Insights into the Structure and Molecular Basis of Ligand Docking to the G Protein-Coupled Secretin Receptor Using Charge-Modified Amino-Terminal Agonist Probes
Maoqing Dong,
Delia I. Pinon and
Laurence J. Miller
Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
Address all correspondence and requests for reprints to: Laurence J. Miller, M.D. Director, Cancer Center Mayo Clinic in Scottsdale, 13400 East Shea Boulevard, Johnson Research Building, Scottsdale Arizona 85259. E-mail: miller{at}mayo.edu.
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ABSTRACT
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The amino terminus and third loop regions of class B G protein-coupled receptors play critical roles in ligand docking and action. For the prototypic secretin receptor, the hormone amino terminus is spatially approximated with receptor region high in transmembrane segment 6 (TM6), whereas residues ranging from position 6 through 26 label the amino terminus. Here, we focus on the role of charge of the secretin amino terminus, using a series of full-agonist, acetylated probes. Sites of covalent labeling were examined using sequential purification, chemical and enzymatic cleavage, and Edman degradation. High-affinity amino-terminally-blocked probes labeled the distal amino-terminal tail, rather than TM6, while adding a basic residue, again labeled TM6. These data suggest that the secretin amino terminus docks between the amino terminus and TM6 of the receptor, with this region of secretin likely interacting with an acidic residue within the receptor TM6 and the third extracellular loop. To explore this, candidate acidic residues were mutated to Ala (E341A, D342A, E345A, E351A). The E351A mutant markedly interfered with binding, biological activity, and internalization, whereas all others bound secretin and signaled and internalized normally. This supports the possibility that there is a charge-charge interaction between this residue and the amino terminus of secretin that is critical to its normal docking.
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INTRODUCTION
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THE SECRETIN RECEPTOR is a prototypic member of the class B G protein-coupled receptor family that includes receptors for secretin, vasoactive intestinal polypeptide, PTH, calcitonin, pituitary adenylate cyclase-activating peptide, glucagon, and glucagon-like peptide-1 (1). This family includes multiple potentially important targets for drug development. However, due to the natural sparsity and physicochemistry of these receptors, high-resolution structural analysis has not yet been available. Our current understanding of the structures of these molecules is based mainly on observations obtained by approaches such as receptor mutagenesis, ligand structure-activity relationships, and photoaffinity labeling studies.
Secretin, like other natural ligands for class B G protein-coupled receptors, is a moderately large peptide, having 27 amino acids with binding determinants distributed throughout its entire length. The amino-terminal regions of natural ligand peptides for this family are believed to contain key determinants for receptor selectivity, whereas carboxyl-terminal regions contain determinants for high-affinity binding (2, 3). Ligand structure-activity relationship studies have identified critical residues in secretin at the amino terminus (His1), amino-terminal half (Phe6), and midregion (Asp15) (4).
A large number of important receptor residues in extracellular domains of this receptor have been identified, by mutagenesis studies, to be involved in secretin binding (4). In particular, the extracellular amino terminus of the secretin receptor, like that of other members in this family, has been shown to play a critical role (2, 4, 5, 6, 7, 8, 9, 10, 11). In fact, the structure of this domain is one of the distinctive characteristics of this receptor family, exceeding 120 residues in length and containing six conserved Cys residues that have been demonstrated to form intradomain disulfide bonds (12, 13, 14, 15, 16). Nevertheless, the extracellular loop domains of this group of receptors have also been shown, by mutagenesis studies (4, 9, 10, 11), to play complementary roles.
Photoaffinity labeling has emerged in recent years as a powerful tool for the study of interactions between a ligand and its receptor. This technique has been particularly useful for elucidation of the molecular basis of ligand binding to the sparse and hydrophobic G protein-coupled receptors, which continue to be extremely challenging to study by more direct, high-resolution physicochemical methods. We have previously used a series of secretin probes incorporating a photolabile residue in distinct regions of the ligand to explore molecular approximations with this receptor. These include carboxyl-terminal [(BzBz)Lys18, Bpa 22, and Bpa26], midregion [(BzBz)Lys12, Bpa13, and (BzBz) Lys14], and amino-terminal (Bpa6) probes (17, 18, 19, 20, 21, 22). Of interest, all these probes labeled residues within the extracellular amino terminus of the secretin receptor, supporting the critical role of the amino-terminal tail in ligand binding, as identified by mutagenesis. This theme is supported by analogous photoaffinity labeling studies of receptors for vasoactive intestinal peptide (23, 24), PTH (9), and calcitonin (25, 26, 27). More recently, we used secretin probes with the photolabile residue at the amino terminus of the peptide ligand and showed that all these probes labeled a distinct domain predicted to be at the top of the transmembrane segment 6 (TM6) region of the secretin receptor (28).
To closely examine how critical the spatial approximation between the amino terminus of secretin and its receptor may be, we propose to investigate whether the charge of the amino terminus of the ligand might affect the spatial approximations between secretin and its receptor. Therefore, we prepared a series of amino-terminal probes by blocking their amino groups by acetylation to eliminate one positive charge. In some of these, we added back a positive charge with an additional basic residue, while maintaining the blocked amino terminus. This series included [Ac-Bpa1,Tyr10]secretin, [Ac-Bpa1,Tyr10]secretin, [Ac-Bpa2,Gly1,Tyr10]secretin, [Ac-Lys2,Bpa1,Tyr10]secretin, and [Ac-Arg2,Bpa1,Tyr10]secretin. Here, we report the characterization of these probes and their use in photoaffinity labeling studies to further explore their spatial approximations with the secretin receptor. In addition, we have prepared and studied a series of receptor site mutants that may contribute to a charge-charge interaction with the amino terminus of the natural peptide ligand.
