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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Demonstration of a Specific Site of Covalent Labeling of the Human Motilin Receptor Using a Biologically Active Photolabile Motilin Analog

Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic in Scottsdale, Scottsdale, Arizona

Received December 1, 2004; accepted January 25, 2005.

	Abstract

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The motilin receptor belongs to a group of class I G protein-coupled receptors that also includes the growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (a cyanogen bromide fragment extending from the first to the second extracellular loop) using a position 1 photolabile motilin analog. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analog probe having site of covalent attachment in position 5. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild-type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe³³² using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be used in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor and to ultimately be useful in the rational design of drugs acting at this important target.

The class I G protein-coupled receptors that are activated by motilin and ghrelin were recognized only recently (Howard et al., 1996; McKee et al., 1997; Feighner et al., 1999) and cluster together in sequence analysis, having substantial structural similarity. This has typically been a predictor of similarity in ligand structure and mechanism of binding and activation (Kolakowski, 1994). Although these natural ligands have extensive sequence homology, ghrelin requires a unique post-translational modification for its activity that is not present or required for motilin (Kojima et al., 1999). This represents the N-octanoylation of the Ser residue in its third position. Furthermore, motilin has an unusual structure-activity relationship for a peptide receptor in the class I family of G protein-coupled receptors, having amino-terminal rather than the predominant carboxyl-terminal sequence determinants for its selectivity of binding and action (Peeters et al., 1992; Poitras et al., 1992, 1994; Boulanger et al., 1995; Miller et al., 1995). This raises the possibility that the mechanisms of ligand binding and activation of these receptors might be distinct.

We have recently performed the initial photoaffinity labeling of the motilin receptor, using a probe with photolabile site of covalent attachment in position 1 of the peptide (Coulie et al., 2001). That probe labeled a region of this receptor (a cyanogen bromide fragment) that included the predicted second intracellular loop domain, the fourth transmembrane segment, and the second extracellular loop (Coulie et al., 2001). This was potentially quite interesting, since this extracellular loop domain was quite long in the motilin receptor, including a 67-residue insertion that was not present in the otherwise structurally similar receptor activated by ghrelin (Howard et al., 1996). We recently performed extensive mutagenesis on this loop domain and were surprised to find that the entire insertion domain could be deleted without having a negative impact on motilin binding or biological activity (Matsuura et al., 2002). The regions of this loop that were functionally important in that series were at both ends of the loop, adjacent to the plasma membrane.

In the current study, we extended our understanding of the molecular basis of motilin binding by developing a second photolabile agonist probe and using it to localize the first residue-residue approximation constraint for this ligand-receptor pair. This probe incorporates a site of covalent attachment in position 5 of a motilin analog, within the pharmacophoric domain of this peptide (Peeters et al., 1992; Poitras et al., 1992, 1994; Boulanger et al., 1995; Miller et al., 1995). We used a series of specific cleavage reactions and a new receptor mutant to localize the region of labeling to a region within the third extracellular loop domain. This was further refined to demonstrate labeling of Phe³³². Thus, it seems that, like several other peptide ligands (Ji et al., 1998), motilin binds to extracellular loop domains of this receptor. This type of constraint will be quite useful in the initial docking of this peptide to its receptor, as meaningful conformational models become available.

	Materials and Methods

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Materials. The solid phase oxidant N-chlorobenzenesulfonamide (Iodo-beads), cyanogen bromide (CNBr), and 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (skatole) were from Pierce Chemical Co. (Rockford, IL). Fura-2/acetoxymethyl ester was from Molecular Probes (Eugene, OR). Endopeptidase Lys-C (Lys-C) was from Roche Diagnostics (Indianapolis, IN). Endoglycosidase F (Endo F) was prepared in our laboratory, as we previously reported (Pearson et al., 1987). All other reagents were of analytical grade.

