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

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.

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.

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).

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.