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RESULTS
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Probe Characterization
The probes were synthesized by manual solid-phase techniques and purified by reversed-phase HPLC. Their identities were verified by mass spectrometry to demonstrate the expected molecular masses. These probes were functionally characterized by binding to secretin receptor-bearing Chinese hamster ovary (CHO)-SecR cell membranes and by their ability to stimulate cAMP accumulation in these cells. As shown in Fig. 1
, these probes all had lower affinity to bind to the secretin receptor than secretin [inhibition constant (Ki) values in nanomolar concentration of secretin, 5.5 ± 0.7; Ac-Bpa1 probe, 303 ± 45; Ac-Bpa1 probe, 196 ± 34; Ac-Bpa2 probe, 23 ± 8; Ac-Lys2,Bpa1 probe, 293 ± 36; Ac-Arg2,Bpa1 probe, 160 ± 27), consistent with the known importance of the amino terminus of secretin for receptor binding (29). Note that the binding affinity of these probes increased very substantially toward that of natural secretin as the bulky p-benzoyl-phenylalanine (Bpa) moiety was removed further from the critical position of His1. Nevertheless, they were all full agonists, stimulating cAMP accumulation in CHO-SecR cells in a concentration-dependent manner, although with lower potency (EC50 values in nanomolar concentration of secretin, 0.07 ± 0.02; Ac-Bpa1 probe, 32 ± 8; Ac-Bpa1 probe, 12 ± 1.3; Ac-Bpa2 probe, 1.4 ± 0.1; Ac-Lys2,Bpa1 probe, 25 ± 3; Ac-Arg2,Bpa1 probe, 45 ± 9) (Fig. 1
). Again, this agonist activity series paralleled the structure-activity considerations that contributed to binding affinity.

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Fig. 1. Characterization of Photolabile Probes
Left panels demonstrate the abilities of increasing concentrations of secretin or each of the photolabile probes to compete for binding of the radioligand, 125I-[Tyr10]secretin, to CHO-SecR cell membranes. Values represent saturable binding as percentages of maximal binding observed in the absence of competitor. Data are expressed as the means ± SEM of duplicate data from a minimum of three independent experiments. Right panels show concentration-dependent intracellular cAMP responses to these peptides in the CHO-SecR cells. Basal levels of cAMP were 2.8 ± 0.7 pmol/million cells, and maximal levels reached 185 ± 22 pmol/million cells. Values are expressed as the means ± SEM of at least three independent experiments, with data normalized relative to the maximal response to natural secretin.
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Photoaffinity Labeling of the Secretin Receptor
Each of the acetylated probes was used to explore its ability to covalently label the secretin receptor. As shown in Fig. 2
, each probe labeled the secretin receptor specifically and saturably. The labeled bands migrated on a 10% SDS-PAGE gel at an approximate Mr of 70,000 that shifted to Mr 42,000 after deglycosylation with endoglycosidase F, as we had observed previously (17, 18, 19, 20, 21, 22, 28). As expected, the labeling was inhibited by competition using increasing concentrations of unlabeled secretin (IC50 values in nanomolar concentration: Ac-Bpa1 probe, 30 ± 2.2; Ac-Bpa1 probe, 8.0 ± 1.7; Ac-Bpa2 probe, 4.8 ± 0.9; Ac-Lys2,Bpa1 probe, 9.0 ± 1.5; Ac-Arg2,Bpa1 probe, 39 ± 6). No radioactive bands were observed in the affinity-labeled non-receptor-bearing CHO membranes.

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Fig. 2. Photoaffinity Labeling of the Secretin Receptor
Left panels show typical autoradiographs of 10% SDS-polyacrylamide electrophoresis gels used to separate the products of affinity labeling of CHO-SecR cell membranes by each of the probes in the presence of increasing concentrations of secretin. Shown in right panels are the densitometric analyses of three similar independent experiments by each probe (means ± SEM). The receptor labeled with each probe migrated at an approximate Mr of 70,000 and shifted to an approximate Mr of 42,000 after deglycosylation with endoglycosidase (EF). No bands were detected in affinity-labeled non-receptor-bearing CHO cell membranes. These data represent three independent experiments.
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Identification of the Sites of Labeling by the Ac-Bpa1, Ac-Bpa2, Ac-Lys2,Bpa1, and Ac-Arg2,Bpa1 Probes
Cyanogen bromide (CNBr) cleavage was used as the first indication of domain of labeling for each of the probes. As shown in Fig. 3
, CNBr cleavage of the secretin receptor labeled with the Ac-Bpa1,Ac-Bpa2, Ac-Lys2,Bpa1, and Ac-Arg2,Bpa1 probes resulted in bands that migrated on a 10% NuPAGE gel at an approximate Mr of 19,000 and shifted to Mr 10,000 after deglycosylation with endoglycosidase F. Given the molecular masses of the radioiodinated probes (Ac-Bpa1 probe, 3372 Da; Ac-Bpa2 probe, 3429 Da; Ac-Lys2,Bpa1 probe, 3500 Da; Ac-Arg2,Bpa1 probe, 3528 Da) and clear evidence of glycosylation, there were two candidate fragments consistent with these data. Both fragments are at the amino-terminal domain of the secretin receptor, with one at the amino terminus of the receptor (first fragment) and the other (third fragment) adjacent to TM1. Of note, the Ac-Arg2,Bpa1 probe labeled an additional nonglycosylated fragment that migrated at an approximate Mr of 8500 (see below).

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Fig. 3. CNBr Cleavage of the Affinity-Labeled Receptor
A, Diagram illustrating the theoretical fragments resulting from CNBr cleavage of the secretin receptor. B, Representative autoradiographs of 10% NuPAGE gels used to separate the products of CNBr cleavage of the secretin receptor labeled with each of the probes indicated. The CNBr fragment of the secretin receptor labeled with the Ac-Bpa1, Ac-Bpa2, Ac-Lys2,Bpa1, and Ac-Arg2,Bpa1 probes migrated at an approximate Mr of 19,000 that shifted to Mr 10,000 after deglycosylation with endoglycosidase F (EF). The first fragment at the amino terminus of the receptor and the third fragment (bold circles) were the best potential candidates to represent the glycosylated fragment labeled with each probe. An additional nonglycosylated band migrating at Mr 8500 was observed with the Ac-Arg2,Bpa1 probe. CNBr cleavage of the secretin receptor labeled with the Ac-Bpa1 probe resulted in a band migrating at Mr 8500 that did not further shift after deglycosylation. Candidates representing the Mr 8500 fragment labeled with either the Ac-Arg2,Bpa1 or Ac-Bpa1 probe are the fragments His158-Met197 and Arg300-Met344 (highlighted in gray; see Figs. 7 and 8 , respectively, for further identification). These data represent at least three independent experiments.