Peptides. The forms of motilin that were used corresponded to the human sequence, with the noted changes. We synthesized natural human motilin, [Ile¹³]motilin, and [Bpa⁵,Ile¹³]motilin using manual solid-phase techniques (Powers et al., 1988b; Coulie et al., 2001). The latter probe was designed to incorporate a photolabile benzoyl-phenylalanine (Bpa) in the position of Phe⁵ as a site for covalent labeling of the receptor and an Ile residue in the position of Met¹³ to eliminate a site for potential oxidative damage during radiolabeling. This probe contained a Tyr residue that is naturally present in position 7 as a site for radioiodination. These peptides were purified to homogeneity by sequential steps of reversed-phase high-performance liquid chromatography (Powers et al., 1988a). Peptides incorporating the D- and L-Bpa stereoisomers were separated and identified using the technique of Miller and Kaiser (1988). The L-Bpa peptide was used in the present study. The chemical identities of these peptides were established by mass spectrometry.

[Ile¹³]Motilin and [Bpa⁵,Ile¹³]motilin were radioiodinated on residue Tyr⁷ using exposure to the solid-phase oxidant Iodo-beads, as previously described (Powers et al., 1988a). They were purified by reversed-phase high-performance liquid chromatography to yield specific radioactivities of 2000 Ci/mmol (Powers et al., 1988a).

Receptor Preparations. The human motilin receptor cDNA was kindly provided by S. D. Feighner and A. D. Howard of Merck Research Laboratories (Rahway, NJ) (Feighner et al., 1999). Mutant motilin receptor constructs were prepared using an oligonucleotide-directed approach with the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA). The development of two new motilin receptor constructs was necessary for this study. This included a mutant to help localize the domain that was affinity-labeled by replacing motilin receptor residue Gln³³⁴, predicted to be within the seventh transmembrane segment, with Met (Q334M) to incorporate an additional site for CNBr cleavage. It also included a mutant that incorporated two Cys residues in the intracellular carboxyl-terminal tail domain (replacement of Lys³⁶⁸ and Lys³⁷⁴ with Cys residues; K368,374C) for covalent coupling of the affinity-labeled receptor fragment to solid-phase glass beads for radiochemical sequencing. Sequences of these constructs were confirmed by direct DNA sequencing.

COS-1 and Chinese hamster ovary (CHO-K1) cells (American Type Culture Collection, Manassas, VA), which do not naturally express the motilin receptor, were used as cellular background for expression of various motilin receptor constructs. We previously established and characterized a CHO cell line stably expressing the wild-type human motilin receptor (CHO-MtlR) (Coulie et al., 2001). For this work, we also established an additional CHO cell line that stably expressed the dual Cys mutant motilin receptor K368,374C using similar procedures (Coulie et al., 2001). Cells were cultured at 37°C on tissue-culture plasticware in Ham's F-12 medium supplemented with 5% Fetal Clone II (Hyclone Laboratories, Logan, UT). They were passaged approximately twice a week and were lifted mechanically before use.

The Q334M motilin receptor construct was expressed transiently in COS cells after transfection using a modified DEAE-dextran protocol (Lopata et al., 1984). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% Fetal Clone II. For receptor binding studies, cells were lifted mechanically 3 days after transfection and were used to prepare enriched plasma membranes (Hadac et al., 1996). For biological activity studies, cells were studied in an appropriate culture dish 3 days after transfection.

Receptor Binding Assay. Plasma membranes were prepared from receptor-bearing cells using sonication and sucrose gradient centrifugation, as we previously reported (Hadac et al., 1996). For binding characterization, enriched membranes (5–10 µg of protein) were incubated with a constant amount of radioligand ¹²⁵I-[Ile¹³]motilin (3–5 pM) in the presence of increasing concentrations of unlabeled motilin or [Bpa⁵,Ile¹³]motilin (ranging from 0 to 1 µM). Incubations were carried out for 1 h at room temperature in Krebs-Ringer-HEPES medium containing 25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1.2 mM MgSO₄, 2 mM CaCl₂, 0.01% soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 0.2% bovine serum albumin. Bound and free radioligand were separated using a Skatron cell harvester (Molecular Devices, Sunnyvale, CA) with receptor-binding filtermats, and bound radioactivity was quantified in a -spectrometer. The intact cell binding assay was performed in 24-well tissue culture plates. Nonspecific binding was determined in the presence of 1 µM motilin and represented less than 20% of the total radioligand bound. All assays were performed in duplicate and were repeated at least three times in independent experiments.

