β-arrestins negatively control human adrenomedullin type 1-receptor internalization

Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM₁ receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two β-arrestin (β-arr) isoforms, β-arr-1 and β-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, β-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of β-arr-1 and β-arr-2 on human AM₁ receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the β₂-adrenergic receptor (β₂-AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [¹²⁵I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of β-arr-1 or -2 resulted in significant decreases in AM₁ receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) β-arr-1 or -2 also significantly decreased AM-induced AM₁ receptor internalization. In contrast, the AM-induced internalization of the chimeric AM₁ receptor was markedly augmented by the cotransfection of β-arr-1 or -2 and significantly reduced by the coexpression of DN-β-arr-1 or -2. These results were consistent with those seen for β₂-AR. Thus, both β-arrs negatively control AM₁ receptor internalization, which depends on the C-tail of CLR.