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To determine which of the two fragments at the amino terminus of the secretin receptor was the domain of labeling with each of the four probes, two well-characterized hemagglutinin (HA) epitope-tagged receptor mutants, SecR-HA37and SecR-HA79 (18), were used in immunoprecipitation experiments with monoclonal anti-HA antibody. Figure 4
shows that both intact mutant receptors labeled with each of the probes were well recognized by the anti-HA monoclonal antibody. However, after CNBr cleavage, only the first fragment from the SecR-HA37 receptor mutant labeled with each probe was radioactive upon immunoprecipitation. This definitively identified the first CNBr fragment (Ala1-Met51) as the domain of labeling with each of the four acetylated probes.

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Fig. 4. Immunoprecipitation of HA-Tagged Secretin Receptor Fragments Labeled by the Ac-Bpa1, Ac-Bpa2, Ac-Lys2,Bpa1, and Ac-Arg2,Bpa1 Probes
A, Diagram illustrating the theoretical sites of CNBr cleavage of the amino terminus of the HA-tagged secretin receptor constructs (SecR-HA37 and SecR-HA79). B, Both intact receptor mutants were affinity labeled with each probe, and they were well recognized by anti-HA monoclonal antibody. C, After CNBr cleavage, only the immunoprecipitated fragment from the labeled SecR-HA37 was radioactive when performed in the absence of the competing HA peptide. This provides the definitive identification of the fragment at the most distal end of the amino terminus as the affinity-labeled receptor domain for all the probes indicated.
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Three well-characterized secretin receptor mutants, V16M (17), V13M (19), and P8M (21), were used for further localization of the domain that was labeled by each of these probes. As shown in Fig. 5
, all three receptor mutants were specifically labeled with each probe, with the labeled receptor migrating at an approximate Mr of 70,000 and shifting to Mr 42,000 after deglycosylation, consistent with migration of the labeled wild-type secretin receptor. CNBr cleavage of the V16M mutants labeled with each probe yielded a fragment migrating on a 10% NuPAGE gel at an approximate Mr of 5,000 that did not further shift after deglycosylation. This represented the nonglycosylated fragment Ala1-Val16. This labeled domain was further localized to Ala1-Val13 and then to Ala1-Pro8 by CNBr cleavage of the labeled V13M and P8M mutants, respectively (Fig. 5
). Again, it should be noted that a labeled nonglycosylated fragment migrating at an approximate Mr of 8500 resulting from CNBr cleavage of the V16M, V13M, and P8M receptor mutants labeled with the Ac-Arg2,Bpa1 probe was observed. This will be discussed later.

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Fig. 5. CNBr Cleavage of the Labeled Secretin Receptor Mutants
Left panels show representative autoradiographs of 10% NuPAGE gels used to separate the products of photoaffinity labeling of the receptor mutant V16M (A), V13M (C), and P8M (E) by each of the probes indicated. All three mutants were able to be affinity labeled saturably and specifically with each probe. The labeled receptors migrated at an approximate Mr of 70,000 and shifted to Mr 42,000 after deglycosylation with endoglycosidase F (Endo F). Right panels are representative autoradiographs of 10% NuPAGE gels used to separate the products of CNBr digestion of the receptor mutant V16M (B), V13M (D) and P8M (F) labeled by each probe. CNBr cleavage of the V16M, V13M, and P8M receptor constructs labeled by each probe consistently resulted in nonglycosylated fragment bands migrating at Mr values of 5000, 4500, and 4000, respectively. This progressively identified that the segment Ala1-Pro8 at the amino terminus of the receptor contained the site of labeling with each probe. Of note, CNBr cleavage of all three mutants labeled with the Ac-Arg2,Bpa1 probe also yielded a nonglycosylated fragment migrating at Mr 8500 (see Fig. 7 ).
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Manual Edman degradation sequencing of the purified labeled fragment resulting from CNBr cleavage of the labeled secretin receptor was performed to identify the site of labeling with each of these four probes. As shown in Fig. 6
, a radioactive peak eluted consistently in cycle 3 for both Ac-Bpa1 and Ac-Bpa2 probes, and cycle 4 for both Ac-Lys2,Bpa1 and Ac-Arg2,Bpa1 probes. These data indicated that the Ac-Bpa1 and Ac-Bpa2 probes each labeled receptor residue, Thr3, whereas the Ac-Lys2,Bpa1 and Ac-Arg2,Bpa1 probes each labeled an adjacent residue, Val4.

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Fig. 6. Radiochemical Edman Degradation Sequencing
Shown are the profiles of eluted radioactivity from sequencing of the purified CNBr fragment of the secretin receptor labeled with each of the probes indicated. A radioactive peak consistently eluted in cycle 3 in experiments performed with CNBr fragments labeled with either the Ac-Bpa1 (A) or Ac-Bpa2 (B) probe, corresponding to the attachment of each probe to Thr3 of the secretin receptor. In experiments performed with fragments labeled with either the Ac-Lys2,Bpa1 (C) or Ac-Arg2,Bpa1 (D) probe, a radioactive peak consistently eluted in cycle 4, corresponding to the attachment of each probe to Val4 of the secretin receptor. These data represent three independent experiments. a.a., Amino acids.
