The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

The orexin-1 receptor interacts with beta-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-intemalized into acidic endosomes. Truncations from the C-tenninal tail did not prevent agonist-induced intemalization of the orexin-l receptor or alter the pathway of internalization, although such mutants failed to interact with beta-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of beta-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with beta-arrestin-2, studies in wild-type and beta-affestin-deficient mouse embryo fibroblasts confirmed that agonist-induced intemalization of this mutant required expression of a P-arrestin. Although without effect on agonist-mediated elevation.