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Mayo Clinic Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Scottsdale, Arizona
Abstract
Understanding the structures of active and inactive agonist- and antagonist-bound receptor complexes is of great interest. In this work, we focus on position 30 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor. For this, we developed two photoaffinity labeling probes, replacing the naturally occurring tryptophan with p-benzoyl-L-phenylalanine (Bpa) or p-nitro-phenylalanine (NO2-Phe). The Bpa probe was shown to represent an antagonist, whereas the NO2-Phe probe stimulated intracellular calcium as a fully efficacious agonist (EC50 = 81 ± 15 nM). Both ligands bound to the receptor specifically, although with lower affinity than CCK (Ki values: Bpa probe, 270 ± 72 nM; NO2-Phe probe, 180 ± 40 nM). Both probes covalently labeled the receptor in an efficient manner. The Bpa antagonist labeled the receptor in two distinct regions as identified by cyanogen bromide cleavage, with labeled bands migrating at Mr = 25,000 and 4500. The former represented the glycosylated amino-terminal fragment, with the site of attachment further localized by endoproteinase Lys-C cleavage to the region between Asn10 and Lys37. The latter was shown to represent the first extracellular loop using further cleavage and sequencing of the wild-type and a mutant receptor. Following the same approach, the NO2-Phe agonist probe was shown to also label the first extracellular loop region. Radiochemical sequencing identified that the Bpa antagonist probe labeled receptor residue Lys105, whereas the NO2-Phe agonist probe labeled residue Leu99. These data extend our understanding of the molecular basis of binding and the conformational states of this important receptor.
Cholecystokinin (CCK) is a peptide hormone and neurotransmitter that plays important roles in nutrient homeostasis, regulating postcibal gallbladder contraction, pancreatic enzyme secretion, and gastrointestinal tract motility, while also being implicated in control of appetite. These functions support the broad potential of CCK receptors as drug targets. This goal of developing such drugs can be facilitated by a detailed understanding of the structure of the receptor mediating this action and by an understanding of the active and inactive conformations of this receptor.
CCK exhibits its agonist activities through binding to two subtypes of CCK receptors, types A and B, that represent structurally related members of the rhodopsin/-adrenergic receptor family (class I) of G protein-coupled receptors (GPCRs) (Noble et al., 1999; Miller and Lybrand, 2002). The structural specificity of these receptors differ, with the type A receptor being most selective, requiring the carboxyl-terminal heptapeptide-amide of CCK with a sulfated tyrosyl residue, whereas the type B receptor requires only the carboxyl-terminal tetrapeptide for high-affinity binding and potent biological activity. In this work, we focus on the type A CCK receptor.
The molecular basis of ligand binding by the type A CCK receptor has been studied much more extensively than that of the type B CCK receptor, with studies using structure-activity (Bodanszky et al., 1977, 1980; Miller et al., 1981), mutagenesis (Kennedy et al., 1997; Gigoux et al., 1999; Gouldson et al., 2000; Ding et al., 2002), and photoaffinity labeling (Ji et al., 1997; Hadac et al., 1998, 1999; Ding et al., 2001; Arlander et al., 2004) approaches. These studies have suggested a theme analogous to that of many peptide receptors in the class I family of GPCRs (Schwartz, 1994; Strader et al., 1994), in which CCK binds to extracellular loop and amino-terminal tail regions of its receptor. Among all approaches used, photoaffinity labeling is a method that provides direct specific residue-residue spatial approximations between a docked ligand and its receptor. We have previously used CCK agonist probes that incorporated photolabile residues within (in positions 27, 29, and 33) and outside (in position 24) the pharmacophore of the ligand (Ji et al., 1997; Hadac et al., 1998; Ding et al., 2001; Arlander et al., 2004) and demonstrated that they all labeled residues within the amino-terminal tail, and the second and third extracellular loop regions of this receptor. It should be noted that the first extracellular loop region has not been previously labeled by photolabile probes.
The tryptophan in position 30 of CCK is very interesting, located centrally within the pharmacophoric domain of the natural hormone. Spatial approximations between this ligand position and adjacent receptor residues have not previously been explored. Therefore, we have prepared a pair of CCK probes, one having a photolabile p-nitro-phenylalanine (NO2-Phe) and another having a p-benzoyl-L-phenylalanine (Bpa) in this position. Here, we report their characterization and use in photoaffinity labeling studies to further explore their spatial approximations with the CCK receptor.
