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Molecular Endocrinology, doi:10.1210/me.2006-0009
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Molecular Endocrinology 20 (7): 1688-1698
Copyright © 2006 by The Endocrine Society

Differential Spatial Approximation between Secretin and Its Receptor Residues in Active and Inactive Conformations Demonstrated by Photoaffinity Labeling

Maoqing Dong1, Keiko Hosohata1, Delia I. Pinon, Natesa Muthukumaraswamy and Laurence J. Miller

Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics (M.D., K.H., D.I.P., L.J.M.), Mayo Clinic, Scottsdale, Arizona 85259; and Commonwealth Biotechnologies, Inc. (N.M.), Richmond, Virginia 23235

Address all correspondence and requests for reprints to: Laurence J. Miller, M.D., Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, Arizona 85259. E-mail: miller{at}mayo.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([{psi}4, 5]secretin) in which the peptide bond between residues 4 and 5 was replaced by a {psi}(CH2-NH) peptide bond isostere. We have developed a series of [{psi}4, 5]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE MOLECULAR BASIS of natural agonist binding to receptors in the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors follows a variety of themes, based on the structures and physicochemical properties of the ligands and their pharmacophoric domains (1). In general, the smallest ligands bind within the intramembranous confluence of helical segments, whereas the larger ligands interact with extracellular loop and tail domains. For family B G protein-coupled receptors, such as the secretin receptor that is the focus of the current report, the natural ligands are all moderately large peptides with diffuse pharmacophoric domains (2, 3). Evaluation of mechanisms of receptor binding that have used peptide structure-activity series, receptor mutagenesis and chimera studies, and photoaffinity labeling studies has supported the importance of the extracellular amino-terminal tail and extracellular loop regions (4, 5, 6, 7).

Current insights into the conformation of the critically important amino-terminal tail region of the family B G protein-coupled receptors and its interactions with ligands are rather limited. Our understanding of the structure of this region of these receptors has advanced substantially in recent years with the clear establishment of the disulfide bond structure, with three highly conserved intradomain bonds that provide important spatial constraints (8, 9, 10, 11, 12), and with the solution of the nuclear magnetic resonance structure of the corticotrophin-releasing factor receptor amino terminus (13). These data provide a useful crude template to use for docking peptide ligands. Although many natural peptide ligands for these receptors have a preferred conformation in solution (14, 15, 16, 17, 18), it is not clear that this has any relationship to their conformation while docked to their receptors. The most useful data to provide direct spatial constraints for correct docking of a peptide ligand have come from intrinsic photoaffinity labeling studies.

For the prototypic secretin receptor, photoaffinity labeling studies with full agonist probes have established residue-residue approximation constraints originating at photolabile residues situated in positions 1, 6, 12, 13, 14, 18, 22, and 26, of analogs of the natural peptide ligand (19, 20, 21, 22, 23, 24, 25). These span the entire pharmacophoric domain of this peptide ligand. Of note, all of these probes, except for those having site of covalent attachment through the amino-terminal end of secretin, covalently labeled receptor residues within the amino-terminal tail region. These seven photoaffinity labeling constraints and the three constraints coming from intradomain disulfide bonds have all been accommodated within a working model of the active state of the secretin-bound secretin receptor (23).

In the current work, we have begun to study the molecular basis of receptor binding to a series of structurally related peptide antagonists. The only structural difference in the radioiodinated, photolabile antagonist probes used in the current report compared with the radioiodinated, photolabile agonist probes previously used is the presence of a {psi}(CH2-NH) peptide bond isostere between residues 4 and 5, replacing the peptide bond normally present in that position. In this series of studies, the positions of photolabile residues and, therefore, the positions of establishment of covalent bonds and spatial approximation with receptor residues were distributed throughout the pharmacophore of secretin, with one in the amino-terminal half (position 6), one in the carboxyl-terminal half (position 26), and one close to the midregion (position 22). These three positions were also used previously in the full-agonist ligand series (19, 20, 21). This provided the basis for comparing the spatial approximations for antagonist, with the receptor by definition in an inactive state, relative to agonist probes in which the receptor is in an active state.