Biological Activity Characterization. The agonist activity of [Bpa⁵,Ile¹³]motilin was studied using an assay for stimulation of intracellular calcium concentration in cells expressing motilin receptor constructs (Grynkiewicz et al., 1985). Natural human motilin was used as a positive control. In this assay, 2 x 10⁶ receptor-bearing cells were loaded with 5 µM Fura-2/acetoxymethyl ester (Molecular Probes) in calcium-free Krebs-Ringer-HEPES medium for 20 min at 37°C. After washing, cells were stimulated with varying concentrations of peptide ligand at 37°C. Fluorescence was quantified in a PerkinElmer LS50B luminescence spectrometer (PerkinElmer Life and Analytical Sciences, Boston, MA). Excitation was performed at 340 and 380 nm, and emission was quantified at 520 nm, with calcium concentration calculated from the ratio as described by Grykiewicz et al. (1985). The peak intracellular calcium transient was used to determine the agonist concentration-dependence of this biological response. Basal levels of calcium were determined by calculating the intracellular calcium concentration in the absence of agonist stimulation, whereas maximal levels were determined as the peak intracellular calcium concentration achieved in the presence of 1 µM agonist. Stimulated values of intracellular calcium were determined by the subtraction of the basal levels from the maximal levels of intracellular calcium concentration in a given set of experiments. All assays were performed in duplicate and were repeated at least three times in independent experiments.

Photoaffinity Labeling of the Motilin Receptor. For this experiment, enriched plasma membranes (100 µg) were incubated for 1 h in the dark at 25°C with 100 pM ¹²⁵I-[Bpa⁵,Ile¹³]motilin in the absence or presence of increasing concentrations of nonradiolabeled motilin. After binding, membranes were exposed to photolysis for 30 min at 4°C using a Rayonet Photochemical Reactor (Southern New England Ultraviolet Co., Bradford, CT) equipped with 3500-Å lamps. Membranes were then washed, solubilized, and separated by gel electrophoresis on 10% SDS-PAGE gels using conditions described by Laemmli (1970). Labeled products were visualized by autoradiography. For selected experiments, the affinity-labeled motilin receptor and its relevant fragments were deglycosylated with endoglycosidase F under the conditions we previously described (Hadac et al., 1998).

Chemical and Enzymatic Cleavage of the Labeled Motilin Receptor. Gel-purified, affinity-labeled native and deglycosylated motilin receptors were digested separately or sequentially with CNBr and/or endopeptidase Lys-C, as we previously described (Dong et al., 1999). Further cleavage of the CNBr cleaved fragment was performed with 2 mg/ml skatole in 70% (v/v) acetic acid, according to the method previously reported (Dong et al., 1999). The products of cleavage were separated on 10% NuPAGE gels (Invitrogen, Carlsbad, CA) using 2-(N-morpholino)ethanesulfonic acid running buffer, with labeled products visualized by autoradiography. The apparent molecular masses of radiolabeled receptor fragments were determined by interpolation on a plot of the mobility of Multimark protein standards (Invitrogen) versus the log values of their apparent masses.

Identification of Site of Covalent Attachment. After achieving definitive identification of the receptor fragment that was labeled with [Bpa⁵,Ile¹³]motilin, its site of attachment was determined using radiochemical Edman degradation sequencing, as we previously described (Dong et al., 1999). This procedure involved in the use of cross-linking through the Cys residues within a protein fragment to N-(2-aminoethyl-1)-3-aminopropyl glass beads (Sigma-Aldrich, St. Louis, MO). For this, we used the K368,374C receptor construct expressed on a CHO cell line. This mutant receptor was affinity-labeled with [Bpa⁵,Ile¹³]motilin and cleaved by CNBr. The gel-purified radiolabeled cleavage product was covalently coupled through its Cys residues to maleimidobenzoyl succinimide-activated N-(2-aminoethyl-1)-3-aminopropyl glass beads. This was followed by repetitive cycles of manual Edman degradation with quantitation of radioactivity released in each cycle. This procedure was performed at least three times in independent experiments.

