are only four contact constraints for the receptor-secretin complex, no final conclusions can be made at this point about the conformation of the complex. However, these studies provide a viable model that can be used to design additional experiments to further probe the nature of the receptor-hormone interaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It is important to note that each of the photolabile probes used in this series of studies has been a secretin analog that retains most of the determinants of binding and activation intrinsic to the natural hormone. Additionally, each has been shown to represent a full agonist acting at the SecR. This ensures that the molecular mechanisms of binding to the receptor and inducing conformational change in that target remain intact. This further supports the physiological significance of the residue-residue approximations that have been experimentally determined in these studies. In photoaffinity labeling, there is a clear requirement for spatial approximation to establish a covalent bond between a photolabile residue in the probe and a receptor residue.
Further evidence for the functional importance of the SecR residue covalently labeled in the current work was the demonstration that its modification by alanine-replacement mutagenesis (R14A) had substantial negative impact on the binding affinity of natural secretin (400-fold reduction) and on the biological activity (127-fold reduction) of secretin to act on this receptor. Further, the replacement of the Arg residue in the next position along the chain of amino acids with Ala (R15A) had markedly reduced functional impact, relative to wild-type receptor. While loss of function in receptor mutagenesis studies can be explained by both direct effects or by indirect allosteric effects, complementation of photoaffinity labeling studies with receptor mutagenesis studies can be quite useful and informative.
The large number and broad distribution of photolabile residues in the ligand that have been used to establish spatial approximation with residues within a focused subdomain of this receptor is of great interest. These sites of covalent attachment are spread throughout both the amino-terminal and carboxyl-terminal helical domains and the intermediate turn domain of secretin (14, 15). One might expect that such a series of constraints would be of great utility in establishing the site of docking secretin to its receptor. Unfortunately, there is not currently an accepted, meaningful conformational model for the entire amino-terminal tail domain of the SecR to provide the setting for docking this ligand.
The amino-terminal tail domain of secretin family receptors represents one of the key signature domains for these membrane glycoproteins. This domain is always greater than 100 residues in length and contains six highly conserved cysteine residues and functionally important intradomain disulfide bonds (1, 16). While such bonds could provide additional constraints for molecular modeling, they have not yet been directly mapped for the SecR. Further, there may be differences in the disulfide-bonding patterns that have recently been proposed for two other members of this receptor family (3, 17).
A disulfide-bonding pattern was recently established using chemical techniques for the amino-terminal fragment of the PTH receptor expressed in Escherichia coli as inclusion bodies and exposed to oxidative refolding (3). Unfortunately, that expression system precluded working with glycosylated material, and this posttranslational modification has been shown to be functionally important for several receptors in this family (18, 19, 20). Disulfide bonds were observed between cysteine residues in PTH receptor positions 108 and 148, 131 and 170, and 117 and 48. Alignment of this domain with the amino-terminal domain of the SecR suggests that these bonds are analogous to bonds between the third and sixth cysteine residues of the SecR, between the fifth and seventh cysteine residues of the SecR, and between the fourth cysteine residue of the SecR and a residue that weakly aligns with the second cysteine residue in the SecR sequence.
A distinct structure has also recently been proposed for the amino-terminal tail domain of the vasoactive intestinal polypeptide receptor based on sequence homology with the Protein Data Bank structure of a subdomain of yeast lipase B (17). Residues 8–117 of this receptor have 27% sequence identity and 50% sequence similarity with this yeast protein. This region of homology does not include key conserved cysteine residues, with component cysteine residues in the template not aligning with cysteine residues in the receptor. Using this lipase structure as a structural template, the only vasoactive intestinal polypeptide receptor cysteine residues that were sufficiently close to form a disulfide bond were in positions 72 and 86 (analogous to the fourth and fifth cysteine residues in the SecR).
We have recently experimentally established that there are three intradomain disulfide bonds within the amino-terminal tail domain of the SecR, and that this domain is not disulfide-bonded to cysteine residues within the body of the SecR (4). There are seven cysteine residues in the amino-terminal tail domain of the SecR, with the first cysteine residue not conserved in this receptor family. We established that the three disulfide bonds involved the six conserved cysteine residues; however, we have not yet been able to directly and definitively map these bonds (4).
Given the uncertainty provided by existing data for the conformation of the entire amino-terminal domain of the SecR, we elected to focus on the subdomain of the receptor amino terminus for which we had direct experimental constraint data for our molecular modeling efforts. We have attempted to dock the solution conformation of secretin that was determined by NMR (21) to an extended conformation of the first 40 residues of the SecR, using as constraints the spatial approximations determined by the extant series of photoaffinity labeling data. It is noteworthy that all four experimentally derived residue-residue approximations were easily accommodated in the refined model of this complex, with essentially no perturbation of the secretin solution structure. While this result does not prove that the secretin peptide retains its solution conformation in the receptor complex, it does indicate that a docked receptor-peptide complex that satisfies the photoaffinity labeling contacts can be generated with a low-energy secretin conformation, such as the experimentally determined solution conformation. At this stage, the model for the receptor-secretin complex is still severely underdetermined. However, the current model can be used to design future experiments that will yield additional information about specific receptor-hormone contacts. These additional experimental constraint data can be incorporated into subsequent structural refinement calculations to provide a more detailed and definitive model for the secretin complex with the amino-terminal receptor fragment.
