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Am J Physiol Gastrointest Liver Physiol 294: G460-G466, 2008. First published November 21, 2007; doi:10.1152/ajpgi.00244.2007
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HORMONES AND SIGNALING

Mutational analysis of predicted intracellular loop domains of human motilin receptor

Hitoo Tokunaga,1 Bunzo Matsuura,1 Maoqing Dong,2 Laurence J. Miller,2 Teruhisa Ueda,1 Shinya Furukawa,1 Yoichi Hiasa,1 and Morikazu Onji1

1Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Tohon, Ehime, Japan; and 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona

Submitted 1 June 2007 ; accepted in final form 21 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63–66, 135–137, and 296–301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.

erythromycin; G protein-coupled receptor; mutagenesis

MOTILIN, a 22-residue peptide hormone, is secreted by endocrine cells of the intestinal mucosa. It plays an important role in the regulation of gastrointestinal motility, stimulating gastric emptying and initiating the interdigestive migrating motor complex. Agonists acting at the motilin receptor hold promise as a new class of drugs for the therapy of various gastrointestinal dysmotility states. However, the molecular basis for the binding of motilin and the nonpeptidyl agonist erythromycin to the motilin receptor and for the activation of that receptor has not been fully examined. A clear understanding of these events can provide insights that could be useful in the rational development and refinement of drugs acting at this important receptor.

The motilin receptor belongs to the class I family of guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), which also includes growth hormone secretagogue receptors (9). We recently reported (8, 16) affinity labeling of this receptor with photolabile motilin analogs with sites of covalent attachment through ligand positions 1 and 5. Both probes labeled receptor residues predicted to reside within extracellular loop regions of this receptor. We also used mutagenesis to identify critical residues within the predicted extracellular perimembranous loop and tail regions of this receptor (17, 18). These residues were shown to be critical for the action of motilin but not for erythromycin, presumably because of distinct modes of receptor binding by these chemically distinct agonists. These data are consistent with the major determinant of peptide binding in extramembranous domains, while the nonpeptidyl agonist likely binds to determinants within the lipid bilayer in the helical bundle domain.

It is noteworthy that in another report mutation of predicted transmembrane segments of this receptor was found to interfere with the binding and action of both motilin and erythromycin (28); however, this is compatible with either a direct or an indirect effect. It is even possible that this represents a direct effect on one ligand, such as the nonpeptidyl agonist erythromycin, and an indirect effect on the other, such as the peptide that appears to bind to extracellular loop regions.

In the present study, we have examined the predicted intracellular regions of this receptor that would not be expected to interact directly with either type of motilin receptor agonist. Instead, these regions represent candidates for interaction with the proximal effector molecules, such as heterotrimeric G proteins. Included in these evaluations was the Glu/Asp-Arg-Tyr residue triplet (the "E/DRY motif"), which is highly conserved at the boundary between the third transmembrane segment and the second intracellular loop of many class I GPCRs. It has been postulated to play an important role in the transition between conformational states of some of these receptors (10, 22, 26).

We have studied sequential deletions of residues within the predicted first, second, and third intracellular loop domains of the motilin receptor and have utilized alanine, phenylalanine, and histidine replacement for each residue within functionally important segments. Tyr66, Arg136, and Val299 were found to be critical for biological responses to both motilin and erythromycin. These data support a common mechanism for activation of this receptor by both peptide and nonpeptidyl agonists that are known to bind to distinct regions of this receptor, and they provide the first indication of specific residues to represent candidates for functionally important sites, such as regions of G protein interaction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Enzymes used for receptor mutagenesis were purchased from Roche Applied Science (Tokyo, Japan), except for Pfu Turbo DNA polymerase, which was from Stratagene (La Jolla, CA). Fura-2 acetoxymethyl ester (AM) was from Molecular Probes (Eugene, OR). Motilin, erythromycin, and nonenzymatic cell dissociation solution were from Sigma (Indianapolis, IN). Expression vector pcDNA3.1(–) was from Invitrogen (Carlsbad, CA). The human motilin receptor cDNA was kindly provided by Drs. S. D. Feighner and A. D. Howard of Merck Research Laboratories (Rahway, NJ; Ref. 9).