Statistical Analysis. All observations are expressed as means ± S.E.M. Binding curves were analyzed using the LIGAND program of Munson and Rodbard (1980) and plotted using the nonlinear regression analysis in the Prism software package (GraphPad Software, San Diego, CA).

	Results

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Characterization of the Probe. [Bpa⁵,Ile¹³]Motilin was synthesized and purified to homogeneity. It was characterized both chemically by mass spectrometry and functionally in binding and biological activity assays. This analog bound to the motilin receptor saturably and specifically but displayed a lower affinity (K_i = 50 ± 7 nM) than natural motilin (K_i = 2.3 ± 0.4 nM) (Fig. 1). The binding of the peptide that incorporated the D-Bpa residue displayed a much lower affinity (K_i = 420 ± 67 nM) than the L-Bpa peptide (K_i = 50 ± 7 nM) and was therefore not considered for further use. The lower affinity of the [D-Bpa⁵,Ile¹³]motilin supports the previous interpretation that the fifth residue of motilin is involved in stabilizing the bioactive conformation of the peptide (Peeters et al., 1992).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Functional characterization of the [Bpa5,Ile13]motilin probe. Shown in the left panel is the competition-binding curve, reflecting the ability of increasing concentrations of unlabeled peptide to displace the binding of 125I-[Ile13]motilin to membranes from CHO-MtlR cells. Values represent percentages of maximal saturable binding that were observed in the absence of competitor. They are expressed as means ± S.E.M. of duplicate data from three independent experiments. Shown in the right panel are intracellular calcium responses to increasing concentrations of [Bpa5,Ile13]motilin in CHO-MtlR cells. The basal level of intracellular calcium was 138 ± 20 nM, with the maximal stimulated level reaching 198 ± 32 nM. Values are expressed as means ± S.E.M. of data from three independent experiments, with data normalized relative to the maximal response to motilin.

Photoaffinity Labeling of the Motilin Receptor. Like the [Bpa¹,Ile¹³]motilin probe that we previously reported (Coulie et al., 2001), the [Bpa⁵,Ile¹³]motilin analog covalently labeled two membrane proteins from receptor-bearing CHO-MtlR cells. The major band migrated on a 10% SDS-polyacrylamide gel at an apparent M_r = 78,000 with the minor band at M_r = 58,000 (Fig. 2). Photoaffinity labeling of each of these bands was inhibited in parallel in a concentration-dependent manner with increasing concentrations of unlabeled motilin. Like the previous study (Coulie et al., 2001), both bands represented distinct glycoforms of motilin receptor, since deglycosylation of each of the bands with endoglycosidase F resulted in bands that migrated similarly at approximately M_r = 45,000, corresponding with the expected mass of the core receptor protein (Fig. 2). Bands of this size were absent in samples prepared from nonreceptor bearing parental CHO-K1 cell membranes (data not shown). Figure 2 also includes the densitometric analysis of the labeling of the M_r = 78,000 band in three similar experiments. The IC₅₀ value for this competition was between 1 and 10 nM.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Photoaffinity labeling of the motilin receptor. Shown is a representative autograph of a 10% SDS-PAGE gel used to separate the products of photoaffinity labeling of the motilin receptor with 125I-[Bpa5,Ile13]motilin in the absence and presence of increasing concentrations of unlabeled motilin (top). Glycoforms of the affinity-labeled receptor migrated at approximately Mr = 78,000 and Mr = 58,000. After deglycosylation with Endo F, both bands shifted to an apparent Mr = 45,000 (bottom). Shown in the middle panel is the densitometric analysis of labeling of the major band in three similar experiments, with data points expressed as means ± S.E.M.