Because the antagonist probes were structurally related to the full-agonist natural secretin ligand, one would expect the determinants of docking to be similar, and for both to attach to similar and overlapping regions of the receptor. Indeed, for these three probes, the sites of covalent attachment to the secretin receptor were each in the general regions of their analogous agonist probes. There was one probe, however, that labeled a receptor residue 16 residues away from that labeled by the analogous agonist probe (position 26 agonist probe labeled Leu36, whereas the antagonist probe labeled Glu20). This suggests that the carboxyl-terminal portion of secretin or the region of the secretin associated with its docking is quite distinct in active and inactive conformations. This provides the first clues to the dynamic changes in conformation of family B G protein-coupled receptors in response to activation. Refinement will be dependent on collection of several additional constraints.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Probe Characterization
A series of [{psi}4,5,Tyr10]rat secretin analogs was developed for photoaffinity labeling studies that each included an isosteric peptide bond replacement [{psi}(CH2-NH)] between the residues in positions 4 and 5 (Fig. 1Go). Each probe incorporated a Tyr at position 10 for radioiodination and a photolabile p-benzoyl-L-phenylalanine (Bpa) into position 6, 22, or 26. The precedent for replacing the Leu residue in position 10 of natural secretin with a Tyr residue for radioiodination is well established (26, 27), and this analog is known to be fully biologically active and to bind to the secretin receptor with high affinity. Probes were synthesized by manual solid-phase techniques, purified to homogeneity by reversed-phase HPLC, and had their identities confirmed by mass spectrometry.


Figure 1
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  		Fig. 1. Sequences of Synthetic Secretin Analogs Used for Current Photoaffinity Labeling Studies

These probes incorporated a photolabile Bpa residue into positions 6, 22, and 26, respectively. They all incorporated a Tyr into position 10 for radioiodination and had a pseudopeptide bond (-CH2NH-) between residues Gly4 and Thr5.

 
All three of these probes were characterized using radioligand-binding assay and tested for measurable agonist activities using an assay for stimulation of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary (CHO)-SecR cells. Although having lower affinity than secretin, all three photolabile secretin probes bound to the secretin receptor specifically and saturably, with similar affinity to that of their parental peptide, [{psi}4,5,Tyr10]rat secretin (Table 1Go). No measurable agonist activity was observed at 1 nM concentrations of these probes, representing a concentration 10 times higher than that used for photoaffinity labeling studies (Table 1Go).


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  		Table 1. Functional Properties of Secretin Analogs in Secretin Receptor-Bearing CHO-SecR Cells

 
Photoaffinity Labeling of the Secretin Receptor
To explore the ability of the three photolabile probes to covalently label the secretin receptor, they were used in photoaffinity labeling experiments using CHO-SecR cell plasma membranes in the presence and absence of competing secretin. As shown in Fig. 2Go, labeling of the secretin receptor was competed in a concentration-dependent manner for each of the probes. The labeled receptor migrated at relative molecular mass (Mr) = 70,000 that shifted to Mr = 42,000 after deglycosylation with endoglycosidase F. This pattern of migration is identical with all the three probes and similar to labeling of the secretin receptor by the analogous Bpa6, Bpa22, and Bpa26 agonist probes (19, 20, 21). No radioactive band was present in the affinity-labeled non-receptor-bearing CHO cell membranes (data not shown).


Figure 2
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  		Fig. 2. Photoaffinity Labeling of the Secretin Receptor

Shown are the representative autoradiographs of 10% SDS polyacrylamide gels used for separating products of photoaffinity labeling of the CHO-SecR cell plasma membranes with each noted probe in the presence of increasing concentrations of competing secretin. The labeled receptor migrated at Mr = 70,000, which shifted to Mr = 42,000 after deglycosylation with endoglycosidase F (EF). Data are representative of at least three independent experiments.