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As mentioned above, apart from labeling the distal amino terminus of the secretin receptor, the Ac-Arg2,Bpa1 probe also labeled a CNBr fragment migrating at Mr 8500 that did not shift after deglycosylation (Figs. 3
and 5
). Giving the molecular mass of the probe (3528 Da) and evidence of nonglycosylated nature of the labeled fragment, there were two candidates that best match these data. These represent the region His158-Met197 including TM2 and the first extracellular loop (ECL1), and the fragment Arg300-Met344 spanning the third intracellular loop (ICL3), TM6, and the beginning of ECL3 (Fig. 3
). Of note, the latter was the domain of labeling for unblocked amino-terminal photolabile secretin probes (28).
To further identify which fragment was most relevant, two previously characterized secretin receptor constructs, A175M and I334M (28), which contained a Met in each of these two candidate fragments, were used. These constructs were specifically and saturably labeled by the Ac-Arg2,Bpa1 probe, with similar efficiency as labeling the wild-type receptor (Fig. 7A
). Both constructs were then used for CNBr cleavage (Fig. 7B
). As shown, CNBr cleavage of the A175M mutant receptor labeled with the Ac-Arg2,Bpa1 probe resulted in a band migrating at Mr 8500, similar to that from the wild-type receptor. However, CNBr cleavage of the labeled I334M mutant yielded a fragment migrating at an approximate Mr of 4500, distinct in size from the fragment resulting from cleavage of the labeled wild-type or A175M receptor constructs (Mr = 8500). This identified that the site of labeling with the Ac-Arg2,Bpa1 probe was within the fragment Arg 300-Met344 spanning the ICL3, TM6, and the beginning of ECL3. Based on the size of the fragment, the carboxyl-terminal segment (Val335-Met344) was likely the domain of labeling. This was confirmed by the fact that this fragment was not cleavable with endoproteinase Lys-C (Fig. 7C
). In conclusion, the Ac-Arg2,Bpa1 probe labeled the segment Val335-Met344 spanning the top of TM6 and the beginning of the ECL3, the same segment as the unblocked amino-terminal Bpa2 and Bpa1 probes (28).

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Fig. 7. Identification of the Second Site Labeled by the Ac-Arg2,Bpa1 Probe
A, Shown is the ability for the Ac-Arg2,Bpa1 probe to affinity label the A175M and I334M secretin receptor mutants. B, CNBr cleavage of the labeled A175M mutant yielded a labeled fragment migrating at an approximate Mr of 8500, similar to that resulting from cleavage of the wild-type receptor. However, CNBr cleavage of the labeled I334M receptor yielded a labeled fragment migrating at an approximate Mr of 4500, distinct in size from the fragment from the wild-type receptor. This identified that the fragment Arg300-Met344 contained the site of labeling with the Ac-Arg2,Bpa1 probe. C, Endoproteinase Lys-C (Lys-C) cleavage of the labeled Mr 8500 CNBr fragment from the wild-type receptor resulted in a fragment migrating at an approximate Mr of 5000, representing segment Ser321-Met344. However, the labeled Mr 4500 fragment from the I334M mutant receptor was not cleavable by Lys-C. This identified the fragment Val335-Met344 at the top of TM6 and the beginning of the ECL3 as the domain of labeling for the Ac-Arg2,Bpa1 probe. D, The CNBr fragment from the labeled I334M/M344I secretin receptor migrated on a 10% NuPAGE gel at an approximate Mr of 14,500, representing the fragment Val335-Ile427, distinct in size from the Mr 4,500 CNBr fragment (Val335-Met344) from the I334M mutant receptor. These data further confirmed the identification of the region of labeling by the Ac-Arg2,Bpa1 probe. Of note, the Mr 19,000 band in panels B and D represents the labeled CNBr fragment at the amino terminus of the secretin receptor (see Fig. 3 ). E, The profile of eluted radioactivity of radiochemical Edman degradation sequencing of purified CNBr fragment (Val335-Ile427) from the I334M/M344I receptor labeled with the Ac-Arg2,Bpa1 probe. A peak appeared in cycle 1, which corresponds with covalent labeling of receptor residue Val335 with this probe. Data represent the means ± SEM of three independent experiments. Endo F, Endoglycosidase F; WT, wild type.
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To identify the specific residue labeled by the Ac-Arg2,Bpa1 probe by radiochemical sequencing, a previously characterized secretin receptor mutant construct I334M/M344I was used (28). This mutant was saturably and specifically labeled by the Ac-Arg2,Bpa1 (data not shown). CNBr cleavage of the labeled I334M/M344I mutant resulted in a band migrating at an approximate Mr of 14,500, representing the fragment Val335-Ile427, distinct in migration from the Mr 4,500 CNBr fragment resulting from cleavage of the labeled I334M receptor (Fig. 7D
). Radiochemical sequencing of the labeled fragment Val335-Ile427 identified Val335 as the site of labeling by the Ac-Arg2,Bpa1 probe (Fig. 7E
).
Identification of the Site of Labeling by the Ac-Bpa1 Probe
As shown in Fig. 3
, CNBr cleavage of the secretin receptor labeled with the Ac-Bpa1 probe also yielded a nonglycosylated fragment that migrated at an approximate Mr of 8500. This pattern of migration was distinct from that of the regions labeled by probes described above. Given the molecular mass of the probe (3232 Da) and evidence of the nonglycosylated nature of the labeled fragment, the candidates representing this migration are the region His158-Met197, including TM2 and ECL1, and the fragment Arg300-Met344 spanning ICL3, TM6, and the beginning of ECL3 (Fig. 3
). It should be noted that the latter was the domain of labeling for unblocked amino-terminal photolabile secretin probes (28) and one of the domains of labeling for the Ac-Arg2,Bpa1 probe as described above.