Materials and Methods
Materials. Synthetic CCK-8 (CCK-26-33) was purchased from Peninsula Laboratories (Belmont, CA). Wheat germ agglutinin-agarose was from E. Y. Labs (San Mateo, CA) and Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR). Cyanogen bromide (CNBr) and m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester were from Pierce Chemical (Rockford, IL), and endoproteinase Lys-C was from Roche Diagnostics (Indianapolis, IN). N-(2-Aminoethyl-1)-3-aminopropyl glass beads were form Sigma-Aldrich (St. Louis, MO). Endoglycosidase F (EF) was prepared in our laboratory as reported previously (Pearson et al., 1987). All other reagents were analytical grade.
Peptides. The photolabile CCK analogs used in this study were des-amino-Tyr-Gly-[(Nle28,31,Bpa30)CCK-26-33] (Bpa30 probe) and des-amino-Tyr-Gly-[(Nle28,31,NO2-Phe30)CCK-26-33] (NO2-Phe30 probe) (Fig. 1). The former incorporated a photolabile Bpa moiety in position 30 to replace the native residue Trp, whereas the latter incorporated a photolabile NO2-Phe in the same position. Each peptide also had an amino-terminal extension of Tyr-Gly to provide a site for radioiodination, and Met residues at position 28 and 31 were replaced with oxidation-resistant norleucine (Nle) residues. Each probe had its amino terminus blocked to permit radiochemical sequencing of the labeled receptor fragments without concomitant probe cleavage during cycles of Edman degradation. These modifications were well tolerated as we demonstrated previously (Ji et al., 1997; Hadac et al., 1998; Ding et al., 2001). These probes, together with the radioligand D-Tyr-Gly-[(Nle28,31)CCK-26-33] that was used for receptor-binding assays (Fig. 1), were synthesized by solid-phase techniques and purified by reversed-phase high-performance liquid chromatography (HPLC) (Powers et al., 1988). Their identities were confirmed by mass spectrometry. They were radioiodinated oxidatively with Na125I upon exposure to the solid-phase oxidant N-chloro-benzenesulfonamide (IODO beads; Pierce, Rockford, IL), for 15 s and purified by reversed-phase HPLC to yield specific radioactivities of 2000 Ci/mmol (Powers et al., 1988).
Receptor Sources. The receptor-bearing Chinese hamster ovary (CHO) cell line that was previously established to stably express the rat type A CCK receptor (CHO-CCKR) (Hadac et al., 1996) was used as the source of wild-type receptor in the current study. Another CHO cell line stably expressing the L104M mutant CCK receptor that was prepared and fully characterized previously (Arlander et al., 2004) was used for mapping the sites of labeling in this study. Cells were grown on tissue culture plasticware in Ham's F-12 medium supplemented with 5% fetal clone 2 (Hyclone Laboratories, Logan, UT) in a humidified atmosphere with 5% CO2 at 37°C. Cells were passaged approximately twice a week and lifted mechanically before use. Plasma membranes were prepared from receptor-expressing cells using methods reported previously (Hadac et al., 1996), homogenized in Krebs-Ringers-HEPES (KRH) medium containing 25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1 mM KH2PO4, 0.01% soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride and stored at eC80°C until ready for use.
Biological Activity Assay. The biological activity of the photolabile probes was studied using a well characterized assay for stimulation of intracellular calcium accumulation in CHO-CCKR cells (Hadac et al., 1996). In brief, approximately 2 x 106 cells were loaded with 5 e Fura-2 acetoxymethyl ester in Ham's F-12 medium for 30 min at 37°C before being washed and stimulated with increasing concentrations of either the Bpa30 or NO2-Phe30 probe or CCK at 37°C. Fluorescence was quantified in a PerkinElmer Life and Analytical Sciences LS50B luminescence spectrometer. Excitation was performed at 340 and 380 nm, and emission was quantified at 520 nm, with calcium concentration calculated from the ratios, as described by Grynkiewicz et al. (1985). The peak intracellular calcium transient was used to determine agonist concentration dependence of the biological responses. Basal levels of calcium were measured in the absence of agonist stimulation, whereas maximum levels were determined as the peak intracellular calcium concentrations measured in the presence of a 1 e CCK. Stimulated values were calculated as percentages of the range of calcium concentrations between basal and maximal levels. Treatment of cells with the Bpa30 probe alone elicited no calcium response. The antagonist activity of this probe was examined by its inhibition of the CCK-induced calcium response that was demonstrated by treating CHO-CCKR cells in the presence of 0.1 nM CCK in the presence of varied concentrations of the Bpa30 probe.
