Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling.

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC(50) = 1.5 +/- 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M(r) = 6,000 and M(r) = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.     

Molecular identification and characterization of the dog motilin receptor

Motilin, a 22-amino acid peptide hormone secreted by endocrine cells of the intestinal mucosa, plays an important role in the regulation of gastrointestinal motility. The actions of motilin agonists have been extensively investigated in dogs due to physiological similarities between the dog and human alimentary tracts. The amino acid sequence of the dog motilin receptor, however, was previously unknown. We have cloned a cDNA from dog stomach corresponding to the motilin receptor. The deduced protein shared 71% and 72% sequence identity with the human and rabbit motilin receptors, respectively. Expression of the dog motilin receptor in CHO cells promoted the typical cellular responses to the agonists, motilin and erythromycin. The rank order of potency determined for these agonists was similar to that found for the human motilin receptor, with motilin being more potent than erythromycin. Immunohistochemistry of the dog stomach revealed that the motilin receptor was localized in neuronal cell bodies and fibers. This is the first study detailing the cloning, expression, and functional characterization of the dog motilin receptor. Determination of the full sequence and functional properties of the dog motilin receptor will provide useful information enabling us to interpret previous and future studies of motilin agonists in dogs.     

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.     

N-terminal portion of motilin determines its biological activity.

This study aimed to identify the portion of the 22 amino acid sequence of motilin responsible for the biological activity of the peptide. The contraction of rabbit duodenal muscle in vitro was measured when exposed to synthetic fragments of motilin corresponding to various sequences of the C- or N-terminal portions of the molecule. Fragments 2-22 or 3-22 (where the initial amino acids of the N-terminal ending were removed) were more than 1000 times less potent than the native molecule 1-22. Fragment 1-9 (where the last 13 amino acids located at the C-terminal side of motilin were removed) was devoid of any contractile capacity, while synthetic fragments whose C-terminal structure extended beyond the 1-9 motilin sequence maintained almost complete biological activity. N-terminal amino acid sequence 1-9 is therefore an essential determinant of the contractile activity of motilin.     

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.     

Distribution and characterization of motilin receptors in the cat

We demonstrate binding of [¹²⁵I][Nle¹³-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (B_max in fmol/mg protein): 0 (fundus); 12 ± 2 (corpus); 22 ± 3 (antrum); 55 ± 12 (duodenum); 44 ± 10 (jejunum); 17 ± 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pK_d = 8.84 ± 0.06) and in the small intestine (pK_d = 8.58 ± 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle¹³-po]motilin bound to cat duodenal receptor with potencies (pK_d) of 5.47 ± 0.23, 7.60 ± 0.24, and < 4.3, respectively. Studies with [Leu¹³-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC₅₀: 8.29 ± 0.08; 7.12 ± 0.12; 5.99 ± 0.15). The compounds had a comparable intrinsic activity (83 ± 3%; 80 ± 5%; 82 ± 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.     

9-Dihydroerythromycins as non-antibiotic motilin receptor agonists

A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.     

The erythromycin derivative EM-523 is a potent motilin agonist in man and in rabbit

Erythromycin may stimulate gastrointestinal motor activity via its effect upon motilin receptors. We have studied the ability of the derivative EM-523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal] to induce contractions in duodenal smooth muscle strips and to displace labeled motilin bound to antral smooth muscle, in man and in rabbit. In both species EM-523 approached the potency of motilin for inducing contractions. Thus pED50 values were 7.84 +/- 0.11 and 8.69 +/- 0.12 for motilin in, respectively, man and rabbit, against 6.08 +/- 0.13 and 8.19 +/- 0.10 for EM-523. In rabbit the efficacy of both compounds decreased in parallel aborally, the responses to EM-523 could not be blocked by atropine (10(-7) M) or TTX (10(-7) M), and both compounds were unable to further enhance the maximum effect to the other compound. In binding studies the order of potency was the same as in the contraction studies. The pIC50 values were: motilin (8.84 +/- 0.31, 9.17 +/- 0.20) greater than EM-523 (7.89 +/- 0.1, 8.40 +/- 0.10). A Schild plot revealed that EM-523 was a competitive inhibitor of motilin receptor binding in man and in rabbit. We conclude that EM-523 is a potent motilin agonist.     