It will ultimately be critical to extend the experimental and modeling studies to include the full receptor construct. The resulting models will provide insight into how the present fragment complex relates to the conformation of the remainder of the amino-terminal domain, as well as the extracellular loops of the receptor. Secretin binding results in a conformational change in the receptor that is transmitted to the cytosolic surface of the plasma membrane, where G protein association occurs. A detailed understanding of the molecular basis for this process will be key for the rational design and modification of SecR ligands. Because the themes for structure and functional importance of the amino-terminal domains of many of the receptors in the SecR family are conserved (8, 22, 23, 24), it is hoped that such insights will also be relevant to many other potentially important new drug targets as well.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Photoaffinity Labeling Probe
The probe, [Tyr10,(BzBz)Lys18]rat secretin-27 [(BzBz)Lys18 analog or probe], was designed to contain a Tyr residue in position 10 as a site for radioiodination and a photolabile residue, (-p-benzoyl-benzoyl)lysine, in position 18 as a site for covalent labeling of the receptor. This photolabile residue was chosen due to the basic nature of the underlying residue in this position (Arg), as opposed to the hydrophobic nature of the secretin residues previously replaced by photolabile benzoyl-phenylalanine (Phe6, Leu22, and Leu26). As such, this was believed to likely be better tolerated for binding and biological activity. It was synthesized by manual solid-phase techniques and purified to homogeneity by reversed-phase HPLC using techniques that we previously described (26). The chemical identity of the probe was established by mass spectrometry. It was radioiodinated oxidatively with Na125I upon exposure to an Iodobead for 15 sec, and it was purified by reversed-phase HPLC to yield a specific radioactivity of 2000 Ci/mmol (26).
Receptor Preparations
Receptor-bearing CHO cell lines stably expressing wild-type (CHO-SecR), HA-tagged (CHO-SecR-HA37 and CHO-SecR-HA79), and V16M mutant (CHO-SecR-V16M-HA37) rat SecRs, which have been previously established and characterized (11, 13, 27), were used as sources of receptors for the current study. Another CHO cell line was established for the present study that stably expressed the previously characterized V13M mutant SecR (SecR-V13M-HA37) (12). These stable CHO cell lines were cultured at 37 C in a 5% CO2 environment on Falcon tissue culture plasticware in Ham’s F-12 medium supplemented with 5% fetal clone-2 (HyClone Laboratories, Inc., Logan, UT). Cells were passaged twice a week and lifted mechanically before use. Enriched plasma membranes were prepared from these cell lines using methods that we previously reported (28).
Additionally, new SecR mutants were prepared and characterized. These included the alanine replacement mutants, R14A and R15A, that were prepared in analogous manner to those constructs described above, using oligonucleotide-directed mutagenesis (29), with sequences verified by direct DNA sequencing (30). Each of these constructs was additionally prepared to incorporate an HA epitope tag in position 37 within the amino-terminal tail region to provide a mechanism to demonstrate appropriate biosynthesis and cellular trafficking to the plasma membrane. Constructs were used to transfect COS cells using the DEAE-dextran method (6). Cells were harvested mechanically after 72 h and were used in radioligand binding, photoaffinity labeling, and biological activity studies.
Biological Activity Assay
The agonist activity of the (BzBz)Lys18 probe was studied for stimulation of cAMP in CHO-SecR cells using a competitive-binding assay (Diagnostic Products Corp., Los Angeles, CA). Cells were stimulated with natural secretin or this secretin analog at 37 C for 30 min, and reactions were stopped by adding ice-cold perchloric acid. After adjusting the pH to 6 with KHCO3, cell lysates were cleared by centrifugation at 3000 rpm for 10 min, and the supernatants were used in the assay, as previously described (31). Radioactivity was quantified by scintillation counting in a Beckman Coulter, Inc. LS6000 (Fullerton, CA).
Ligand Binding
Binding to SecRs was characterized in a standard assay using membranes from the CHO-SecR cell line as the source of receptor. Membranes (5–10 µg protein) were incubated with a constant amount of radioligand, 125I-[Tyr10]rat secretin-27 (3–5 pM), in the presence of increasing concentrations of nonradiolabeled [Tyr10,(BzBz)Lys18]rat secretin-27 (0–1 µM) for 1 h at room temperature. Incubations were performed in Krebs-Ringers-HEPES medium containing 25 mM HEPES, pH 7.4; 104 mM NaCl; 5 mM KCl; 1 mM KH2PO4; 1.2 mM MgSO4; 2 mM CaCl2; 1 mM phenylmethylsulfonylfluoride; 0.01% soybean trypsin inhibitor; and 0.2% BSA. 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. Bound radioactivity was quantified in a 
-spectrometer. Nonspecific binding was determined in the presence of 1 µM secretin and represented less than 20% of total binding.