Binding Assay. The binding activity of the photoaffinity labeling probes was studied using a standard radioligand competition-binding assay using CHO-CCKR membranes, as we described previously (Hadac et al., 1996). In brief, 5 e of plasma membranes were incubated with increasing concentrations (0eC1 e) of either the Bpa30 or NO2-Phe30 probe or CCK in the presence of a constant amount of the radioiodinated radioligand D-Tyr-Gly-[(Nle28,31)CCK-26-33] (5 pM), for 1 h at room temperature in KRH medium containing 0.2% bovine serum albumin. Bound and free radioligand were separated using a Skatron cell harvester (Molecular Devices, Sunnyvale, CA) with glass fiber filtermats, with bound radioactivity quantified in a gamma counter. Nonspecific binding was determined in the presence of 1 e CCK and represented less than 20% of total binding.
Photoaffinity Labeling. This was done using an established procedure that we described previously (Ding et al., 2001). In brief, 50 e of plasma membranes from receptor-bearing CHO cells expressing wild-type or mutant receptors or from control noneCreceptor-bearing parental CHO cells was incubated with 0.1 nM radioiodinated Bpa30 or NO2-Phe30 probe in the absence or presence of increasing concentrations of competing nonradioactive CCK (0eC1 e) for 1 h at room temperature in the dark. After incubation, the reactions were transferred to a ice-cold siliconized glass tubes (12 x 75 mm) for photolysis. This was performed for 30 min at 4°C in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) equipped with 3500- (for the Bpa30 probe) or 3000- (for the NO2-Phe30 probe) lamps. Membranes were then solubilized with 1% Nonidet P-40 in KRH medium before wheat-germ agglutinin-agarose affinity chromatography and subsequent SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). Radiolabeled bands were visualized by autoradiography.
Peptide Mapping. To map the regions of receptor labeling using chemical cleavage and subsequent sequencing, it was necessary to prepare affinity-labeled receptor in larger scale. For this, 200 e of receptor-bearing membranes and 0.5 nM 125I-Bpa30 or 125I-NO2-Phe30 probe were incubated in the absence of competing CCK before photolysis. After electrophoresis, radioactive protein bands were excised from polyacrylamide gels, eluted with water, lyophilized, ethanol-precipitated, and cleaved with 2.5 mg of CNBr in 70% formic acid, as described in detail previously (Hadac et al., 1998). Samples were then washed twice with water and dried in a vacuum centrifuge before being resolved on a 10% NuPAGE gel using MES running buffer (Invitrogen, Carlsbad, CA). Deglycosylation was performed on gel-purified receptor or fragment using conditions described previously (Hadac et al., 1996), and the products were analyzed on 10% SDS-polyacrylamide gels (for intact receptor) or 10% NuPAGE gels (for receptor fragments), and labeled bands were visualized by autoradiography.
Identification of the domain of labeling of the CCK receptor by the Bpa30 probe was also achieved by Edman degradation sequencing of the purified CNBr fragments of the labeled receptor using strategies described previously (Hadac et al., 1998; Dong et al., 2004). In brief, 10 mg of CHO-CCKR plasma membranes photoaffinity labeled with 0.1 e nonradioactive Bpa30 probe were mixed with 1 mg of membranes labeled with 1 nM 125I-Bpa30 probe and were gel-purified by the procedure described above. The labeled receptor was further cleaved by CNBr, and the resultant fragments were purified by the procedure described that included 10% NuPAGE gel electrophoresis and microbore C18 HPLC before being subjected to Edman degradation sequencing using an Applied Biosystems automated instrument (Hadac et al., 1998; Dong et al., 2004).