Receptor constructs. Constructs representing a series of mutations of the predicted intracellular loop regions of the motilin receptor were prepared (Fig. 1). These represented deletions of segments ranging in length from 2 to 6 amino acid residues and substitutions of single amino acid residues in domains of interest with alanine, the neutral amino acid, and phenylalanine or histidine, the amino acids most structurally similar to the original residues. Mutant motilin receptors were constructed by an oligonucleotide-directed approach. PCR was performed in a thermal cycler with Pfu Turbo DNA polymerase, running 18 cycles of 95°C for 30 s, 65°C for 1 min, and 68°C for 14 min. Products of the PCR and restriction enzyme digestion were separated on 1% agarose gels and purified with a Qiagen kit (Valencia, CA). Receptor constructs were subcloned into pcDNA3.1(–). The sequences of all constructs were confirmed by direct DNA sequencing with an ABI Prism DNA Sequencer (Foster City, CA).


Figure 1
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  		Fig. 1. Primary structures of the human motilin receptor constructs used in the present report. Shown is a schematic diagram of the amino acid sequence and possible membrane topology (based on hydropathy) of the motilin receptor, along with the design of the segmental deletions (in numbered brackets) and Ala or Phe or His-replacement constructs (areas of dark circles with white lettering).

 
Receptor expression. Receptor constructs were expressed transiently in COS-1 cells (American Type Culture Collection), which do not naturally express the motilin receptor. Cells (0.5 x 106) plated on plastic culture ware were transfected with 3 µg of DNA by a modification of the DEAE-dextran method. Cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. Cells were harvested with nonenzymatic cell dissociation solution 72 h after transfection for biological activity assay and radioligand binding assay.

Intracellular calcium biological activity assay. The abilities of each motilin receptor construct to respond to motilin and erythromycin were studied with a well-established assay for intracellular calcium signaling in fura-2 AM-loaded transfected COS cells. For this, COS cells expressing the receptor constructs were lifted with nonenzymatic cell dissociation solution, washed. and loaded with 5 µM fura-2 AM in serum-free culture medium by incubation at 37°C for 20 min. Cells were then washed twice with Krebs-Ringer-HEPES (KRH) medium (in mM: 25 HEPES pH 7.4, 104 NaCl, 5 KCl, 1.2 MgSO4, 1 KH2PO4, and 2 CaCl2, with 0.2% bovine serum albumin and 0.01% soybean trypsin inhibitor) before being suspended in KRH medium at a density of 1.0 x 106/ml. Approximately 2.0 x 106 cells per assay were stimulated with varied concentrations of motilin at 37°C, with fluorescence quantified in a PerkinElmer LS55 Luminescence Spectrometer (Beaconsfield, UK). Excitation was performed at both 340 and 380 nm, with emission determined at 520 nm and calcium concentration calculated from the ratios as described by Grynkiewicz et al. (12). The peak intracellular calcium concentration that was transiently achieved was used to determine the agonist concentration dependence of the biological responses.

Receptor binding assay. Radioligand binding assays were performed with the various motilin receptor-bearing cells, 125I-labeled motilin (3–5 pM radioligand), and KRH medium. Incubations were for 60 min at 25°C. The binding assays were performed in 24-well tissue culture plates. Nonspecific binding was determined in the presence of 1 µM motilin and represented <20% of total binding.