 
Identification of the Specific Photoaffinity Labeled Receptor Residues
CNBr, which cleaves at the carboxyl termini of the Met residues in a protein sequence, was used as the first indication of the domains of labeling with each of the three probes. Theoretically, CNBr cleavage of the secretin receptor would yield 10 fragments ranging in molecular mass from 1–11 kDa, with three of the fragments also containing potential sites of N-linked glycosylation (see diagram in Fig. 3Go). As shown in Fig. 3Go, CNBr cleavage of the secretin receptor labeled with each probe yielded a band migrating at Mr = 19,000 that further shifted to Mr = 10,000 after deglycosylation with endoglycosidase F. Given the molecular mass of the probes (Bpa6 probe, 3291 Da; Bpa22 probe, 3325 Da; Bpa26 probe, 3325 Da) and evidence of glycosylation of the labeled fragments, the first and third fragments at the amino terminus of the receptor were the best potential candidates to represent the glycosylated fragments labeled with each probe (Fig. 3Go).


Figure 3
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  		Fig. 3. CNBr Cleavage of the Labeled Secretin Receptor

Left panel shows a diagram illustrating the theoretical sites of CNBr cleavage of the secretin receptor. Right panel shows a representative autoradiograph of 10% NuPAGE gel used for separating the products of CNBr cleavage of the native and deglycosylated secretin receptors labeled with each of the noted probe. As shown, CNBr cleavage of the secretin receptor labeled with each probe yielded a band migrating at Mr = 19,000, which further shifted to Mr = 10,000 after endoglycosidase F (EF) treatment. The two glycosylated fragments (highlighted in bold circles) at the amino terminus of the receptor were the best potential candidates to represent the fragments labeled with each probe.

 
To further identify which receptor fragment was most relevant, a well-characterized hemagglutinin (HA)-tagged secretin receptor mutant (SecR-HA37) (20) was used in immunoprecipitation experiments. As shown in Fig. 4Go, the Mr = 19,000 CNBr fragment labeled by each probe that was immunoprecipitated by anti-HA antibody was radioactive, and this band was completely competed off in the presence of excess HA peptide. This definitively identified the fragment (Ala 1-Met51) at the most distal end of the amino terminus as the affinity-labeled receptor region for each of the three probes.


Figure 4
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  		Fig. 4. Immunoprecipitation of the Epitope-Tagged Secretin Receptor CNBr Fragment

Upper panel shows the graphic representation of the sites of CNBr cleavage of the amino terminus of the epitope-tagged SecR-HA37 secretin receptor construct. Lower panel shows representative autoradiographs of 10% NuPAGE gels used for separating products of immunoprecipitation with monoclonal anti-HA antibody of CNBr fragments from cleavage of HA37-tagged secretin receptor labeled with each of the three probes in the presence and absence of competing HA peptide.

 
To further refine the domain of labeling, labeled receptors were submitted to endoproteinase Lys-C treatment that cleaves at the carboxyl termini of Lys residues in a protein sequence. As shown in Fig. 5Go, endoproteinase Lys-C cleavage of the secretin receptor labeled with each probe yielded a nonglycosylated fragment migrating at approximate Mr = 6000. The cleavage patterns were identical for all three probes. This further confirmed that the Ala1-Lys30 fragment at the distal end of the amino terminus of the secretin receptor represented the region of labeling by each probe.


Figure 5
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  		Fig. 5. Endoproteinase Lys-C Digestion of the Labeled Secretin Receptor

Upper panel shows theoretical sites of Lys-C cleavage and masses of expected fragments of the affinity-labeled CNBr fragment 1 (Ala1–Met 51). Lower panel shows typical autoradiographs of 10% NuPAGE gels used to separate the products of Lys-C digestion of both native and deglycosylated secretin receptor labeled with each of the three probes. Both resulted in fragments migrating similarly at approximate Mr = 6000. This is most consistent with labeling the fragment representing the region of the receptor between residue Ala1 and Lys30.