Radiochemical Sequencing. Gel-purified CNBr fragments from cleavage of labeled wild type and L104M mutant CCK receptor constructs were used for radiochemical Edman degradation sequencing to localize the receptor residues labeled by both the Bpa30 and NO2-Phe30 probes. Sequencing was performed on cysteine-containing CNBr fragments (Pro96-Met121 from the wild-type receptor and Lys105-Met121 from the L104M mutant receptor) of the receptor that were immobilized through the thiol group of Cys114 using the bifunctional cross-linker m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester and N-(2-aminoethyl-1)-3-aminopropyl glass beads. Edman degradation was manually repeated in a manner that has been reported previously in detail (Ji et al., 1997; Hadac et al., 1998), and the radioactivity released in each cycle was quantified in a gamma spectrometer.
Statistical Analysis. All observations were repeated at least three times in independent experiments and are expressed as mean ± S.E.M. Binding data were analyzed and plotted using the nonlinear regression analysis routine for radioligand binding in the Prism software package (GraphPad Software Inc., San Diego, CA), and binding kinetics was determined by analysis with the LIGAND program of Munson and Rodbard (1980).
Results
Probe Characterization. The photoaffinty labeling probes used in this study were des-amino-Tyr-Gly-[(Nle28,31,Bpa30)CCK-26-33] (Bpa30 probe) and des-amino-Tyr-Gly-[(Nle28,31,NO2-Phe30)CCK-26-33] (NO2-Phe30 probe) (Fig. 1). Both incorporated a photolabile residue at position 30 to replace the naturally occurring Trp residue, with one probe having a Bpa and the other having a NO2-Phe. Both probes were synthesized by solid-phase techniques and purified by reversed-phase HPLC. They were characterized to demonstrate the expected molecular masses by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.
Probe usefulness in photoaffinity labeling studies was first evaluated by characterization of binding and biological activity. As shown in Fig. 2, both probes bound to the CCK receptor specifically and saturably, although with lower affinity than natural CCK (Ki values: CCK, 0.9 ± 0.1 nM; Bpa30 probe, 270 ± 72 nM; and NO2-Phe30 probe, 180 ± 40 nM). Although less potent than natural CCK, the NO2-Phe30 probe was a full agonist, stimulating intracellular calcium accumulation in CHO-CCKR cells in a concentration-dependent manner (EC50 values: CCK, 9.2 ± 2.3 pM; and NO2-Phe30 probe, 81 ± 15 nM). However, the Bpa30 probe was an antagonist, having no endogenous agonist activity at a concentration as high as 1 e (Fig. 2). It inhibited 0.1 nM CCK-stimulated calcium signaling in a concentration-dependent manner (IC50 = 2.0 ± 0.1 e, Fig. 2). Thus, we have developed a pair of structurally related photoaffinity labeling probes having their photolabile site of covalent attachment in the same position within the pharmacophore of CCK, but having distinct biological activities, one representing a full agonist and the other an antagonist.
Photoaffinity Labeling of the CCK Receptor. Both the Bpa30 and NO2-Phe30 probes were used to examine their ability to label the CCK receptor. Figure 3 shows that both probes covalently labeled the CCK receptor specifically and saturably, with the labeling inhibited by natural CCK in a concentration-dependent manner (IC50 values: Bpa30 probe, 15.5 ± 2.9 nM; and NO2-Phe30 probe, 36 ± 4 nM). Consistent with previous photoaffinity labeling studies of this receptor (Ji et al., 1997; Hadac et al., 1998; Ding et al., 2001; Arlander et al., 2004), the band labeled by each probe migrated at approximate Mr = 85,000 to 95,000 and shifted to approximate Mr = 42,000 after deglycosylation with endoglycosidase F. Attempts to label noneCreceptor-bearing CHO cell membranes did not yield visible radioactive bands on the gel.