Identification of the Domain of Labeling. CNBr cleavage was used to gain initial insight into the domain of labeling of the motilin receptor with the [Bpa⁵,Ile¹³]motilin probe. CNBr cleavage of the motilin receptor would theoretically result in 10 fragments ranging in molecular weight from less than 1 to 12.5 kD, with two fragments containing glycosylation sites (Fig. 3). As shown in Fig. 3, CNBr cleavage of the intact labeled motilin receptor yielded a band migrating at approximately M_r = 14,000 that did not shift after deglycosylation with endoglycosidase F. Considering the mass of the covalently bound ligand probe (2677 Da) and the nonglycosylated nature of the labeled fragment, two candidate fragments best fit these data. The first candidate represents the fragment spanning the fifth transmembrane segment, the third intracellular loop, the sixth transmembrane segment, and the third extracellular loop (fragment 7). The other candidate represents the fragment incorporating the seventh transmembrane segment, with extracellular and intracellular domains on either side (fragment 9).

View larger version (48K): [in this window] [in a new window]   Fig. 3. Chemical and enzymatic cleavage of the affinity-labeled motilin receptor. Shown in the left panel is a diagram of the predicted sites of CNBr cleavage of the human motilin receptor, along with the masses of protein cores of these fragments. Shown in the middle panel is a typical autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the motilin receptor that had been labeled with 125I-[Bpa1,Ile13]motilin. Cleavage of the native receptor yielded a band migrating at Mr = 14,000, which was not affected by deglycosylation with Endo F. Fragments 7 and 9 were the best candidates to fit these data. The right panel reflects the migration of the products of further cleavage of this band using endoproteinase Lys-C and skatole. The band shifted to Mr = 5000 after Lys-C cleavage but was not affected by skatole cleavage. These data suggest that fragment 9 contained the site of labeling, likely within the region Tyr331-Lys360.

To further establish this identity and localize the site of labeling, a receptor mutant was constructed with Gln³³⁴ changed to Met (Q334M) to introduce an additional site for CNBr cleavage within the expected fragment. This was expressed transiently in COS cells. Figure 4 shows that this construct bound motilin with normal affinity (K_i = 0.85 ± 0.19 nM) and was able to initiate normal signaling (EC₅₀ for motilin stimulation of an intracellular calcium response of 8.4 ± 4.9 nM). The [Bpa⁵,Ile¹³]motilin probe efficiently and saturably affinity-labeled the Q334M motilin receptor construct expressed in COS cells (Fig. 4). Shown also is its labeling of the wild-type motilin receptor expressed in the same cells, since the glycosylation of the motilin receptor on these cells was different from that observed in the CHO-MtlR cells (Coulie et al., 2001). As expected, the labeled M_r = 14,000 CNBr fragment from the wild-type receptor shifted to an approximate M_r = 4000 band in the Q334M receptor construct (Fig. 4). These results definitively establish the identity of the labeled CNBr fragment of the motilin receptor while at the same time helping to refine the labeled domain. The domain labeled includes the region between receptor residues Tyr³³¹ and Gln³³⁴.

View larger version (53K): [in this window] [in a new window]   Fig. 4. Further localization of the ligand binding domain by CNBr cleavage of the affinity-labeled Q334M motilin receptor mutant. Shown in the top panel are motilin competition-binding data and intracellular calcium concentration response data for the Q334M motilin receptor construct expressed in COS cells. The basal level of intracellular calcium was 140 ± 18 nM, with the maximal stimulated level reaching 209 ± 28 nM. Values are illustrated as in Fig. 1, representing means ± S.E.M. of data from three independent experiments performed in duplicate. Shown in the bottom panel are the saturable affinity labeling of the Q334M construct, the wild-type motilin receptor, and the differential migration of glycoforms in COS and CHO cells. Shown also in the bottom panel is an autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the affinity-labeled Q334M construct. As in CHO cells, the CNBr cleavage of the wild-type receptor expressed in COS cells yielded a band migrating at approximately Mr = 14,000. This band shifted to Mr = 4000 in the Q334M motilin receptor, indicating the site of labeling being within the region Tyr331 and Gln334.