 
Two secretin receptor mutants, V16M and V13M, which have previously been shown to bind secretin with normal affinity and to respond to secretin with normal cAMP responses (19, 21), were used for further localization of the labeling domain for each of the probes. These receptor constructs provided the ability to quickly narrow down the region of covalent attachment within the larger CNBr segment and also provided a labeled fragment with the site of covalent attachment close enough to the amino terminus to allow sequential Edman degradation to ultimately identify the labeled residue. As shown in Fig. 6Go, both intact mutant receptors were labeled specifically and saturably by each noted probe. CNBr cleavage of the V16M construct labeled with the Bpa6 probe resulted in a nonglycosylated fragment band migrating at Mr = 4500, indicating the site of labeling for this probe was within the first 16 residues. CNBr cleavage of the V13M and V16M constructs labeled with the Bpa22 probe identified the glycosylated Leu17-Met51 fragment as the domain of labeling (Fig. 6Go). Combined with the above endoproteinase Lys-C cleavage data (Fig. 5Go), the domain of labeling for this probe was further localized within the segment Leu17–Lys30. This fragment was also identified as the domain of labeling for the Bpa26 probe by CNBr cleavage of the labeled V16M construct (Fig. 6Go).


Figure 6
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  		Fig. 6. CNBr Cleavage of the Labeled Mutant Secretin Receptors

Shown in upper left panel is a typical autoradiograph of a 10% SDS-polyacrylamide gel used to separate products of labeling of SecR-V16M and SecR-V13M receptor-bearing membranes in the presence and absence of excess competing unlabeled secretin (1 µM) by each of the noted probes. Like labeling the wild-type receptor, the affinity-labeled secretin receptor migrated at approximate Mr = 70,000. Shown in upper right panel are typical autoradiographs of 10% NuPAGE gels used to separate products of CNBr cleavage of both mutant constructs labeled by each noted probe. Lower panel shows theoretical sites of CNBr cleavage of expected fragments of the CNBr fragment 1 (Ala1–Met51) of the V13M and V16M receptor mutants labeled with each of the noted probes. As shown, CNBr cleavage of both native and deglycosylated V16M mutant receptor labeled with the Bpa6 probe yielded fragments migrating similarly at Mr = 4500, indicating the site of labeling being within the first 16 amino acids. CNBr cleavage of the V13M and V16M mutant receptors labeled with the Bpa22 probe both yielded a fragment migrating at Mr = 19,000 and shifting to Mr = 9,000 after deglycosylation with endoglycosydase F (EF), representing the glycosylated fragment Arg14–Met51 and Leu17–Met51, respectively. This is also the pattern observed for CNBr cleavage of the V16M mutant receptor labeled with the Bpa26 probe, suggesting the site of labeling for the Bpa26 probe was also within the fragment Leu17–Met51.

 
Manual radiochemical sequencing of the Ala1–Met51 fragment resulting from CNBr cleavage of the wild-type secretin receptor was performed to identify the site of labeling with the Bpa6 probe. As shown in Fig. 7Go, a radioactive peak eluted in cycle 2, corresponding to attachment of this probe to His2 of the receptor. Sequencing of the Arg14–Met51 and Leu17–Met51 fragments resulting from CNBr cleavage of the respective V13M and V16M constructs labeled with the Bpa22 probe identified Leu17 of the receptor as the site of labeling (Fig. 7Go). Receptor residue Glu20 was identified as the site of labeling with the Bpa26 probe by sequencing of the Leu17–Met51 fragment resulting from CNBr cleavage of the labeled V16M construct (Fig. 7Go).