Mapping the Receptor Ligand-Binding Domain. The receptor regions covalently labeled by the probes were examined using proteolytic cleavage. CNBr was used to provide the first indication because of its high efficiency of cleavage. The CCK receptor contains 15 Met residues, representing the sites of CNBr cleavage, theoretically resulting in 16 fragments with molecular masses ranging from 0.1 to 9.9 kDa, with two fragments containing potential sites for N-glycosylation (Fig. 4). Figure 4 shows the CNBr cleavage patterns for CCK receptor labeled by each of the probes. As shown, the Bpa30 probe labeled two fragments, a glycosylated fragment migrating at approximate Mr = 25,000 and shifting to approximate Mr = 8500 after deglycosylation, and a nonglycosylated fragment migrating at approximate Mr = 4500. Considering the mass of the attached probe (1378 Da) and the nature of glycosylation, the glycosylated fragment represented the segment Asn10-Met72 that contained the amino terminus and the first transmembrane domain of the receptor (see below for further identification). The candidates for the nonglycosylated fragment included all three extracellular loop fragments because of their similar masses.
Distinct from labeling by the Bpa30 probe, the NO2-Phe30 probe only labeled a nonglycosylated receptor fragment migrating at approximate Mr = 4500. Considering the molecular mass of the attached probe (1357 Da), the expected fragment would be in the range of 2 to 3 kDa, and candidates also included fragments within all the extracellular loop domains.
The CNBr fragment from the CCK receptor labeled by the Bpa30-probe was extensively purified to chemical homogeneity as described under Materials and Methods and was subjected to Edman degradation sequencing. This resulted in the identification of the sequence Pro-Phe-Asn-Leu-Ile-Pro-Asn-Leu-Leu in cycles 1 to 9. This sequence exists only in the rat type A CCK receptor and corresponds with the first nine residues of the CNBr fragment spanning the first extracellular loop (Pro96-Met121). This provided definitive identification of the fragment Pro96-Met121 as one of the two regions labeled by the Bpa30 probe. This was further confirmed by CNBr cleavage of the L104M CCK receptor mutant (Arlander et al., 2004) labeled by the Bpa30 probe, which resulted in a small but significant shift of the approximate Mr = 4500 fragment in the labeled wild-type receptor to approximate Mr = 4000 in the L104M mutant (Fig. 5).
Likewise, the L104M receptor construct was also used to test whether the NO2-Phe30 probe labeled this CNBr fragment. As shown in Fig. 5, CNBr cleavage of the L104M receptor mutant labeled with the NO2-Phe30 probe yielded a significant shift to approximate Mr = 3000 from the approximate Mr = 4500 fragment in the labeled wild-type receptor. This confirmed that the NO2-Phe30 agonist probe labeled the CNBr fragment Pro96-Met121 that included the first extracellular loop, the same fragment as that labeled by the Bpa30 antagonist probe, although likely at a different site.
Of note, the Bpa30 probe also labeled the glycosylated fragment Asn10-Met72 as described above. Further localization was achieved with subsequent endoproteinase Lys-C treatment. As shown in Fig. 6, cleavage of the labeled Mr = 25,000 CNBr fragment with this protease yielded a band migrating at approximate Mr = 22,000 that further shifted to approximate Mr = 4500 after deglycosylation. This further refined the region of labeling with the Bpa30 probe as between Asn10 and Lys37 within the extracellular amino terminus of the CCK receptor.
Identification of Distinct Sites of Labeling by the Bpa30 Antagonist and NO2-Phe30 Agonist Probes. Radiochemical sequencing was used to identify the specific receptor residues labeled by each probe. For this, CNBr fragments from both the wild type (Pro96-Met121) and L104M mutant (Lys105-Met121) receptors labeled by each probe were purified to radiochemical homogeneity and coupled to N-(2-aminoethyl-1)-3-aminopropyl glass beads through Cys114 for manual Edman degradation sequencing. As shown in Fig. 7, a radioactive peak eluted in cycle 1 when sequencing the Lys105-Met121 fragment from the L104M mutant labeled by the Bpa30 antagonist probe, whereas no peak was found when sequencing the Pro96-Met121 fragment from the wild-type receptor labeled with the same probe. This indicated that the probe specifically labeled the residue Lys105 of the receptor. Shown also in Fig. 7 is the radioactivity elution profile of sequencing the radiochemically pure CNBr fragments labeled by the NO2-Phe30 probe. A peak eluted in cycle 4 when sequencing the Pro96-Met121 fragment from the wild-type receptor labeled by the NO2-Phe30 probe, whereas no peak was found when sequencing the Lys105-Met121 fragment from the L104M mutant labeled with the same probe. This identified the receptor residue Leu99 as the site of labeling by the NO2-Phe30 agonist probe.