Identification of the Specific Site of Labeling. For these studies, a CHO cell line was generated that stably expressed a receptor mutant with residues Lys³⁶⁸ and Lys³⁷⁴ both changed to Cys (K368,374C) to introduce residues that would allow it to be covalently coupled via their sulfhydryl groups to glass beads for sequencing. Figure 5 shows that motilin bound with normal affinity (K_i = 0.6 ± 0.1 nM) and stimulated a normal intracellular calcium concentration response (EC₅₀ = 0.3 ± 0.1 nM) in these cells. The [Bpa⁵,Ile¹³]motilin probe efficiently and saturably labeled the K368,374C construct (Fig. 5). As expected, CNBr cleavage of this construct yielded a labeled band that migrated at the same position as the M_r = 14,000 CNBr fragment from the labeled wild-type receptor expressed in CHO-MtlR cells.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Characterization of the K368,374C construct. Shown in the top panel are motilin competition-binding data and intracellular calcium concentration response data for the K368,374C construct stably expressed in CHO cells. The basal level of intracellular calcium was 145 ± 27 nM, with the maximal stimulated level reaching 210 ± 30 nM. Values are illustrated as in Fig. 1, with means ± S.E.M. of data from three independent experiments performed in duplicate. Bottom panel shows the saturable affinity labeling of the K368,374C construct, along with its CNBr cleavage to yield a band migrating at Mr = 14,000, similar to that of the CNBr fragment from the wild-type motilin receptor.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Edman degradation sequencing of the fragment labeled with [Bpa5,Ile13]motilin probe. Shown is the profile of elution of radioactivity from Edman degradation sequencing of the purified CNBr fragment (Tyr331-Met411) of the K368,374C motilin receptor mutant labeled with 125I-[Bpa5,Ile13]motilin. The peak in eluted radioactivity was consistently observed in cycle 2 in three independent experiments. This corresponds to covalent attachment of Bpa5 of the probe to Phe332 of the receptor.

	Discussion

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Motilin is a physiologically important linear 22-residue peptide that stimulates gastrointestinal motility (Janssens et al., 1983; Itoh, 1997). It reaches peak levels in the circulation cyclically through the day, correlating with an increase in interdigestive motor activity in the stomach and small intestine. The prokinetic effect of this hormone is believed to play an important role in the normal housekeeping of the gut by purging any residual luminal material in preparation for the subsequent meal. It also represents a potentially important pharmacological agent to stimulate gastric emptying and intestinal transit in clinical dysmotility states (Janssens et al., 1983; Itoh, 1997). Indeed, this represents the rationale for the therapeutic use of erythromycin, found to represent a weak agonist acting at the motilin receptor (Janssens et al., 1990; Peeters, 1993).

The current work represents part of an experimental strategy to gain insights into the structure of the motilin receptor and its mode of agonist ligand binding, in an effort to ultimately develop the next generation of drugs that can act at the motilin receptor. Little is currently known of the mode of binding of this peptide to its receptor. There is reason to believe that the peptide ligands for the receptors in the motilin receptor family will bind to their receptors in a manner different from the natural peptide ligands for other class I G protein-coupled receptors. This relates to the finite carboxyl-terminal pharmacophoric domain typical of the other peptides that bind to class I G protein-coupled receptors (Kolakowski, 1994), whereas motilin has its major region of selectivity within its amino-terminal decapeptide (Poitras et al., 1992; Feighner et al., 1999). The unique structural characteristics of the natural ligand for another closely related receptor in the motilin receptor family, the ghrelin receptor having a ligand with a functionally critical N-octanoyl moiety at the serine residue in the third position, add to the interest in how the natural ligands for the motilin receptor family might dock with their receptors.