Figure 7
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  		Fig. 7. Radiochemical Sequencing to Identify the Labeled Receptor Residues

Shown are representative radioactivity elution profiles from sequencing of the labeled Ala1–Met51 fragment from the wild-type (WT) secretin receptor for the Bpa6 probe (upper left panel), Leu17–Met51 (from the V16M mutant receptor) and Arg14–Met51 (from the V13M mutant receptor) fragments for the Bpa22 probe (upper right and lower right panels), and Leu17–Met51 fragment from the V16M mutant receptor for the Bpa26 probe (lower left panel). As seen, a peak eluted in radioactivity appeared in cycle 2 for the Bpa6 probe, representing receptor residue His2, in cycles 1 (V16M) and 4 (V13M) for the Bpa22 probe, representing receptor residue Leu17, and in cycle 4 for the Bpa26 probe, representing receptor residue Glu20.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current report provides the first insights into spatial approximations between residues distributed throughout an antagonist ligand and residues within the secretin receptor as these molecules reside in a docked complex. The antagonist probes used in this work are structurally related to the full-agonist secretin peptide probes previously used, except for the presence of a {psi}(CH2-NH) peptide bond isostere replacing the peptide bond normally present between residues 4 and 5. This modification of secretin was previously reported (28) and continues to represent the most potent and selective secretin receptor antagonist yet described. By maintaining intact the structure of as much of the remainder of this 27-residue linear peptide as possible, the key determinants for receptor-docking specificity were retained. Probes contained a tyrosine residue in position 10, replacing the leucine residue normally present in that position, to provide a site for radioiodination. This modification has been previously reported and well tolerated, resulting in a tracer that is fully biologically active and that binds with high affinity (26, 27). Indeed, the results of the current report support normal docking of the antagonist probes by demonstrating that each of the three photolabile antagonists covalently labeled the amino-terminal domain of the secretin receptor, similar to their analogous agonist probes (19, 20, 21). Of note, however, the residue-residue approximations that have been established for these antagonist probes ranged from being identical to the analogous agonist probe to being quite divergent, labeling amino acids that were 16 residues apart (Fig. 8Go).


Figure 8
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  		Fig. 8. Comparison of Secretin Receptor Residues Labeled by Analogous Agonists and Antagonists

As shown, the Bpa6 antagonist and agonist labeled distinct receptor residues, but they are close to each other within the distal end of the amino terminus of the secretin receptor. The Bpa22 antagonist and agonist labeled the same receptor residue Leu17. Bpa26 antagonist labeled a receptor residue (Glu20) that is 16 amino acids away from that labeled by the Bpa26 agonist (Leu36).

 
For the natural peptide ligands of family B G protein-coupled receptors, a general rule has been proposed that suggests that the carboxyl-terminal region is most critical for high-affinity binding, whereas the amino-terminal region is most critical for biological activity (29, 30). Consistent with this, the modification in the structure of the peptide bond to change a secretin agonist into an antagonist that was used in the current studies was in its amino-terminal region (28). Amino-terminal truncation of natural ligands for several members of this family has also resulted in antagonists (31, 32, 33). With this in mind, it is interesting that two of the three probes having their sites of covalent attachment at the amino terminus and midregion of secretin labeled residues that were the same or very close to those residues labeled by their analogous agonist probes, whereas the probe that labeled a distinct residue had its position of covalent attachment at the carboxyl terminus of the ligand (Fig. 8Go).

Spatial approximation established in photoaffinity labeling studies can be affected by site of ligand probe docking, conformation of the ligand, and conformation of the receptor. The structural similarities between antagonist and agonist probes and their similarities in the general receptor domain that was labeled support similar initial docking. Indeed, the importance of amino-terminal domains of receptors in this family for peptide ligand docking has been well established by receptor-truncation and mutagenesis studies, as well as photoaffinity labeling studies (4, 5, 6, 7). By first principles, these long linear peptides would be expected to be highly flexible and to assume conformations upon docking that could have little relationship to their preferred conformations in solution. It is also quite likely that active and inactive conformations of the receptors are distinctly different. Indeed, that is the basis for G protein association with this superfamily of receptors and the resultant initiation of their signaling cascades.