Discussion
Understanding the molecular basis of ligand binding to physiologically important receptors and the conformational changes associated with activation of these receptors is critical for rational structure-based drug design. GPCRs represent the largest group of receptors in the body that includes many important drug targets. Although high-resolution biophysical techniques such as NMR or X-ray crystallography are the preferred methods for gaining insights into the detailed structural features of receptor-ligand complexes, these have been difficult to apply to this group of molecules, because of their natural sparsity, extreme hydrophobicity, and difficulty in purification and crystallization. The only extant high-resolution three-dimensional crystal structure of an intact member of this superfamily is that of rhodopsin in its inactive state (Palczewski et al., 2000).
The CCK receptor represents a potentially important drug target, having diverse physiological actions at multiple targets along the gastrointestinal tract and brain. Because it is structurally related to rhodopsin, belonging to the class I family of GPCRs, the confluence of transmembrane helices probably resembles that structure. However, the extracellular loop and tail domains of the CCK receptor that have been implicated as playing important roles in natural peptide ligand recognition are probably very different from rhodopsin (Ding et al., 2002). Current insight into these regions of the CCK receptor is based largely on the indirect observations provided by receptor mutagenesis and by ligand structure-activity relationships (Bodanszky et al., 1977, 1980; Miller et al., 1981; Kennedy et al., 1997; Gigoux et al., 1999; Gouldson et al., 2000; Ding et al., 2002) and on direct observations of residue-residue approximations determined using photoaffinity labeling studies with docked photolabile ligand probes (Ji et al., 1997; Hadac et al., 1998; Hadac et al., 1999; Ding et al., 2001; Arlander et al., 2004).
Photoaffinity labeling is a powerful tool to establish spatial approximations between specific residues within a ligand and within its receptor as complexed together. Using this approach, we have previously established such constraints for residues at the carboxyl terminus (position 33), midregion (positions 29), and extension of the amino terminus (position 24) of the pharmacophores of full agonist CCK analogs. The position 33 probe labels Trp39 within the amino terminus of the CCK receptor (Ji et al., 1997), whereas position 29 probe labels His347 and Leu348 within the third extracellular loop (Hadac et al., 1998), and the position 24 probe labels a dominant site within the third loop (Glu345)as well as a minor site in the amino terminus of the receptor, between Asn10 and Lys37 (Ding et al., 2001).
These constraints have been used to build and subsequently refine molecular models of the CCK agonist-bound receptor (Ding et al., 2001). These have resulted in a model in which the carboxyl terminus of CCK is sited near receptor amino terminal residue Trp39 just above the first transmembrane segment and the amino terminus of the natural peptide ligand is oriented away from the lipid bilayer (Dawson et al., 2002). This is in distinct contrast with another model proposed by the Fourmy group that is based on receptor mutagenesis data, in which the peptide is docked with its carboxyl terminus situated within the confluence of transmembrane helices adjacent to transmembrane segment six (Escrieut et al., 2002). Our proposed model for the active complex of agonist docked to this receptor has recently been further validated by fluorescence resonance energy transfer analysis, with distances from CCK determined to each of the extracellular loop regions (Harikumar and Miller, 2002). No models have yet been proposed for the peptide antagonist-bound CCK receptor in its inactive state.
We recently provided the first insights into the changes in conformation associated with active and inactive states of the CCK receptor using photolabile position 27 agonist and antagonist probes (Arlander et al., 2004). In that work, direct photoaffinity labeling with a full agonist probe confirmed the spatial approximation of this residue with receptor residue Arg197 within the second loop in the active complex. However, the analogous photolabile position 27 antagonist probe was shown to label a distinct region within the receptor amino terminus. These data demonstrated a key structural difference in active and inactive complexes of the ligand-bound CCK receptor.