Statistical analysis. Binding kinetics were determined with the LIGAND program of Munson and Rodbard (20). Binding curves and biological activity curves were analyzed and plotted with the nonlinear regression analysis program in the Prism software package (GraphPad Software, San Diego, CA). All assays were repeated at least four times. All results are expressed as means ± SE. Data were two-tailed analyzed with two-way analysis of variance followed by Newman-Keuls test for multiple comparisons. A P < 0.05 was regarded as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mutagenesis of predicted first intracellular loop of motilin receptor. In this series of studies, we sequentially deleted segments in the predicted first intracellular loop domain. Segments four amino acid residues in length were deleted. Of all constructs tested, only one first loop deletion mutant demonstrated a marked reduction in motilin- and/or erythromycin-stimulated intracellular calcium responses. This represented deletion of the segment including residues 63 through 66, situated at the amino-terminal end of the first loop that reduced the signaling response to both types of agonists (Fig. 2, A and B, Table 1). To identify potentially important residues in this region, we mutated each of the four residues to Ala. Figure 2, C and D, show that I63A, G64A, and R65A mutants signaled in response to motilin or erythromycin similarly to the wild-type motilin receptor (Table 1). In contrast, mutation of Tyr66 to either Ala or Phe resulted in dramatic decreases of intracellular calcium responses to both motilin (Fig. 2C, Table 1) and erythromycin (Fig. 2D, Table 1). The maximal calcium responses to motilin and erythromycin for these two Tyr66 mutants were only 30% of that for the wild-type motilin receptor.


Figure 2
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  		Fig. 2. Biological activities of the mutant constructs in the predicted first intracellular loop of the motilin receptor. Shown are intracellular calcium responses to motilin (A and C) and erythromycin (B and D) in COS cells transfected with each of the receptor constructs indicated. A and B: deletion mutants. The only segment that had a negative impact on the intracellular calcium responses was the deletion construct {Delta}63–66. C and D: single residue replacement mutants. Only mutation of Tyr66 to Ala or Phe had substantial negative impact on the calcium responses. Values are expressed as means ± SE of data from 4 independent assays, with these values normalized relative to the maximal response to motilin or erythromycin in cells expressing the wild-type receptor.

 

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  		Table 1. Biological activity data

 
Mutagenesis of predicted second intracellular loop of motilin receptor. In this region, segments ranging in length from two to six amino acid residues were deleted. Of the eight deletion constructs tested, only the {Delta}135–137 mutant situated within the amino-terminal half of this loop had a significant reduction in intracellular calcium responses, with this being true for both motilin and erythromycin stimulation (Fig. 3, A and B, Table 1). This segment represents the conserved E/DRY motif. The maximal calcium responses were only ~30% of that of the wild-type receptor (Fig. 3, A and B, Table 1). To further localize the functionally critical residues, we mutated each residue between Glu135 and Tyr137 to Ala or His. Of all site mutants tested, only mutation of Arg136 to Ala or His resulted in significant loss of intracellular calcium responses to motilin and erythromycin (by ~30%) (Fig. 3, C and D, Table 1).


Figure 3
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  		Fig. 3. Biological activities of the mutant constructs in the predicted second intracellular loop of the motilin receptor. Shown are intracellular calcium responses to motilin (A and C) and erythromycin (B and D) in COS cells transfected with each of the receptor constructs indicated. A and B: deletion mutants. The only segment that had a negative impact on the intracellular calcium responses was the deletion construct {Delta}135–137. C and D: single residue replacement mutants. Only mutation of Arg136 to Ala or His had substantial negative impact on the calcium responses. Data are illustrated as in Fig. 2 from 4 independent assays.

 
Mutagenesis of predicted third intracellular loop of motilin receptor. In this region, segments ranging in length from four to six amino acid residues were deleted. Of the five third loop deletion mutants tested, only the {Delta}296–301 mutant at the carboxy-terminal end of this loop displayed reduced intracellular calcium responses to motilin and erythromycin (Fig. 4, A and B, Table 1). Mutation of each of the residues within this segment to Ala revealed that only the V299A mutant displayed markedly reduced intracellular calcium responses, with this again true for both motilin and erythromycin (Fig. 4, C and D, Table 1).