The distinct spatial approximations between residue 26 of the secretin agonist and antagonist probes and adjacent receptor residues most likely reflect this conformational change. It is noteworthy that the major difference resides at the amino terminus of secretin, whereas spatial approximations with the carboxyl terminus are relatively stable. Based on current hypotheses for the molecular basis of activation of receptors in this family, one would expect substantial change in the environment of the amino terminus of a secretin ligand in active and inactive conformations (13, 34). The proposed docking of peptide ligands for members of this receptor family, including PTH (35, 36), calcitonin (37), and corticotropin-releasing factor (13), places the carboxyl terminus of the ligands adjacent to the amino-terminal tail of their respective receptors, whereas the amino terminus of the ligands is proposed to dip down within the confluence of intramembranous helices upon activation, a position where it can exert tension on the body of the receptor to expose the region of G protein association. Analogous localization, adjacent to the top of the sixth transmembrane segment, has also been proposed for the amino terminus of secretin (24). It should be noted that the probe used in the current studies labeled the receptor through position 6, rather than position 1. Whereas these data reflect the spatial approximation for the amino-terminal half of the peptide ligand, they certainly do not reflect the absolute change in approximation of the amino-terminal residue and, therefore, cannot be used to refute or support this aspect of the molecular basis of receptor activation.

The three intradomain disulfide bonds that are fully conserved within the amino-terminal tail region of G protein-coupled receptors in the B family contribute to an important architectural platform for peptide ligand binding (8, 9, 10, 11, 12). The recently reported NMR structure of the corticotrophin-releasing factor receptor (13) adds two pairs of antiparallel ß-sheet structures to further stabilize this platform. The current studies provide insights into spatial approximations between three residues distributed along secretin and its analogs that are adjacent to receptor residues in this platform. These data support major differences only at the carboxyl-terminal region of the agonist and antagonist ligands. This provides intriguing new insights, but is not yet adequate to provide a credible working model of the antagonist-occupied conformation of this receptor. As additional experimentally derived constraints are added, the differences in active, and inactive conformations of this portion of the secretin receptor will be quite interesting and important.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Cyanogen bromide (CNBr) and the solid-phase oxidant, N-chlorobenzenesulfonamide (Iodo-beads), were purchased from Pierce Chemical Co. (Rockford, IL). Endoproteinase Lys-C and a monoclonal antibody against HA epitope were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Endoglycosidase F was prepared in our laboratory as we reported previously (38). Other reagents were analytical grade.

Synthesis of Peptides
The peptides used in this study were [{psi}4,5,Tyr10]rat secretin, [{psi}4,5, Bpa6,Tyr10]rat secretin (Bpa6 analog or probe), [{psi}4, 5,Tyr10, Bpa22]rat secretin (Bpa22 analog or probe), and [{psi}4,5,Tyr10,Bpa26]rat secretin (Bpa26 analog or probe) (Fig. 1Go). They each contained a Tyr residue in position 10 for radioiodination, and the photoaffinity labeling probes (Bpa6, Bpa22, and Bpa26 probes) each incorporated a p-benzoyl-L-phenylalanine (Bpa) in position 6, 22, or 26, respectively. The peptide bond between residues 4 and 5 was replaced by a {psi}(CH2-NH) peptide bond isostere (a reduced peptide bond) in each of the antagonist peptides.