The I334M secretin receptor mutant was used to identify whether the segment Arg300-Met344 was also the domain of the Ac-Bpa1 probe. It was labeled saturably and specifically by this probe (data not shown). CNBr cleavage of the labeled receptor I334M receptor mutant yielded a labeled band migrating at an approximate Mr of 4500, distinct in migration from the Mr 8500 CNBr fragment resulting from cleavage of the labeled wild-type receptor (Fig. 8A
). This identified that the segment Arg300-Met344 contained the site of labeling by the Ac-Bpa1 probe. Figure 8B
shows that the Mr 4500 CNBr fragment from cleavage of the labeled I334M mutant was not cleavable by endoproteinase Lys-C cleavage, suggesting that the segment Val335-Met344 contained the site of labeling. This was further confirmed by CNBr cleavage of the labeled I334M/M344I mutant that yielded a band migrating at an approximate Mr of 14,500, representing the fragment Val335-Ile427. Radiochemical sequencing of the CNBr fragment from the I334M/M344I mutant identified Val335 as the site of labeling by the Ac-Bpa1 probe (Fig. 8D
).

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Fig. 8. Identification of the Site of Labeling by the Ac-Bpa1 Probe
A, CNBr cleavage of the labeled I334M secretin mutant yielded a fragment migrating on a 10% gel at an approximate Mr of 4500, distinct in size from the Mr 8500 fragment from CNBr cleavage of the wild-type receptor. This identified the receptor fragment Arg300-Met344 spanning ICL3, TM6, and ECL3 as the domain of labeling by the Ac-Bpa1 probe. B, The Mr 4500 fragment from the labeled I334M receptor was not cleavable by endoproteinase Lys-C, indicating that the segment Val335-Met344 contained the site of labeling. C, CNBr cleavage of the labeled I334M/M344I receptor yielded a fragment migrating at Mr 14,500, distinct in size from the Mr 4,500 fragment from CNBr cleavage of the I334M mutant receptor. This served to further confirm the domain identification described above. D, Identification of receptor residue Val335 as the site of labeling by the Ac-Bpa1 probe by radiochemical Edman degradation sequencing of purified CNBr fragment (Val335-Ile427) from the labeled I334M/M344I receptor. Data represent the means ± SEM of three independent experiments. a.a., Amino acids; Lys-C, endoproteinase Lys-C; WT, wild type.
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Secretin Receptor Charge Mutants
Each of the acidic residues present between TM6 and TM7 (ECL3 region) of the secretin receptor was mutated to an uncharged Ala residue. The mutated receptor constructs were transfected into COS cells, where immunohistochemistry was used to examine cell surface expression (Fig. 9
). Indeed, each mutant receptor construct was shown to be expressed on the cell surface in density similar to the wild-type receptor. An appropriate negative control represented the COS cells transfected with the empty pcDNA3 vector.

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Fig. 9. Effects of Mutation of Charged Residues on Secretin Receptor Binding and Activation
Top panel, Immunohistochemical evidence for normal cell surface expression of the HA-epitope-tagged secretin receptor constructs having acidic residues changed to neutral Ala residues. Shown are COS cells transfected with wild-type (WT) secretin receptor (A), the empty pcDNA3 eukaryotic expression vector (B), or the secretin receptor mutants (C, E341A; D, D342A; E, E345A; and F, E351A). Images are representative of three independent experiments. Bottom panel, Secretin radioligand binding experiments (A) and concentration dependency of secretin-stimulated cAMP (B) of these constructs expressed in intact COS cells. Data represent the means ± SEM of three independent experiments.
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Of these mutant secretin receptors, all except for the E351A construct were observed to bind secretin normally, with E351A not exhibiting any saturable binding of this hormone (Fig. 9
). The Ki values for the E341A, D342A, and E345A constructs were not significantly different from that of the wild-type receptor (values in nanomolar concentration: wild type, 6.7 ± 1.2; E341A, 7.0 ± 1.4; D342A, 5.6 ± 1.0; E345A, 5.8 ± 0.9). Secretin-stimulated biological activity was also studied using a standard cellular cAMP assay. Here too, cellular responses to E341A, D342A, and E345A were not different from those in wild-type receptor, whereas the E351A receptor-bearing cells exhibited only a small partial response to very high concentrations of secretin (Fig. 9
). EC50 values to stimulate cAMP were the following (in picomolar concentrations of secretin): wild type, 64 ± 8; E341A, 73 ± 11; D342A, 66 ± 6; E345A, 87 ± 12; whereas cells expressing the E351A construct reached only 21% of the maximal cAMP response at a secretin concentration of 1 µM.
Each of the mutant receptors was further tested in receptor internalization studies in COS cells. As shown in Fig. 10
, like the wild-type receptor, the E341A, D342A, and E345A mutant receptor constructs on the cell surface were clearly occupied by fluorescent secretin after a 1-h preincubation period at 4 C. Upon warming to 37 C, the fluorescent labeling patterns changed rapidly. After 2 min of warming, the fluorescence displayed a more punctate pattern at or near the plasmalemma. By 5 min, most fluorescence was present in vesicular structures in the perinuclear region, with this pattern persisting through the 30-min time point. There was no consistent or quantifiable difference in the pattern of internalization of the fluorescent secretin-receptor complex between these constructs and that of the wild-type receptor. It should be noted that the time-dependent pattern of internalization of agonist-occupied secretin receptors transiently expressed in COS cells was similar to that previously reported for CHO-SecR cells stably expressing the wild-type secretin receptor (30). Consistent with its secretin binding characterization, the E351A construct was not labeled by the fluorescent Alexa-secretin ([rat secretin-27]-Gly-Cys-Alexa488), making it impossible to assess agonist-stimulated internalization of this construct using this method (Fig. 10
, upper panel). We, therefore, also evaluated receptor internalization using immunohistochemistry of HA-tagged constructs (Fig. 10
, lower panel). For this, COS cells expressing the HA-tagged E351A and wild-type receptor constructs were exposed to the same experimental protocol, except using nonfluorescent secretin. At each time point, the receptors were localized by immunostaining of permeabilized cells with anti-HA antibody. After preincubation for 1 h, receptors were localized to the cell surface. The wild-type receptor was shown to internalize normally after exposure to secretin, whereas the immunostaining of the E351A receptor construct remained at the cell surface.

