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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).
Source publication
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 region...
Contexts in source publication
Context 1
... of predicted second intracellular loop of moti- lin receptor. In this region, segments ranging in length from two to six amino acid residues were deleted. Of the eight The construct V299A was the only one that had substantial negative impact on calcium re- sponses. Data are illustrated as in Fig. 2 from 4 independent assays. deletion constructs tested, only the 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 be- tween 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 ...
Context 2
... 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 1.70.8 9431 ...
Context 3
... 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 (Valen- cia, 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, ...
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Citations
... These findings may be of direct relevance to treatment because individuals with the identified mutation may respond differently to gastroparesis treatments especially those targeting the MLNR protein such as motilin receptor agonists. Interestingly, previous experiments examined the mutations within predicted intracellular loop regions of MLNR and their effects on motilin-and erythromycin-stimulated activity (7). The authors identified functionally relevant residues: deletions of receptor residues 63-66, 135-137, and 296-301 that each resulted in significant loss of intracellular calcium responses on stimulation by motilin and erythromycin (7). ...
... Interestingly, previous experiments examined the mutations within predicted intracellular loop regions of MLNR and their effects on motilin-and erythromycin-stimulated activity (7). The authors identified functionally relevant residues: deletions of receptor residues 63-66, 135-137, and 296-301 that each resulted in significant loss of intracellular calcium responses on stimulation by motilin and erythromycin (7). It is noteworthy that our identified frameshift causes out-of-frame translation starting at amino acid 202 (leucine), leading to premature termination at 260. ...
Introduction:
Gastroparesis is a serious medical condition characterized by delayed gastric emptying and symptoms of nausea, vomiting, bloating, fullness after meals, and abdominal pain.
Methods:
To ascertain the genetic risk factors for gastroparesis, we conducted the largest thus far whole-genome sequencing study of gastroparesis. We investigated the frequency and effect of rare loss-of-function variants in patients with both idiopathic and diabetic gastroparesis enrolled in a clinical study of gastroparesis.
Results:
Among rare loss-of-function variants, we reported an increased frequency of a frameshift mutation p.Leu202ArgfsTer105, within the motilin receptor gene, variant rs562138828 (odds ratio 4.9). We currently replicated this finding in an independent large cohort of gastroparesis samples obtained from patients participating in the ongoing phase III gastroparesis clinical study.
Discussion:
Motilin receptor is an important therapeutic target for the treatment of hypomotility disorders. The identified genetic variants may be important risk factors for disease as well as may inform treatments, especially those targeting motilin receptor.
... The human ghrelin and motilin receptors share 52% overall amino acid identity, including 86% identity in the seven-transmembrane region [2][3][4][5][6]. Matsuura et al. have reported the molecular binding mechanism of motilin and EM to MLNR, as well as that of ghrelin to the growth hormone secretagogues receptor (GHSR) [7][8][9][10][11][12][13]. Similarities also are suggested by ghrelin and motilin's predominant distribution in the upper gastrointestinal tract and their release during hunger to influence gastrointestinal functions. ...
Gastrointestinal motility is regulated by neural factors and humoral factors. Both motilin and ghrelin improve gastrointestinal motility, but many issues remain unclear. We prepared human motilin receptor transgenic (Tg) mice and performed experiments evaluating the effects of motilin, erythromycin (EM), and ghrelin. EM and ghrelin promoted gastric emptying (GE) when administered either peripherally or centrally to Tg mice. Atropine (a muscarinic receptor antagonist) counteracted GE induced by centrally administered EM, but not that induced by peripherally administered EM. The administration of EM in this model promoted the effect of mosapride (a selective serotonin 5-hydroxytryptamine 4 (5-HT4) receptor agonist), and improved loperamide (a μ-opioid receptor agonist)-induced gastroparesis. The level of acyl-ghrelin was significantly attenuated by EM administration. Thus, we have established an animal model appropriate for the evaluation of motilin receptor agonists. These data and the model are expected to facilitate the identification of novel compounds with clinical potential for relieving symptoms of dyspepsia and gastroparesis.