The synthetic strategy to prepare the peptidic analogs followed a variation of the technique described by Meyer et al. (39). In this, we used solid-phase synthetic techniques with Fmoc/tert-butyl protection, except for the introduction of the aldehyde that was prepared from Bpoc-Gly at the amino terminus. The reduced bond was introduced via reductive alkylation of the amino-terminal amino group of the growing peptide. In this approach, the aldehyde of the Bpoc-Glyaldehyde was obtained by treatment with LiAlH4 in dry tetrahydrofuran and after hydrolysis of excess LiAlH4 with 0.35 M KHSO4. Ether was added and sequentially extracted with saturated KHSO4, NaCl, NaHCO3, and NaCl, and the product was dried by evaporation and immediately used in the synthesis. The N-terminal free peptide resin was washed three times with 1% acetic acid (HAC)/dimethylformamide (DMF) to remove any residual traces of piperidine after Fmoc deprotection. The Bpoc-Gly-N,O-dimethyl hydroxamate was dissolved in 1% HAC/DMF and added to the peptide resin, and NaCNBH3/DMF was immediately added. The reaction was followed until the ninhydrin test was negative. The secondary amine formed was protected by reacting the peptide resin overnight with di-tert-butyl dicarbonate and N-ethyldiisopropylamine. The Bpoc peptide resin was deprotected with 0.5% trifluoroacetic acid (TFA) in CH2Cl2. The remainder of the synthesis followed usual Fmoc procedures with active esters of the remaining amino acids. The peptides were cleaved from the resin using 95% TFA for 3 h and filtered, and the resin was washed with TFA and CH2Cl2. The combined filtrates were evaporated and precipitated in ether. The crude peptides were then dissolved in approximately 10% CH3CN/H2O, lyophilized, and cleaned using semipreparative HPLC.

Radioiodination of Probes
Each of the peptides described above was radioiodinated oxidatively as reported previously (40). Each was exposed to the solid-phase oxidant, N-chlorobenzenesulfonamide (Iodo-beads, Pierce), in the presence of Na125I (PerkinElmer, Boston, MA) for 15 sec and was purified to homogeneity by reversed-phase HPLC. This yielded specific radioactivities of 2000 Ci/mmol.

Receptor Preparations
CHO cells stably expressing the wild-type rat secretin receptor (CHO-SecR), hemagglutinin (HA) epitope-tagged receptor (SecR-HA37), and mutant receptors (SecR-V13M-HA37 and SecR-V16M-HA37), which were established and characterized previously (19, 20, 21, 41), were used as sources of receptors for the current study. Cells were cultured at 37 C in an environment with 5% CO2 in tissue culture flasks in Ham’s F-12 medium supplemented with 5% Fetal Clone-2 (Hyclone Laboratories, Logan, UT). Cells were passaged twice a week and were harvested mechanically before membrane preparation. Enriched plasma membranes were prepared from these cell lines as we described previously (38, 41).

Receptor Binding Studies
The binding properties of [{psi}4,5,Tyr10]rat secretin analogs at the rat secretin receptor were determined in a standard radioligand competition-binding assay (41). In brief, membranes (5–10 µg) prepared from CHO-SecR cells were incubated in Krebs-Ringers-HEPES (KRH) buffer [25 mM HEPES (pH 7.4), 104 mm NaCl, 5 mM KCl, 1 mm KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 0.01% soybean trypsin inhibitor] containing 0.2% BSA and 1 mM phenylmethylsulfonyl fluoride with a constant amount of [125I-Tyr10]rat secretin (3–5 pM) (41) and increasing concentrations (0–1 µM) of nonradiolabeled [{psi}4,5,Tyr10]rat secretin analogs for 1 h at room temperature. Membrane-bound and free radioligand were separated by filtering through glass fiber filters that were presoaked in 0.3% Polybrene using a Skatron cell harvester (Molecular Devices Corp., Sunnyvale, CA). Nonspecific binding was determined in the presence of 1 µM secretin.

Biological Activity Studies
The biological activities of [{psi}4,5,Tyr10]rat secretin analogs were demonstrated by measuring cAMP accumulation in CHO-SecR cells using a kit from Diagnostic Products Corp. (Los Angeles, CA). As we reported previously (42), intact cells were incubated in KRH buffer containing 0.2% BSA, 0.1% bacitracin, and 1 mM 3-isobutyl-1-methylxanthine with increasing concentrations of each of the [{psi}4,5,Tyr10]rat secretin analogs for 30 min at 37 C. The stimulation was terminated by removing the medium and lysing the cell pellet in ice-cold 6% (wt/wt) perchloric acid for 15 min with vigorous shaking. The resulting lysates were adjusted to pH 6 with 30% KHCO3 before being used in the cAMP competition-binding assay. The radioactivity was quantified using a Beckman LS6000 scintillation counter.

