Elsevier

Biochemical Pharmacology

Volume 70, Issue 6, 15 September 2005, Pages 879-887
Biochemical Pharmacology

Motilin and erythromycin-A share a common binding site in the third transmembrane segment of the motilin receptor

https://doi.org/10.1016/j.bcp.2005.06.022Get rights and content

Abstract

The motilin receptor (MTLR) represents a clinically useful pharmacological target, as agonists binding to the MTLR have gastroprokinetic properties. In order to compare the molecular basis for interaction of the MTLR with motilin and with the non-peptide motilin agonist, erythromycin-A (EM-A), the negatively charged E119 located in the third transmembrane (TM3) region was mutated to D (E119D) and Q (E119Q), respectively, and changes in activity of the mutant receptors were verified.

Methods:

Each mutant receptor was stably transfected in CHO-cells containing the Ca2+ indicator apo-aequorin. Receptor activation in response to motilin, EM-A and their analogues was assessed by Ca2+-luminescense.

Results:

In the E119Q mutant, the Ca2+ response to motilin and EM-A was abolished while in the E119D mutant it was reduced with 62% (motilin) and 81% (EM-A). The pEC50 values were shifted from 9.65 ± 0.03 to 7.41 ± 0.09 (motilin) and from 6.63 ± 0.12 to 4.60 ± 0.07 (EM-A). Acetylation of the N-terminal amine group as in [N-acetyl-Phe]1 mot (1–14), decreased the potency 6.3-fold (WT-MTLR) and 148-fold (E119D). Acetylation of EM-A enol ether induced a more pronounced shift in potency: 7943-fold (WT-MTLR) and 1413-fold (E119D).

Conclusion:

The comparable loss of affinity of the mutant receptors for motilin and EM-A indicate that these agonists both interact with the TM3 domain of the MTLR. The results with acetylated derivatives support an ionic interaction between E119 of the MTLR with the N+ of the desosamine sugar in EM-A, but not with the N+ of the free amine group in motilin.

Introduction

Gastrointestinal motility is a coordinated neuromuscular process that transports nutrients through the digestive system. Impaired gastrointestinal motility, which can lead to gastroesophageal reflux disease, gastroparesis (diabetic and post-surgical), irritable bowel syndrome and constipation, is one of the largest health care burdens of industrialized nations. Motilin has long been recognized as an important endogenous peptide regulator of gastrointestinal motor function and there is considerable interest into the therapeutic applications of motilin agonists [1]. This interest arose after the demonstration that the antibiotic erythromycin accelerates gastric emptying in patients with diabetic gastroparesis by interacting with the motilin receptor [2], [3]. Pharmaceutical companies have developed erythromycin derivatives without anti-bacterial activity but with good gastrointestinal motor stimulating properties. These compounds are currently under investigation for the treatment of hypo-motility disorders. Chemically, these compounds, now called motilides, show little structural resemblance with motilin, raising the question of how they interact with the motilin receptor.

In 1999, an orphan receptor, originally isolated from the thyroid gland, was identified as the motilin receptor [4]. The amino acid sequence of the motilin receptor is for 44% identical with the human growth hormone secretagogue receptor (GHS-R), even for 86% in the predicted transmembrane regions. Both receptors stimulate pulsatile biological activity and are now considered a new family within class A of G-protein coupled receptors (GPCRs). The full-length human motilin receptor complementary DNA encodes a polypeptide of 412 amino acids (type 1a). Alternative splicing may produce a truncated version of the motilin receptor (type 1b) of 386 amino acids which is biologically inactive. In a CHO-cell line expressing the cloned motilin receptor (type 1a) and the Ca2+ indicator apo-aequorin, the potencies of motilin agonists to induce Ca2+ fluxes strongly correlates with their potencies to induce contraction in rabbit intestinal tissue, suggesting that the cloned motilin receptor is indeed the receptor responsible for the contractile effects [5].

While extensive structure–activity studies have been performed to delineate the pharmacophore of motilin and erythromycin [6], [7], [8], [9], [10], mutagenesis studies to reveal the receptor domains involved in the interaction of the ligands with the motilin receptor are scarce. A better understanding of the relationship between the molecular structure and function of the receptor may provide important insights for drug development. They are also of fundamental importance to clarify key concepts of receptor biology. With the development of non-peptide agonists for peptide receptors, it is becoming increasingly clear that the simple lock-and-key concept may be insufficient to explain structural changes leading to receptor activation.

In several GPCRs, there is a growing body of evidence implicating that transmembrane (TM) helixes 3, 5 and 6 and extra-cellular loops 2 and 3 are involved in ligand binding [11]. Alignment of the sequences of the receptors belonging to the same subclass as the motilin receptor (thyrotropin-releasing hormone and secretagogue) within the class A rhodopsin like family of GPCRs, shows that the glutamic acid in the TM3 region is well conserved. Mutagenesis studies revealed that this is an important binding site for activation of the GHS-R to which motilin is most related [12].

The aim of the present study was to evaluate the effect of mutating the negatively charged glutamic acid (Glu, E) at position 119 of the TM3 to aspartic acid (E119D mutant), which puts the negative charge closer to the receptor backbone, and to glutamine (E119Q mutant), which removes the negative charge, on the response of the mutant receptors stably expressed in CHO-cells to motilin and EM-A. In addition we aimed at delineating the structural elements of motilin and EM-A involved in the interaction with E119 by measuring the Ca2+ luminescent response upon stimulation with motilin analogues and motilides in the wild-type receptor (WT-MTLR) and in the mutant receptors.

Section snippets

Materials

Norleucine13-porcine-motilin (1–22) was purchased from Eurogentec, Namur, Belgium. The N-terminal (1–14) fragment of porcine [leu13]motilin and the analogues of the (1–14) fragments were synthesized as previously described [7], [8]. A Chinese hamster ovary-K1 (CHO-K1) cell line stably expressing the wild-type human motilin receptor and the mitochondrially targeted apo-aequorin was obtained from Euroscreen (Belgium). The human WT-MTLR coding region, inserted into the mammalian expression vector

Activity of CHO-cells expressing the WT-MTLR, the E119D mutant or the E119Q mutant

The potency of motilin and of EM-A to mobilize intra-cellular Ca2+ causing concomitant Ca2+-induced aequorin bioluminescence, was measured in CHO-K1 cells expressing either the WT-MTLR, the E119D mutant or the E119Q mutant.

In the E119D mutant, the maximal Ca2+ response to motilin and EM-A was reduced from 82 ± 5 and 88 ± 7% of the maximal Triton-induced Ca2+ luminescence in the WT-MTLR to 31 ± 2 and 17 ± 1% in the E119D mutant, respectively (Fig. 1). Also, the pEC50 values were shifted from 9.65 ± 0.03

Discussion

Our results indicate that motilin and the non-peptide motilin agonist, EM-A, share a common binding site in the third transmembrane region of the motilin receptor. This conclusion is based on the comparable loss of affinity of the mutated receptors for their native ligand and for EM-A. Thus, both motilin and motilides were unable to activate the E119Q mutant, in which there is no negative charge on the side chain of residue 119 and both compounds had a decreased potency in the E119D mutant, in

Acknowledgments

Supported by grants from the Fund for Scientific Research—Flanders (Belgium) (contract FWO G.0144.04), the Belgian Ministry of Science (GOA 03/11 and IUAP P5/20) and by grants from the Bilateral Scientific Cooperation Flanders—China (project 01/13).

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