Figure 4
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  		Fig. 4. Biological activities of the mutant constructs in the predicted second intracellular loop of the motilin receptor. Shown are intracellular calcium responses to motilin (A and C) and erythromycin (B and D) in COS cells transfected with each of the receptor constructs indicated. A and B: deletion mutants. The only segment that had a negative impact on the intracellular calcium responses was the deletion construct {Delta}296–301. C and D: single residue replacement mutants. The construct V299A was the only one that had substantial negative impact on calcium responses. Data are illustrated as in Fig. 2 from 4 independent assays.

 
Radioligand binding assay. Each of the functionally impaired site mutants (Y66A, Y66F, R136A, R136H, and V299A) was tested for its ability to bind motilin. For this, COS cells expressing each of these constructs were used in assays in which increasing concentrations of motilin were used to compete for binding of the radioligand 125I-motilin. Figure 5 shows that each of these constructs bound motilin in a saturable manner, with their affinities similar to that of the wild-type motilin receptor (Table 2). This also provides evidence of levels of cell surface expression for each of the functionally impaired receptor constructs similar to that of the wild-type motilin receptor.


Figure 5
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  		Fig. 5. Binding activities for motilin. Shown are the curves for motilin competition for binding of the 125I-motilin radioligand to COS cells expressing the wild-type motilin receptor or key mutant motilin receptors Y66A, Y66F, R136A, R136H, or V299A. These mutant constructs bound motilin with affinities similar to that of the wild-type receptor. Values represent % of maximal saturable binding that were observed in the absence of motilin and are expressed as means ± SE of values from 4 independent studies.

 

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  		Table 2. Receptor binding data

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The motilin receptor belongs to a group of class I GPCRs that also includes growth hormone secretagogue receptors (9). In this work, we have focused on the predicted intracellular loop domains of the motilin receptor and have utilized receptor mutagenesis and intracellular calcium responses to systematically scan for residues that might be critical for motilin- and erythromycin-stimulated biological responses. Three residues, Tyr66 in the predicted first intracellular loop, Arg136 in the predicted second intracellular loop, and Val299 in the predicted third intracellular loop, were shown to be critical for calcium signaling, with each mutant affected similarly for stimulation by the natural peptide agonist motilin and the nonpeptidyl agonist erythromycin.

There have been only a small number of reports identifying roles for G protein coupling within the first intracellular loop of receptors in this family. Liu and Wu (14) reported that all three intracellular loops of the endothelin receptor are involved in G protein coupling, with residues Met128-Arg129-Asn130 in the first intracellular loop specifically required for activation of G{alpha}13. Geng et al. (11) reported that residue Arg60 in the thromboxane A2 receptor (TP{alpha}R) and residue Lys60 in the same position in other prostanoid receptors are involved in coupling to G{alpha}q protein. The mutant motilin constructs in which the Tyr66 residue was replaced with Ala or Phe exhibited marked reductions in motilin- and erythromycin-stimulated responses.

The E/DRY motif at the interface between transmembrane segment three and the second intracellular loop of many class I GPCRs has been shown to be functionally important (10, 22, 26). Indeed, in the motilin receptor this was also shown to represent a functionally important region. In fact, this was shown to represent the only functionally important segment within the second intracellular loop region of the motilin receptor. Of interest, the roles for the residues within this motif seem to be distinct in the motilin receptor relative to other family members that have been studied. While the role of the Tyr of the typical E/DRY motif remains unclear, the Glu/Asp and Arg residues of this motif have been shown to regulate GPCR conformational states and G protein coupling/recognition of other class I receptors (22). It is interesting that in this study Arg136 was identified to be the only functionally critical residue within the motilin receptor sequence.

Mutation of this residue (Arg136) to Ala and His in the motilin receptor resulted in markedly impaired responses to both peptide and nonpeptidyl agonist ligands, while the agonist binding affinity remained normal. Nonconservative mutations of the Arg residue in the E/DRY motif in other class I GPCRs, such as rhodopsin, adrenergic (AR), histamine, and muscarinic cholinergic (AChR) receptors, have been characterized as "loss-of-function phenotypes" (1, 2, 5, 7, 23, 29). Mutation of this Arg in the vasopressin type II receptor (V2R) produces a "constitutively desensitized phenotype," with decreased expression at the plasma membrane (3). For a small subgroup of these receptors, including the {alpha}2A-AR and muscarinic M1 and M5 AchR, nonconservative mutations of this Arg decrease agonist binding affinity, as well as impairing signaling (5, 7, 29). Capra et al. (6) also reported mutations of the Arg in the E/DRY motif of the TP{alpha}R that decrease its ligand binding, signaling, and G protein interaction.