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Fig. 10. Effects of Mutation of Charged Residues on Secretin Receptor Internalization
Top panel, Time course of internalization of the Alexa-secretin-occupied secretin receptor constructs in COS cells. Cells were preincubated at 4 C in 50 nM Alexa-secretin, washed, and warmed to 37 C for 1, 5, and 30 min. Aliquots were fixed with paraformaldehyde at time 0 or after warming for the noted times. Images are representative of three independent experiments. Bottom panel, Time course of internalization of the secretin-occupied wild-type and E351A mutant receptor in COS cells. Cells were incubated in 50 nM nonfluorescent secretin for 1 h at 4 C and then warmed for the noted periods of time. Shown are representative immunofluorescent images of cells incubated with anti-HA antibody after permeablization with 0.1% saponin, with reactivity demonstrated using Alexa488-conjugated goat antimouse IgG. Images are representative of three independent experiments. WT, Wild type.
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DISCUSSION
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The Class B secretin receptor family of G protein-coupled receptors contains numerous potentially important drug targets. Understanding of the themes for agonist binding and activation of these receptors will help us move closer toward rational design of receptor-active drugs. The current work extends our efforts to explore the spatial approximations between the amino terminus of secretin and its receptor using photoaffinity labeling and receptor mutagenesis. Recently, we used a series of secretin agonists incorporating photolabile residues at the amino terminus (Bpa1) and at its extension (Bpa1 and Bpa2) of the peptide ligands and demonstrated that they all labeled residues within the top of TM6 of the secretin receptor (28). In this work, we acetylated these probes (Ac-Bpa1, Ac-Bpa2, and Ac-Bpa1) to eliminate the charged amino termini, characterized them, and explored their molecular approximations with the receptor. In addition, we used two new probes that had a positively charged residue Lys or Arg at the extension of the Bpa1 probe (Ac-Lys2,Bpa1 and Ac-Arg2, Bpa1).
These analogs were all shown to be full agonists, and bound to the secretin receptor specifically and saturably, although moving the photolabile Bpa moiety toward the position of His1 resulted in substantial interference with binding affinity and biological potency. The probes were shown to covalently label the secretin receptor efficiently. The sites of labeling of the highest affinity acetylated probes having a Bpa at the extension (position 1 and 2) of the peptide ligand were identified as either residue Thr3 or Val4 within the amino-terminal tail domain of the receptor, a region distinct from the domain of labeling by their parental probes. The acetylated Bpa1 probe labeled Val335 at the top of TM6 of the receptor, the same domain labeled by its parental probe; however, the low affinity binding and low biological potency of this probe raises some concerns about the implications of this labeling relative to that of the highest affinity and potency probes in this series. Of note, the Ac-Arg2,Bpa1 probe also labeled a second site at the same domain and same residue (Val335) as the Ac-Bpa1 probe.
The long and complex amino-terminal domain of the secretin receptor has been shown to be critical for ligand binding based on receptor mutagenesis and chimeric receptor studies (2, 5), as well as extensive photoaffinity labeling studies (17, 18, 19, 20, 21, 22). Notably, almost all of the photoaffinity labeling studies performed to date resulted in covalent attachments to this domain. It is notable that photolabile residues distributed widely throughout secretin each labeled the same receptor domain. We have previously shown that photolabile residues incorporated in the amino-terminal region (in position 6), midregion (in positions 12, 13, and 14) and carboxyl-terminal region (in positions 18, 22, and 26) of this hormone each labeled residues within the amino terminus of the secretin receptor. All of these studies point to the amino terminus of the secretin receptor as providing a structurally important platform for binding the natural agonist peptide ligand. Finally, we were able to demonstrate that the amino terminus of secretin lies in spatial approximation with a part of the body of this receptor (28). In that work, receptor residues high in TM6 were labeled through the position His1 of secretin or amino-terminal extensions in positions 1 and 2. These insights seemed to support a mechanism for receptor activation similar to that proposed for the structurally related PTH receptor (31, 32), where the agonist peptide might act as a tether. This can then pull together two distinct domains of the receptor, providing a mechanism to change the conformation of the receptor, affecting the cytosolic domain of coupling with G proteins. This may be a common theme for the class B G protein-coupled receptors, as further supported by recent analogous observations with the calcitonin (25, 26, 27) and vasoactive intestinal polypeptide type 1 (VPAC1) (23, 24) receptors.
The current data with the acetylated amino-terminal probes that labeled the extracellular amino terminus of the secretin receptor suggest that charge is critical for the positioning of the peptide amino terminus adjacent to TM6 of the receptor. With their
-amino groups being blocked by acetylation to eliminate one positive charge, these amino-terminal photolabile probes still retained their binding affinity and full biological activity, while moving their sites of covalent labeling away from the receptor TM6. This may suggest that finite and critical interactions for the tethering role may be less critical than previously suggested. As an initial effort to identify residues that might be involved in charge pairing with the amino terminus of natural secretin, we performed Ala-replacement mutagenesis of receptor acidic residues Glu341, Asp342, and Glu345 within the top of TM6 and the beginning of ECL3. However, we did not observe any negative impact of these mutations on secretin-stimulated biological activity or secretin binding affinity. Other candidate acidic residues that may be spatially close to the covalently labeled area included the mutation of Glu351, at the end of ECL3 near TM7. Of note, this mutation completely eliminated secretin binding and ultimate receptor internalization, while mediating only a small partial biological response to secretin. This response in intact cells established that the receptor was able to traffic to the cell surface to interact with the large peptide ligand, but that this receptor mutation markedly interfered with its binding and action. Other candidate residues for charge pairing may be identified as our molecular models improve further.