The most extensive literature contributing to our understanding of mechanisms of ligand binding to a receptor come from structure-activity studies, including receptor mutagenesis. To date, the only published mutagenesis data for any receptor in the motilin receptor family is limited to a detailed analysis of the predicted very large second extracellular loop region of the motilin receptor (Matsuura et al., 2002). An extensive series of deletion and replacement mutations throughout that region revealed that, in addition to the cysteine residue in this region (Cys²³⁵) that is likely involved in the heavily conserved disulfide-bond linking the first and second loops of most G protein-coupled receptors, alanine-replacement mutagenesis of only a few of the residues that are predicted to be at the membranous interface (Val¹⁷⁹, Leu²⁴⁵, and Arg²⁴⁶) had any negative functional impact on natural motilin binding or biological activity. Of interest, the same mutations had no negative effect on biological responses to the nonpeptidyl agonist erythromycin. The functional role of this large second extracellular loop of the motilin receptor is, therefore, currently indeterminate. Other regions of the motilin receptor have not yet been systematically analyzed by this methodology.

Photoaffinity data, such as that provided by the current studies, can contribute direct insights into spatial approximation between distinct residues within a docked ligand and within its receptor. However, prior to this, such studies have been limited to the affinity labeling of the entire intact receptor or of large regions within this receptor (Coulie et al., 2001). Previous use of a probe with site of covalent attachment in position 1 labeled a region of this receptor (a cyanogen bromide fragment) that included the predicted second intracellular loop domain, the fourth transmembrane segment, and the second extracellular loop (Coulie et al., 2001). The current data, using a position 5 probe, established spatial approximation with a distinct receptor residue within the third extracellular loop region. This supports the general theme of other members of this superfamily, in which large peptides having diffuse pharmacophoric regions bind to extracellular epitopes of the receptor. The generation of residue-residue approximation constraints will have to be replicated for additional loci throughout the pharmacophoric region of motilin to contribute to the meaningful docking of this ligand to its receptor.

As data from both receptor mutagenesis studies and photoaffinity studies accumulate, a more complete picture of critical regions and sites of spatial approximation between ligands and this receptor will emerge. These can contribute not only to effective ligand docking but also to the refinement of our understanding of the conformation of the receptor itself. This will become effective once such sets of data are comprehensive and representative of the entire ligand pharmacophore and the full surface of the receptor.

	Acknowledgements

 
We acknowledge the strong support of Professor Morikazu Onji of Ehime University and the excellent technical assistance of E. M. Hadac and E. Holicky.

	Footnotes

 
This work was supported by the Mayo Clinic and Foundation and by grants from the Japanese Ministry of Education and Science and the Fiterman Foundation.

doi:10.1124/jpet.104.081562.

ABBREVIATIONS: CNBr, cyanogen bromide; Bpa, benzoyl-phenylalanine; CHO, Chinese hamster ovary; CHO-MtlR, Chinese hamster ovary cell lines stably expressing the wild-type human motilin receptor; PAGE, polyacrylamide gel electrophoresis.

¹ Current address: Third Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan.

² Current address: Johnson and Johnson Pharmaceutical Research and Development, Beerse, Belgium.

Address correspondence to: Dr. Laurence J. Miller, Director, Cancer Center, Mayo Clinic in Scottsdale, 13400 East Shea Boulevard, Scottsdale, AZ 85259. E-mail: miller{at}mayo.edu

	References

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Boulanger Y, Khiat A, Chen Y, Gagnon D, Poitras P, and St-Pierre S (1995) Structural effects of the selective reduction of amide carbonyl groups in motilin 1–12 as determined by nuclear magnetic resonance. Int J Pept Prot Res 46: 527–534.

Coulie B, Matsuura B, Dong M, Hadac EM, Pinon DI, Feighner SD, Howard AD, and Miller LJ (2001) Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling. J Biol Chem 276: 35518–35522.[Abstract/Free Full Text]

Dong M, Wang Y, Hadac EM, Pinon DI, Holicky E, and Miller LJ (1999) Identification of an interaction between residue 6 of the natural peptide ligand and a distinct residue within the amino-terminal tail of the secretin receptor. J Biol Chem 274: 19161–19167.[Abstract/Free Full Text]

Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, MacNeil D, Cascieri MA, et al. (1999) Receptor for motilin identified in the human gastrointestinal system. Science (Wash DC) 284: 2184–2188.[Abstract/Free Full Text]