... Nevertheless, Xu et al. (2005b) demonstrated that motilin and EM-A share a common binding site on Glu 119 of the transmembrane region. In addition both agonists recognize the same residues in the predicted intracellular (cytosolic) region of the receptor that may represent functionally important sites involved in G-protein coupling (Tokunaga et al., 2008). ...
Motilin is a hormone released by the endocrine cells of the duodenal mucosa during fasting to stimulate gastrointestinal motility. Ghrelin, the closest family member of motilin, was discovered 10 years ago from the rat stomach as the long-awaited endogenous ligand of the growth hormone secretagogue receptor. Ghrelin has now emerged as a multifunctional hormone with important effects on energy homeostasis but also on gastrointestinal motility. Like motilin, it induces hunger contractions in the fasting state and acts postprandially to accelerate gastric emptying. While the development of motilin agonists for the treatment of hypomotility disorders has been going on for more than 15 years, the development of ghrelin agonists is still in its infancy. The failure of the first generation of motilin agonists in clinical trials has been largely due to problems of desensitization and worsening of symptoms due to effects on gastric accommodation. These issues are being taken care of with the second generation of motilin agonists that are currently under evaluation. Ghrelin agonists have the same potential as motilin agonists to treat hypomotility disorders but their effects on appetite may even be a bonus to treat disorders such as functional dyspepsia while ghrelin's anti-inflammatory effects may make it superior to motilin to treat post-operative ileus. Nevertheless the important endocrine activities of ghrelin may result in side effects which are not encountered with motilin. Future studies will need to point out whether the motilin–ghrelin receptor family will make it as a new class of gastroprokinetics.
G protein-coupled receptors (GPCRs) are the largest family of membrane receptors involved in the regulation of almost all known physiological processes. Dysfunctions of GPCR-mediated signaling have been shown to cause various diseases. The prevalence of obesity and type 2 diabetes mellitus (T2DM), two strongly associated disorders, is increasing worldwide, with tremendous economical and health burden. New safer and more efficacious drugs are required for successful weight reduction and T2DM treatment. Multiple GPCRs are involved in the regulation of energy and glucose homeostasis. Mutations in these GPCRs contribute to the development and progression of obesity and T2DM. Therefore, these receptors can be therapeutic targets for obesity and T2DM. Indeed some of these receptors, such as melanocortin-4 receptor and glucagon-like peptide 1 receptor, have provided important new drugs for treating obesity and T2DM. This review will focus on the naturally occurring mutations of several GPCRs associated with obesity and T2DM, especially incorporating recent large genomic data and insights from structure-function studies, providing leads for future investigations.
The motilin receptor belongs to a family of Class I G protein-coupled receptors, and is an important endogenous regulator of gastrointestinal motor function. 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. The action of these different chemical classes of agonists likely yields a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. In the current work, we studied the predicted transmembrane (TM) domains of this receptor for functional responses to motilin and erythromycin. Motilin receptor constructs were prepared in which each residue in the TM domains was mutated to alanine or valine. Each construct was expressed in COS cells and characterized for motilin and erythromycin binding and intracellular calcium responses stimulated by both agonists. Constructs with mutations of residues, Asp94, Leu95, Arg97 and Try99 in TM2, Ser169 in TM4, and Tyr321 and Glu325 in TM6, were responsible for the negative impact on biological activity stimulated by erythromycin, but had no effect on motilin responses. On the other hand, constructs with mutations of residues, Leu113 in TM3, Pro172 in TM4, Trp250 and Tyr255 in TM5, and Gln334 in TM7, were negatively responsive to both erythromycin and motilin. These data support important roles of new regions in the TM domains of the motilin receptor for erythromycin action, suggesting differential mechanisms of actions by peptidyl and non-peptidyl ligands.