The current work continues our efforts to obtain additional constraints for refining our agonist-bound CCK receptor model and provides additional insights into the differences in molecular approximation between active and inactive states of the CCK receptor by using structurally related antagonist probes. We now focus on the tryptophan in position 30 of CCK. Although this residue has been shown to be important for full agonist activity at the type A CCK receptor, it can be substituted by norleucine (Rolland et al., 1991) or 6-NO2-Trp (Klueppelberg et al., 1989) without loss of affinity or potency. Although the latter peptide (125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33]) was able to covalently label the receptor, the efficiency of covalent attachment was not adequate to identify adjacent residues.
In the current work, Trp30 was replaced by a photolabile NO2-Phe moiety, and the resultant probe retained full efficacy of biological activity and also yielded high-efficiency covalent attachment to permit such identification. In another analog of this peptide, Trp30 was replaced by a photolabile Bpa moiety, resulting in an antagonist, rather than an agonist. Because the Bpa moiety is larger than either the 6-NO2-Trp or NO2-Phe moieties, which are closer in size to the natural Trp present in that position, it is possible that steric hindrance was responsible for the inability of the Bpa analog to achieve an active conformation. It is unclear whether a constrained peptide conformation or interference with the peptide-receptor interaction was responsible, but the net effect was to yield binding affinity similar to the analogous agonist probe, but no intrinsic biological activity. Thus, we have developed a pair of analogous agonist and antagonist photolabile probes, with the only structural difference being the photolabile residue in position 30.
It is of note that the NO2-Phe30 agonist probe in this study labeled CCK receptor residue Leu99 within the predicted first extracellular loop. This is a region that includes residues with substantial impact in mutagenesis studies of both the type A (Silvente-Poirot et al., 1998; Gouldson et al., 2000) and type B (Silvente-Poirot et al., 1998) CCK receptors. This region is clearly distinct from type A CCK receptor regions previously demonstrated to be adjacent to the carboxyl terminus (position 33), mid-region (position 27 and 29), and the amino-terminal extension (position 24) of the pharmacophore of CCK (Ji et al., 1997; Hadac et al., 1998; Ding et al., 2001; Arlander et al., 2004). However, it is consistent with analogous labeling of the type B CCK receptor using the same position 33 and 24 probes (Dong et al., 2005). This spatial approximation is fully compatible with the model we recently proposed for the peptide agonist ligand-occupied CCK receptor (Ding et al., 2001).
It is also notable that one of the regions of labeling by the Bpa30 antagonist probe was similar to that for the NO2-Phe30 agonist probe. This supports the general similarity in docking the structurally related agonist and antagonist probes, as we previously observed for structurally related agonist, partial agonist, and antagonist probes that had a photolabile Bpa situated at their amino terminus (Dong et al., 1999). Although both the agonist and antagonist used in the current study labeled the same receptor region, the specific residues that were labeled were distinct, with the Bpa30 antagonist probe labeling Lys105 and the NO2-Phe30 agonist probe labeling Leu99. Mutagenesis of Lys105 has previously been shown to be well tolerated for agonist action, consistent with absence of direct functional importance (Gouldson et al., 2000).
The Bpa30 antagonist probe also labeled a second receptor region, the receptor amino terminus between Asn10 and Lys37. This region can be truncated without significant negative impact on receptor ligand binding or agonist-stimulated signaling (Kennedy et al., 1995; Ding et al., 2001). This was also the region labeled by position 33 and position 24 agonist probes (Ji et al., 1997; Ding et al., 2001), as well as by the position 27 antagonist probe (Arlander et al., 2004). These data are consistent with the amino terminus covering the peptide-binding domain of this receptor, but not playing a significant direct functional role, as was proposed previously (Ding et al., 2001).
In summary, we have now prepared a pair of structurally-related CCK agonist and antagonist probes that each incorporated a photolabile residue into position 30 of the ligand and have used them to explore the residue-residue approximations as docked at the CCK receptor in active and inactive states. Both labeled the same region of the CCK receptor (the first extracellular loop) that is distinct from regions labeled previously by probes of this receptor, providing further important confirmation of the evolving molecular model. The distinct sites of labeling within this region provide additional insights into the differences between active and inactive conformations of this receptor.
Acknowledgements
We thank Eileen L. Holicky for excellent technical assistance, Dana Huether for secretarial assistance, and Benjamin J. Madden (Mayo Clinic Protein Core Facility) for helping with protein sequencing.
doi:10.1124/mol.105.012179.
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