In conclusion, the amino-terminal domain of the secretin receptor likely folds into a unique structure that is functionally critical for ligand binding and activity. Multiple residue-residue approximations between divergent regions of the secretin pharmacophore and its receptor have been established, with all falling within this domain, except for the amino terminus of secretin that is spatially approximated with the top of TM6 of the receptor. Modification of the charge of the amino terminus of the peptide ligand retains its binding activity and full biological activity, but interferes with this spatial approximation with TM6 of the secretin receptor. Mutation of E351A to eliminate its charge also eliminated natural secretin binding and internalization and dramatically decreased its biological activity. This acidic residue may contribute to the charge pairing with the amino terminus of secretin that is responsible for its normal docking between the receptor transmembrane domains and amino terminus.
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MATERIALS AND METHODS
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Materials
Rat secretin and [Tyr10]secretin were prepared in our laboratory (33). We also synthesized a new, biologically active, fluorescent analog of secretin, [rat secretin-27]-Gly-Cys-Alexa488 (Alexa-secretin), for morphological analysis of cellular handling of the agonist-occupied receptor, based on the design of [rat secretin-27]-Gly-rhodamine that we used previously (30). Synthesis was accomplished using manual, solid-phase techniques analogous to those used previously (33, 34).
CNBr and solid-phase oxidant N-chloro-benzenesulfonamide (Iodo-beads) were purchased from Pierce Chemical Co. (Rockford, IL). Endoproteinase Lys-C and the mouse 12CA5 monoclonal antibody against the HA epitope were from Roche Applied Science (Indianapolis, IN). Alexa488-conjugated goat antimouse IgG was supplied by Molecular Probes, Inc. (Eugene, OR). Endoglycosidase F was prepared in our laboratory (35). All other reagents were analytical grade.
Photoaffinity Labeling Probes
The peptide ligand probes, [Ac-Bpa1,Tyr10]secretin (Ac-Bpa1 probe), [Ac-Bpa1,Tyr10]secretin (Ac-Bpa1 probe), [Ac-Bpa2, Gly1,Tyr10]secretin (Ac-Bpa2 probe), [Ac-Lys2,Bpa1, Tyr10]secretin (Ac-Lys2,Bpa1 probe) and [Ac-Arg2,Bpa1, Tyr10]secretin (Ac-Arg2,Bpa1 probe) were designed to contain a Tyr residue at position 10 of rat secretin-27 for radioiodination, and a photolabile residue, p-benzoyl-phenylalanine (Bpa), in position 1, 1, or 2 for covalent labeling of its receptor, while blocking the
-amino groups by acetylation. These probes were synthesized by manual solid-phase techniques and were purified to homogeneity by reversed-phase HPLC using techniques that were previously described (34). Their chemical identity was established by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. They were radioiodinated oxidatively with Na125I upon exposure to an Iodo-bead (Pierce) for 15 sec and purified by reversed-phase HPLC to yield specific radioactivity of 2000 Ci/mmol (34).
Receptor-Bearing Cell Lines
Receptor-bearing CHO cell lines stably expressing wild-type (CHO-SecR), HA-tagged (CHO-SecR-HA37 and CHO-SecR-HA79), V13M (CHO-SecR-V13M-HA37), and V16M (CHO-SecR-V16M-HA37) mutant secretin receptors, which have been previously established and characterized (17, 18, 19, 20, 33), were used as sources of receptors for the current study. Each of these secretin receptor constructs has been demonstrated to be expressed on the cell surface and to bind with appropriate specificity and high affinity. These stable CHO cell lines were cultured at 37 C in a 5% CO2 environment on Falcon tissue culture plastic ware in Hams F-12 medium supplemented with 5% fetal clone-2 (Hyclone Laboratories, Inc., Logan, UT). Cells were passaged twice weekly and were lifted mechanically before use. Receptor-enriched plasma membranes were prepared from these cell lines using methods that we reported previously (36).
Four additional mutant secretin receptor constructs that either incorporated an additional site for CNBr cleavage or eliminated a naturally occurring Met residue in key positions were used for the current work. These represented P8M, A175M, I334M, and I334M/M344I secretin receptor constructs that have been previously established and characterized (21, 28). They were expressed transiently on COS cells (American Type Cell Collection, Manassas, VA) after transfection using a modification of the diethylaminoethyl-dextran method (37), and cells were harvested mechanically 72 h after transfection.
Four more secretin receptor site mutants were constructed to represent the elimination of acidic residues in the region of the receptor that represented the best candidates for potential charge pairing with the amino terminus of secretin. These included HA-tagged E341A (SecR-E341A-HA37), D342A (SecR-D342A-HA37), E345A (SecR-E345A-HA37), and E351A (SecR-E351A-HA37), which were prepared using an oligonucleotide-directed approach with the QuikChange Site-Directed Mutagenesis kit from Stratagene (La Jolla, CA). These constructs were subcloned into the eukaryotic expression vector, pcDNA3 (Invitrogen, Carlsbad, CA), and their sequences were confirmed by direct DNA sequencing (38). These were also transiently expressed in COS cells and were studied for secretin binding, secretin-stimulated cAMP responses, and internalization.
Biological Activity Assays
The agonist activity of each of the photoaffinity labeling probes was studied for stimulation of cAMP activity in receptor-bearing cells using a competition-binding assay (Diagnostic Products Corp., Los Angeles, CA). In brief, cells grown in 24-well plates were washed in ice-cold PBS and stimulated by increasing concentrations (01 µM) of peptide for 30 min at 37 C in Krebs-Ringer-HEPES medium (25 mM HEPES, pH 7.4; 104 mM NaCl; 5 mM KCl; 1 mM KH2PO4; 1.2 mM MgSO4; 2 mM CaCl2) containing 1 mM 3-isobutyl-1-methylxanthine, 0.01% soybean trypsin inhibitor, 0.1% bacitracin, and 0.2% BSA. The reactions were stopped by adding ice-cold perchloric acid. After the pH was adjusted 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 (39). Radioactivity was quantified by scintillation counting in a Beckman LS6000 counter. Assays were performed in duplicate and repeated in at least three independent experiments.
