d-Amino acid and alanine scans of the bioactive portion of porcine motilin

Peeters, Theo; Macielag, Mark J.; Depoortere, Inge; Konteatis, Zenon; Florance, James R.; Lessor, Ralph A.; Galdes, Alphonse

doi:10.1016/0196-9781(92)90014-t

Cited by 54 publications

(40 citation statements)

References 16 publications

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“…The binding of the peptide that incorporated the D-Bpa residue displayed a much lower affinity (K i ϭ 420 Ϯ 67 nM) than the L-Bpa peptide (K i ϭ 50 Ϯ 7 nM) and was therefore not considered for further use. The lower affinity of the [D-Bpa 5 ,Ile 13 ]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).…”

Section: Resultssupporting

confidence: 75%

“…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, 1992Poitras et al, , 1994Boulanger 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.…”

mentioning

confidence: 95%

“…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, 1992Poitras et al, , 1994Boulanger 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.…”

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confidence: 99%

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

Matsuura

Dong

Coulie

et al. 2005

J Pharmacol Exp Ther

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The motilin receptor belongs to a group of class I G proteincoupled 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 Phe 332 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 detailed understanding of the basis for binding a ligand by a receptor can help refine our understanding of the conformation of the receptor and of the molecular basis for its activation. Although all members of the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors have structural similarities, with seven hydrophobic segments that are believed to traverse the plasma membrane, the details of even this most fundamental characteristic varies within the family of these receptors (Kolakowski, 1994;Ji et al., 1998). This superfamily is also remarkable for the structural diversity of the natural ligands that activate various members, with themes ranging from small photons and biogenic amines that bind within the confluence of transmembrane segments to larger peptide and glycoprotein ligands that bind to extracellular tail and loop domains.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...

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Section: Resultssupporting

confidence: 75%

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confidence: 95%

mentioning

confidence: 99%

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

Matsuura

Dong

Coulie

et al. 2005

J Pharmacol Exp Ther

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“…In contrast, ghrelin does have sequence similarity (36%) with motilin but the pharmacophore of ghrelin, the [1-5] fragment and in particular the octanoyl residue on Ser 3 (Bednarek et al, 2000), and the pharmacophore of motilin, the [1][2][3][4][5][6][7] fragment and in particular Phe 1 , Val 2 , Ile 4 , and Tyr 7 Peeters et al, 1992), show little if any overlap. Interestingly, the weak interaction that we observed seems to be related to the octanoyl group because ghrelin (1-28) oct and ghrelin (1-5) oct have a comparable affinity, which is completely lost when the octanoyl group is removed.…”

Section: Discussionmentioning

confidence: 99%

Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum

Depoortere

Thijs²,

Thielemans³

et al. 2003

J Pharmacol Exp Ther

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The structural relationship between the motilin and the growth hormone secretagogue receptor (GHS-R), and between their respective ligands, motilin and ghrelin, prompted us to investigate whether ghrelin and the GHS-R agonist growth hormonereleasing peptide-6 (GHRP-6), could interact with the motilin receptor. The interaction was evaluated in the rabbit gastric antrum with binding studies on membrane preparations and with contraction studies on muscle strips in the presence of selective antagonists under conditions of electrical field stimulation (EFS) or not. Binding studies indicated that the affinity (pK d ) for the motilin receptor was in the order of ghrelin (4.23 Ϯ 0.07) Ͻ GHRP-6 (5.54 Ϯ 0.08) Ͻ motilin (9.13 Ϯ 0.03). The interaction of ghrelin with the motilin receptor requires the octanoyl group. Motilin induced smooth muscle contractile responses but ghrelin and GHRP-6 were ineffective. EFS elicited on-and off-responses that were increased by motilin already at 10 Ϫ9 M, but not by 10 Ϫ5 M ghrelin. In contrast, GHRP-6 also enhanced the on-and off-responses. The motilin antagonist Phe-cyclo[Lys-Tyr(3-tBu)-␤Ala-] trifluoroacetate (GM-109) blocked the effect of GHRP-6 on the off-responses but not on the on-responses. Under nonadrenergic noncholinergic conditions, the effects of motilin and GHRP-6 on the on-responses were abolished; those on the off-responses were preserved. All responses were blocked by neurokinin (NK) 1 and NK 2 antagonists. In conclusion, ghrelin is unable to induce contractions via the motilin receptor. However, GHRP-6 enhances neural contractile responses, partially via interaction with the motilin receptor on noncholinergic nerves with tachykinins as mediator, and partially via another receptor that may be a GHS-R subtype on cholinergic nerves that corelease tachykinins.

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“…Structureâ ctivity studies in the rabbit have shown that bioactivity is mostly determined by the N-terminal end, especially residues 1, 4, and 7 [14,15], and it is assumed that the C-terminal region (amino acids 10^22) forms an K-helix which stabilizes the interaction of the N-terminal residues at the active site [16]. It remains to be determined how these changes a¡ect bioactivity.…”

Section: Homology With the Motilin Precursors From Other Speciesmentioning

confidence: 99%

Identification and expression of the motilin precursor in the guinea pig

et al. 2001

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Motilin has never been isolated from rodents, the most frequently used laboratory animals, despite several attempts. We have isolated and sequenced the motilin precursor from duodenal mucosa of guinea pig (GenBank accession number AF323752) and studied its expression in several tissues. The percent homology with human motilin is the lowest yet observed due to several unique substitutions in the C-terminal end. As expected, the precursor was present in the gut mucosa with the exception of the gastric corpus. It was also present in medulla oblongata, nucleus of the solitary tract, hypophysis, spinal cord, hypothalamus, and cerebellum but not in the cerebral cortex. For the first time we demonstrated motilin expression in the thyroid. ß

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d-Amino acid and alanine scans of the bioactive portion of porcine motilin

Cited by 54 publications

References 16 publications

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

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

Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum

Identification and expression of the motilin precursor in the guinea pig

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