Photoaffinity Labeling of the Secretin Receptor
Receptor membranes prepared from CHO-SecR-HA37 cells (~100 µg) were incubated in KRH buffer with 100 pM radioiodinated probe in the absence of increasing concentrations of secretin (0–1 µM) for 1 h at room temperature in the dark. For covalent labeling of the receptor, the incubation reaction was 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. Membranes were then pelleted by centrifugation at 14,000 rpm in a bench-top centrifuge for 5 min at 4 C and washed twice with KRH buffer. Membrane proteins were either directly dissolved using sodium dodecyl sulfate (SDS) sample buffer and applied to a 10% SDS-polyacrylamide gel or solubilized in 1% Nonidet P-40 and enriched using adsorption to wheat germ agglutinin-agarose (EY Laboratories, San Mateo, CA) before electrophoresis. Radioactive bands were visualized by autoradiography.

Chemical and Enzymatic Cleavage of the Secretin Receptor
Deglycosylation of the photoaffinity-labeled receptor was performed with endoglycosidase F as we described previously (38). Photoaffinity-labeled native or deglycosylated secretin receptors were purified by SDS-polyacrylamide gels and submitted to chemical and enzymatic cleavage with CNBr and endoproteinase Lys-C. The detailed procedures for these reactions were described previously (20). Cleavage products were resolved on 10% NuPAGE gels (Invitrogen, Carlsbad, CA) and visualized by autoradiography.

Immunoprecipitation
Gel-purified affinity-labeled receptor fragments resulting from cleavage of the HA-tagged secretin receptor (SecR-HA37) were used in immunoprecipitation experiments by incubation with 12CA5 monoclonal anti-HA antibody (Roche Molecular Biochemicals) and subsequently with protein G plus-agarose (Oncogene Research Products, Boston, MA). This procedure has been well described previously (20). The immunoprecipitated fragments were eluted in SDS sample buffer and resolved by NuPAGE gel electrophoresis.

Receptor Labeling Site Identification
Radiochemical sequencing using Edman degradation chemistry was used to identify the specific receptor residue that was covalently labeled. For this, the affinity labeled receptor was cleaved by CNBr and purified on a gel to radioactive homogeneity. The purified receptor fragments were coupled to N-(2-aminoethyl-1)-3-aminopropyl glass beads through the sulfhydryl group of a free Cys residue using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester as a cross-linking reagent (19). Cycles of Edman degradation were repeated manually as we previously reported in detail (43).

Statistical Analysis
All data were calculated from at least three independent experiments and are expressed as means ± SEM. Binding curves were plotted using the nonlinear regression analysis routine for radioligand binding in the Prism software package (GraphPad Software Inc., San Diego, CA), and binding kinetics were determined by analysis with the LIGAND program of Munson and Rodbard (44).


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health (DK46577) and the Fiterman Foundation. We thank L.-A. Bruins for excellent technical assistance and E. M. Posthumus for secretarial assistance.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (DK46577) and the Fiterman Foundation.

All authors (M.D., K.H., D.I.P., N.M., and L.J.M.) listed in this manuscript have nothing to declare.

First Published Online March 2, 2006

1 M.D. and K.H. contributed equally to this work. Back

Abbreviations: Bpa, p-Benzoyl-L-phenylalanine; CHO, Chinese hamster ovary; CHO-SecR, secretin receptor-bearing CHO cells; DMF, dimethylformamide; HA, hemagglutinin; KRH, Krebs-Ringers-HEPES; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid.

Received for publication January 6, 2006. Accepted for publication February 24, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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