Grynkiewicz G, Poenie M, and Tsien RY (1985) A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450.[Abstract/Free Full Text]

Hadac EM, Ghanekar DV, Holicky EL, Pinon DI, Dougherty RW, and Miller LJ (1996) Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation. Pancreas 13: 130–139.[Medline]

Hadac EM, Pinon DI, Ji Z, Holicky EL, Henne RM, Lybrand TP, and Miller LJ (1998) Direct identification of a second distinct site of contact between cholecystokinin and its receptor. J Biol Chem 273: 12988–12993.[Abstract/Free Full Text]

Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, et al. (1996) A receptor in pituitary and hypothalamus that functions in growth hormone release. Science (Wash DC) 273: 974–977.[Abstract]

Itoh Z (1997) Motilin and clinical application. Peptides 18: 593–608.[CrossRef][Medline]

Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Muls E, and Bouillon R (1990) Improvement of gastric emptying in diabetic gastroparesis by erythromycin. Preliminary studies. N Engl J Med 322: 1028–1031.[Abstract]

Janssens J, Vantrappen G, and Peeters TL (1983) The activity front of the migrating motor complex of the human stomach but not of the small intestine is motilin-dependent. Regul Pept 6: 363–369.[CrossRef][Medline]

Ji TH, Grossmann M, and Ji I (1998) G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem 273: 17299–17302.[Free Full Text]

Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, and Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature (Lond) 402: 656–660.[CrossRef][Medline]

Kolakowski LF (1994) GCRDb: a G-protein-coupled receptor database. Recept Channels 2: 1–7.[Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227: 680–685.[CrossRef][Medline]

Lopata MA, Cleveland DW, and Sollner-Webb B (1984) High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucl Acids Res 12: 5707–5717.[Abstract/Free Full Text]

Matsuura B, Dong M, and Miller LJ (2002) Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action. J Biol Chem 277: 9834–9839.[Abstract/Free Full Text]

McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, Smith RG, Howard AD, and Van der Ploeg LH (1997) Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 46: 426–434.[CrossRef][Medline]

Miller P, Gagnon D, Dickner M, Aubin P, St-Pierre S, and Poitras P (1995) Structure-function studies of motilin analogues. Peptides 16: 11–18.[CrossRef][Medline]

Miller WT and Kaiser ET (1988) Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label. Proc Natl Acad Sci USA 85: 5429–5433.[Abstract/Free Full Text]

Munson PJ and Rodbard D (1980) LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107: 220–239.[CrossRef][Medline]

Pearson RK, Miller LJ, Hadac EM, and Powers SP (1987) Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor. J Biol Chem 262: 13850–13856.[Abstract/Free Full Text]

Peeters TL (1993) Erythromycin and other macrolides as prokinetic agents. Gastroenterology 105: 1886–1899.[Medline]

Peeters TL, Macielag MJ, Depoortere I, Konteatis ZD, Florance JR, Lessor RA, and Galdes A (1992) D-amino acid and alanine scans of the bioactive portion of porcine motilin. Peptides 13: 1103–1107.[CrossRef][Medline]

Poitras P, Gagnon D, and St-Pierre S (1992) N-terminal portion of motilin determines its biological activity. Biochem Biophys Res Comm 183: 36–40.[CrossRef][Medline]

Poitras P, Miller P, Gagnon D, and St-Pierre S (1994) Motilin synthetic analogues and motilin receptor antagonists. Biochem Biophys Res Comm 205: 449–454.[CrossRef][Medline]

Powers SP, Fourmy D, Gaisano H, and Miller LJ (1988a) Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors D-Tyr-Gly-[Nle^28,31,pNO₂-Phe³³)CCK-26–33). J Biol Chem 263: 5295–5300.[Abstract/Free Full Text]

Powers SP, Pinon DI, and Miller LJ (1988b) Use of N,O-bis-Fmoc-D-Tyr-ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides. Int J Pept Protein Res 31: 429–434.[Medline]

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