Photoaffinity Labeling Studies
For covalent labeling studies, membranes from receptor-bearing CHO cells containing approximately 50 µg protein were incubated with 0.1 nM 125I-[Tyr10,(BzBz)Lys18]rat secretin-27 in the presence of increasing concentrations of secretin (0–1 µM) for 1 h at room temperature. This was then exposed to photolysis for 30 min at 4 C in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) equipped with 3500-Å lamps. To scale up for receptor purification, larger amount of membranes (
150–200 µg protein) were incubated with 0.5 nM radioligand in the absence of competing secretin. After photolysis, membranes were washed, pelleted, solubilized in SDS sample buffer, and resolved by electrophoresis on a 10% SDS-polyacrylamide gel (32). Radiolabeled bands were detected by autoradiography.
Radioactive receptor bands were excised from the gel and eluted by homogenization in a Dounce homogenizer in water. They were then lyophilized and precipitated with ethanol. Purified material was used for chemical and enzymatic cleavage experiments. CNBr and endoproteinase Lys-C were used to separately or sequentially cleave the labeled SecR and its fragments, using procedures previously described (11). 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 an intensifying screen at -80 C. Aliquots of affinity labeled SecR and relevant receptor fragments were deglycosylated with endoglycosidase F, as we have described (12, 25).
Identification of the affinity labeled SecR fragments was achieved by immunoprecipitation of the affinity labeled, HA-tagged receptor constructs and their CNBr fragments using anti-HA monoclonal antibody, as we previously described (13).
Radiochemical Sequencing
For this, the purified radiolabeled fragment from CNBr cleavage of the secretin V13M mutant receptor was coupled to N-(2-aminoethyl-1)-3-aminopropyl glass beads through the sulfhydryl side chain of an intrinsic cysteine residue. Cycles of Edman degradation were manually repeated, in a manner that has been previously reported in detail (23), and the radioactivity released in each cycle was quantified in a -spectrometer.
Molecular Modeling
Three-dimensional models were generated for secretin bound to the first 40 residues of the mature SecR amino terminus (after cleavage of the signal peptide sequence), using available photoaffinity labeling data for spatial constraints. The NMR-derived solution structure for secretin (21) was used for initial models of the peptide-receptor complex. An extended conformation for the amino-terminal fragment of the SecR was used in initial models, and the peptide hormone and receptor fragment were aligned approximately parallel to each other. In the absence of specific information about the conformation of the amino-terminal receptor fragment, a fully extended starting conformation is most appropriate, as it minimizes bias in the starting model of the complex.
The initial models were subjected to 100 steps of energy minimization in vacuo with a distance-dependent dielectric constant and no constraints, followed by structural refinement with molecular dynamics using a generalized Born model to account for solvent effects. During structural refinement of the complex, the secretin peptide was maintained close to its solution conformation with a collection of harmonic restraints, and four distance constraints were applied to impose the photoaffinity labeling contacts. Molecular dynamics simulations were started at 10 K, and the temperature of the system was gradually increased to 300 K over a 100- psec interval, with a force constant of 5.0 kcal/mol/Å for the distance constraints from photoaffinity labeling data. The simulation temperature was maintained at 300 K for an additional 100 psec and the constraint force constants were increased gradually to 40.0 kcal/mol/Å. Structures from the end of the molecular dynamics trajectories were energy minimized without constraints to generate final structures for the complex. All energy minimization and molecular dynamics simulations were performed using the AMBER 5.0 suite of programs (24).
Statistical Analysis
All observations were repeated at least three times in independent experiments and are expressed as the means ± SEM. Binding curves were analyzed and plotted using the nonlinear regression analysis routine for radioligand binding in the Prism software package (GraphPad Software, Inc., San Diego, CA). Binding kinetics were determined by analysis with the LIGAND program of Munson and Rodbard (33).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: Ala, Alanine; Arg, arginine; Bpa, p-benzoyl-L-phenylalanine; BzBz, benzoyl-benzoyl; CHO, Chinese hamster ovary; CNBr, cyanogen bromide; HA, hemagglutinin; Leu, leucine; Lys, lysine; Lys-C, endoproteinase Lys-C (enzyme that cleaves carboxylic peptide bond of lysine); Mr, molecular weight; NMR, nuclear magnetic resonance; SDS, sodium dodecyl sulfate; SecR, secretin receptor; Tyr, tyrosine; Val, valine.
Received for publication March 19, 2002. Accepted for publication July 26, 2002.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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