The Glu/Asp of the E/DRY motif in the second intracellular loop of various receptors can have distinct regulatory effects. It is suggested that mutation of Glu/Asp of the E/DRY motif produces at least two distinct phenotypes in class I GPCRs (22). The first phenotype is characterized by an increase of agonist-independent constitutive receptor activity while maintaining high-affinity agonist binding and G protein coupling. The examples are rhodopsin, {alpha}1B-AR, β2-AR, V2R, and histamine H2 receptors (H2R) (19, 21, 24, 25, 27). The second phenotype does not exhibit increased constitutive receptor activity and is often characterized by a loss of high-affinity agonist binding and a loss of G protein coupling. The examples are {alpha}2A-AR, M1 AchR, M5 AchR, and TP{alpha}R (5, 6, 7, 15). In this work, mutation of motilin receptor Glu135 to Ala did not result in either of these effects, with no evidence for constitutive activity or for any effects on agonist binding or biological activity.

There are many examples of effects of GPCR third loop residues on G protein coupling. Bernstein et al. (4) reported that regulators of G protein signaling (RGS) proteins bind directly to the third intracellular loop in the M1, M3, and M5 AChR. Hague et al. reported (13) that in {alpha}1A-AR, RGS protein binds directly and selectively to the third intracellular loop and Lys219, Ser220, and Arg238 in the third intracellular loop are essential for this interaction. In our studies on the motilin receptor, only Val299 in the third intracellular loop was found to be important for agonist signaling. Further investigation of a possible interaction between this Val residue and RGS proteins seems to be important.

In the present studies, we demonstrated that not only the Arg136 residue in the second intracellular loop E/DRY motif but also new residues, Tyr66 and Val 299, in the first and third intracellular loops play important roles in motilin receptor agonist-stimulated signaling. The same residues are similarly important for both motilin- and erythromycin-stimulated signaling. Using an analogous receptor mutagenesis approach, we previously demonstrated (17, 18) the functional importance of predicted extracellular perimembranous residues for motilin-stimulated action, but not for erythromycin-stimulated action. Those effects were likely mediated by direct inhibition of peptide binding. A similar study that evaluated the transmembrane region of this receptor identified mutants that interfered with the action of both motilin and erythromycin (28). That work did not determine whether the effect was direct or indirect, via allosteric changes in conformation. It is likely that erythromycin binds to a region within the helical bundle, based on the absence of effect of mutagenesis of extracellular regions of this receptor. At this point, we cannot be certain whether the interference with motilin action by these mutations in the transmembrane segments reflected motilin coming in contact with those residues or whether it reflected an indirect effect.

Our present findings provide important new insights into the molecular basis of signaling at the motilin receptor. It seems clear that the natural peptide agonist motilin and the nonpeptidyl agonist erythromycin bind to distinct regions of this receptor, yet both are full agonists. We now recognize that the same residues in the cytosolic face of the motilin receptor are important for the signaling by these chemically distinct agonist ligands. This likely reflects a common active conformation of the G protein effector face of this receptor, independent of agonist. These residues also provide important leads to focus on as we build a molecular understanding of the mode of G protein coupling to this receptor.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant (No. 18590683, 2006–7) from the Japanese Ministry of Education, Culture, Sports, Science and Technology and by the Mayo Clinic and Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Matsuura, Dept. of Gastroenterology and Metabology, Ehime Univ. Graduate School of Medicine, Shitsukawa 454, Tohon, Ehime 791-0295, Japan (e-mail: bmatsu{at}m.ehime-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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