The motilin receptor (MR) belongs to a family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptor (GHSR). Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the MR were important for responses to the natural peptide ligand, motilin, and the transmembrane domains of the MR were important for a non-peptidyl ligand, erythromycin. We also reported that the perimembranous residues in the second extracellular loop of the GHSR were critical for natural ligand ghrelin binding and activity. The MR is 52% identical to GHSR, with 86% sequence identity in the transmembrane domains. In the current work, to gain insight into a relationship between MR and GHSR, we studied functional responses to motilin, erythromycin and ghrelin of expression cells of chimeric constructs of MR and GHSR and co-expression cells of both MR and GHSR. We also generated human MR transgenic mice, and clarified a relationship between motilin and ghrelin. MR(1-62)/GHSR(68-366) construct responded only to ghrelin, MR(1-102)/GHSR(108-366) responded to ghrelin and erythromycin, and MR(1-129)/GHSR(135-366) and MR(1-178)/GHSR(184-366) responded to erythromycin, while GHSR(1-183)/MR(179-412) responded to neither motilin, erythromycin nor ghrelin. MR and GHSR co-expression cells have no additional responses to these ligands. Motilin or erythromycin administration to human MR transgenic mice resulted in a decrease of serum acyl-ghrelin levels, while MR and GHSR mRNA expression in the gastrointestinal tracts were not changed. These data suggested that in species expressing both motilin-MR and ghrelin-GHSR, there is a compensatory relationship in vivo.
The Class A family of guanine nucleotide-binding protein (G protein)-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue (GHS) has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights helpful in the development of such drugs. We previously reported that Cys residues and the perimembranous residues in the extracellular loops and amino-terminal tail of the motilin receptor are critical for peptide ligand, motilin, binding and biological activity. In the current work, we focused on the predicted extracellular domains of the human GHS receptor 1a, and identified functionally important residues by using sequential deletions ranging from one to twelve amino acid residues and site-directed replacement mutagenesis approach. Each construct was transiently expressed in COS cells, and characterized for ghrelin- and growth hormone releasing peptide (GHRP)-6-stimulated intracellular calcium responses and ghrelin radioligand binding. Cys residues in positions 116 and 198 in the first and second extracellular loops and the perimembranous Glu¹⁸⁷ residue in the second extracellular loop were critical for ghrelin and GHRP-6 biological activity. These results suggest that Cys residues in the extracellular domains in this family of Class A G protein-coupled receptor is likely involved in the highly conserved and functionally important disulfide bond, and that the perimembranous residues contribute peptide ligand binding and signaling.
The motilin receptor (MTLR) is an important therapeutic target for the treatment of hypomotility disorders but desensitization may limit its clinical utility. The aim of this study was to investigate the role of the C-terminal tail of the MTLR in the desensitization, phosphorylation and internalization process. Three MTLR mutants, C-terminally truncated from amino acid 412 till 384 (MTLRDelta385), 374 (MTLRDelta375) or 368 (MTLRDelta369), were constructed and C-terminally tagged with an EGFP and stably expressed in CHO cells co-expressing the Ca(2+) indicator apoaequorin. Activity and desensitization were studied by measuring changes in motilin-induced luminescent Ca(2+) rises. Receptor phosphorylation was investigated by immunoprecipitation and MTLR-EGFP internalization was visualized by fluorescence microscopy. Truncation only reduced MTLR affinity and the efficacy to induce Ca(2+) luminescent responses of the MTLRDelta375-EGFP mutant. Furthermore, the region between amino acid 375 and 368 seems to be important for proper cell surface expression of the MTLR since receptors of the MTLRDelta369-EGFP mutant but not of the other mutants were found intracellularly in vesicles. Truncation of the receptor till amino acid 384 or 374 did neither affect desensitization nor internalization. In contrast phosphorylation of the MTLRDelta385-EGFP mutant was reduced by 80% but was not affected in the MTLRDelta375-EGFP mutant. In conclusion, MTLR desensitization and internalization is not dependent on the presence of the C-terminal tail. Truncation favors internalization via either phosphorylation-independent pathways or via phosphorylation of alternative sites in the receptor.
