Desensitization and internalization of the human motilin receptor is independent of the C-terminal tail.

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 (MTLRΔ385), 374 (MTLRΔ375) or 368 (MTLRΔ369), were constructed and C-terminally tagged with an EGFP and stably expressed in CHO cells co-expressing the Ca2+ indicator apoaequorin. Activity and desensitization were studied by measuring changes in motilin-induced luminescent Ca2+ 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 Ca2+ luminescent responses of the MTLRΔ375-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 MTLRΔ369-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 MTLRΔ385-EGFP mutant was reduced by 80% but was not affected in the MTLRΔ375-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.