Ligand Binding Studies
Binding of the secretin analogs to their receptors was characterized in a standard assay using membranes from receptor-bearing cells (typically the CHO-SecR cells, unless otherwise noted) as source of receptor. Membranes (5 µg) were incubated with a constant amount of radioligand, 125I-[Tyr10]secretin (5 pM), in the presence of increasing concentrations of each of the probes (01 µM) for 1 h at room temperature in Krebs-Ringer-HEPES medium containing 1 mM phenylmethylsulfonyl fluoride, 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 filter mats that had been soaked in 0.3% Polybrene for 1 h, and 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, receptor-bearing membranes from receptor-bearing CHO cells containing approximately 50 µg protein were incubated with approximately 0.1 nM radioiodinated probe in the presence of increasing concentrations of secretin (01 µM) for 1 h at room temperature before 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 receptor purification, larger amounts of membrane (
200 µg) were incubated with each of the above radiolabeling probes (
0.5 nM) in the absence of competing secretin. After photolysis, membranes were washed, pelleted, solubilized in sodium dodecyl sulfate (SDS) sample buffer, and applied to a 10% SDS-polyacrylamide gel for electrophoresis (40). Radiolabeled bands were detected by autoradiography.
Radioactive receptor bands were cut out of the gel and homogenized in a Dounce homogenizer in water, followed by gel purification, elution, lyophilization, and ethanol precipitation. Materials purified to radiochemical homogeneity were directly used for chemical or enzymatic cleavage experiments. CNBr and endoproteinase Lys-C were used separately and in sequence to cleave the labeled secretin receptor and its fragments using procedures previously described (18). The products of cleavage were resolved on 10% NuPAGE gels using 2-(N-morpholino)ethanesulfonic acid running buffer (Invitrogen). Labeled bands were identified by exposure to x-ray film with intensifying screens at 80 C. Aliquots of affinity-labeled secretin receptor and relevant receptor fragments were deglycosylated with endoglycosidase F, as described previously (19).
Identification of the affinity-labeled secretin receptor fragments was achieved by immunoprecipitation of the affinity-labeled, well-characterized HA-tagged receptor constructs and their CNBr fragments using anti-HA monoclonal antibody, as we previously described (17).
Radiochemical Sequencing
For this, the purified radiolabeled fragments from either CNBr cleavage of the wild-type secretin receptor or the I334M/M344I mutant receptor were coupled to N-(2-aminoethyl-1)-3-aminopropyl glass beads (Sigma Chemical Co., St. Louis, MO) through sulfhydryl groups within Cys residues (for wild-type receptor, these represented Cys11, Cys24, and Cys44; for the I334M/M344I mutant receptor, this represented Cys367). Cycles of Edman degradation were repeated manually in a manner that has been previously reported in detail (41), and the radioactivity released in each cycle was quantified in a
-spectrometer.
Immunofluorescence Microscopy
For morphological assessment of receptor expression on the cell surface, COS cells transfected with wild-type and ECL3 mutant secretin receptors were replated to grow on glass coverslips. After washing with PBS [1.5 mM NaH2PO4; 8 mM Na2HPO4; 145 mM NaCl (pH 7.4)], intact cells were fixed in 2% paraformaldehyde in PBS for 30 min, washed twice with PBS, and incubated for 1 h with 1% normal goat serum in PBS to block nonspecific binding sites and an additional hour with mouse monoclonal anti-HA antibody (1:500). After washing three times with PBS containing 1% normal goat serum, cells were incubated with Alexa488-conjugated goat antimouse IgG (1:200) for 1 h. Cells were again washed three times with PBS, mounted, and examined with a Zeiss Axiovert 200M inverted microscope (Carl Zeiss, Oberkochen, Germany) equipped for epifluorescence. All above procedures were performed at room temperature.
Internalization Studies
Internalization of agonist-occupied wild-type and mutant secretin receptors was assayed morphologically, using the fluorescent Alexa-secretin analog described above, and following experimental protocols that we described previously in detail (42). The Alexa-secretin analog was characterized to bind with similar affinity and signal with similar potency to natural secretin (data not shown). Briefly, transfected COS cells grown on glass coverslips were washed twice with cold PBS containing 0.1 mM MgCl2 and 0.08 mM CaCl2 and labeled with 50 nM Alexa-secretin for 1 h at 4 C. The coverslips were then rinsed in cold PBS and incubated in PBS at 37 C for the time points indicated before being fixed in 2% paraformaldehyde, washed, and mounted on slides. Additionally, internalization of COS cells transfected with the wild-type and E351A secretin receptor constructs was also studied using an alternative procedure. For this, transfected COS cells grown on coverslips were incubated with 50 nM nonfluorescent secretin in place of Alexa-secretin for 1 h at 4 C before being warmed to the time points indicated. After fixation, cells were permeabilized with 0.1% saponin in PBS for 10 min, followed by immunostaining with mouse anti-HA antibody and Alexa488-conjugated goat antimouse IgG as described above. Labeled cells were examined with a Zeiss Axiovert 200M inverted microscope equipped for epifluorescence.
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, San Diego, CA). Binding kinetics was determined by analysis with the LIGAND program of Munson and Rodbard (43).
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ACKNOWLEDGMENTS
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We thank L.-A. Bruins, E. Holicky and E. M. Hadac for excellent technical assistance, and D. Huether for secretarial assistance. We thank Drs. K. G. Harikumar and C. S. Lisenbee for their helpful discussions.
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FOOTNOTES
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This work was supported by National Institutes of Health Grant DK46577 and the Fiterman Foundation.
First Published Online February 24, 2005
Abbreviations: Alexa-secretin, [Rat secretin-27]-Gly-Cys-Alexa488; Bpa, p-benzoyl-phenylalanine; (BzBz)Lys, benzoyl-benzoyl lysine; CHO, Chinese hamster ovary; CHO-SecR, secretin receptor-bearing CHO cells; CNBr, cyanogen bromide; ECL, extracellular loop; HA, hemagglutinin; ICL, intracellular loop; SDS, sodium dodecyl sulfate; TM, transmembrane segment or domain.
Received for publication October 18, 2004.
Accepted for publication February 17, 2005.
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