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.     

Structure-activity studies with fragments and analogues of salmonid melanin-concentrating hormone

A number of cyclic and linear fragments and analogues of MCH were synthesized and their biological potencies tested using the isolated carp scale melanophore assay. In this system, the cyclic portion MCH(5–14) exhibited only 0.1% bioactivity, which was markedly enhanced by the addition of the exocyclic sequences MCH(15–17) and MCH(1–4). The exocyclic sequence itself, MCH(1–4, 15–17), had minimal activity, however. Substitution of Tyr¹¹ with phenylalanine reduced the potency of the ring structure MCH(5–14) by about 4-fold. Substitution of Gly⁸ with D-alanine reduced the potency of MCH(5–14) 16-fold, while both substitutions together caused a still more marked reduction (200-fold) in bioactivity. Linearized fragments of MCH, extending from MCH(15–17) to [Cys(Acm)^5,14]MCH(1–17), showed a progressive increase in potency. The linearized forms of MCH, MCH(5–17) and MCH(5–14), were approximately 100-fold or less potent than their cyclic forms. The significant increases in bioactivity produced by the addition of the C- and N-terminal exocyclic sequence even to these linearized forms further emphasizes the importance of these regions for interaction at the receptor site.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Design and synthesis of novel tetra-peptide motilin agonists

A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH(2), were designed, on the basis of structure-activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue, have agonistic activity. H-Phe-Val-Trp(2'-CH(2)CH(2)OH)-Ile-NH(2)(7), H-Phe-Val-Trp(2'-SCH(3))-Ile-NH(2)(8), and H-Phe-Val-Trp(2'-SCH(2)CH(2)CH(3))-Ile-NH(2)(9), showed an EC(50) for contractile activity in the rabbit smooth muscle of 14.1+/-3.2, 12.9+/-4.1, and 4.6+/-1.6 microM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal transduction via motilin receptor.     

Human TE671 cells express functional motilin receptors

In our search for a cell line expressing endogenous human motilin receptor, we have discovered that theTE671 cell line, a neuron-derived medulloblastoma human line, expresses functional motilin receptors. The cDNA of the receptor was isolated from the cells and its sequence was confirmed to be identical to the previously reported cDNA sequence isolated from human thyroid. The function of the receptor protein was evaluated both for its ability to inhibit the binding of ¹²⁵I-motilin to a crude membrane preparation of TE671 cells and for activation of the phospholipase C signal transduction pathway by calcium mobilization assay. The precise numbers of motilin receptor RNA molecule in TE671 cell and 24 human tissues were quantitatively determined by real-time PCR. TE671 cell line should be a useful tool for the study of motilin receptor-involved signal transduction in humans.     

Comparison of the biological activity of canine and porcine motilin in rabbit

The biological activity of porcine and canine motilin was studied in rabbits by establishing dose-response curves of both peptides using two different methods. The dissociation constant, obtained from the displacement of iodinated porcine motilin by canine motilin was 0.6 +/- 0.3 nM, versus 1.2 +/- 0.4 nM for porcine motilin. For the 13-norleucine and 13-leucine analogues of porcine motilin a value of 0.8 +/- 0.3 nM was obtained. Both motilins were almost equipotent in stimulating the in vitro contractile response of longitudinal smooth muscle strips: half-maximal effect was achieved at a concentration of 1.0 +/- 0.1 nM for canine versus 1.3 +/- 0.2 nM for the 13-norleucine analogue of porcine motilin. We conclude that porcine and canine motilin have a comparable bioactivity in the rabbit, although canine motilin is slightly more effective. The motilin receptor is probably specific for the N-terminal portion which is identical in both molecules.     

Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin

Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.     

The role of the 4'-hydroxyl on motilin agonist potency in the 9-dihydroerythromycin series

The role of the erythromycin 4′′-hydroxyl group has been explored on the motilin agonist potential in the 9-dihydroerythromycin series of motilides. The compounds show potencies 2- to 4-fold superior to the corresponding hydroxylated compounds. The relationship is maintained when the 9-hydroxyl is alkylated to generate the corresponding 4′′-deoxy-9-O-acetamido-9-dihydroerythromycins. However, concomitant with this increase in potency is an increase in hERG inhibition.     

Motilin receptors in the human antrum

Motilin is an intestinal peptide that stimulates contraction of gut smooth muscle. The motilin receptor has not been cloned yet, but motilin-receptor agonists appear to be potent prokinetic agents for the treatment of dysmotility disorders. The aim of this study was to determine neural or muscular localization of motilin receptors in human upper gastrointestinal tract and to investigate their pharmacological characteristics. The binding of (125)I-labeled motilin to tissue membranes prepared from human stomach and duodenum was studied; rabbit tissues were used for comparison. Solutions enriched in neural synaptosomes or in smooth muscle plasma membranes were obtained. Various motilin analogs were used to displace the motilin radioligand from the various tissue membranes. The highest concentration of human motilin receptors was found in the antrum, predominantly in the neural preparation. Human motilin receptors were sensitive to the NH(2)-terminal portion of the motilin molecule, but comparison with rabbit showed that both species had specific affinities for various motilin analogs [i.e., Mot-(1-9), Mot-(1-12), Mot-(1-12) (CH(2)NH)(10-11), and erythromycin]. Motilin receptors obtained from synaptosomes or muscular plasma membranes of human antrum expressed different affinity for two motilin-receptor agonists, Mot-(1-12) and Mot-(1-12) (CH(2)NH)(10-11), suggesting that they correspond to specific receptor subtypes. We conclude that human motilin receptors are located predominantly in nerves of the antral wall, are functionally (and probably structurally) different from those found in other species such as the rabbit, and express specific functional (and probably structural) characteristics dependent on their localization on antral nerves or muscles, suggesting the existence of specific receptor subtypes, potentially of significant physiological or pharmacological relevance.     

5-羟色胺和胃动素在狗小肠移行性复合运动调控中的作用

应用慢性植入小肠腔外应力传感器记录清醒狗小肠移行性复合运动（ＭＭＣ）。在颈外静脉和支配１０￣１５ｃｍ肠段的空肠动脉内分别放置－硅胶管以备灌流药物的取血样品用。本研究观察了静脉和动脉注射药物对小肠ＭＭＣ的影响及ＭＭＣ不同时相血浆５－羟色胺（５－ＨＴ）和胃动素的浓度变化。结果表明：（１）ＭＭＣ不同时相血浆５－ＨＴ和胃动素浓度呈周期性变化,５－ＨＴ峰值在胃动素峰值之前出现。血浆５－ＨＴ浓度在ＭＭＣⅡ相后     

Exon-intron organization, expression, and chromosomal localization of the human motilin gene

The human motilin gene has been isolated and characterized. The gene spans about 9 kilobase pairs (kb) and the 0.7 kb motilin mRNA is encoded by five exons. The 22-amino-acid motilin sequence is encoded by exons 2 and 3. The human motilin gene was mapped to the p21.2----p21.3 region of chromosome 6 by hybridization of the cloned cDNA to DNAs from a panel of reduced human-mouse somatic cell hybrids and by in situ hybridization to human prometaphase chromosomes. RNA blotting using RNA prepared from various regions of the human gastrointestinal tract revealed high levels of motilin mRNA in duodenum and lower levels in the antrum of the stomach; motilin mRNA could not be detected by this procedure in the esophagus, cardia of the stomach, descending colon or gallbladder.     

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.