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

Biochem Biophys Res Commun. 2017 May 27;487(2):438-443. doi: 10.1016/j.bbrc.2017.04.083. Epub 2017 Apr 17.

Abstract

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.

Keywords: Adrenomedullin; Calcitonin receptor-like receptor; Receptor activity-modifying protein 2; Receptor trafficking; Signal transduction; β-arrestins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenomedullin / metabolism*
  • Calcitonin Receptor-Like Protein / metabolism*
  • HEK293 Cells
  • Humans
  • Receptors, Adrenomedullin / metabolism*
  • Receptors, G-Protein-Coupled / metabolism*
  • beta-Arrestin 1 / metabolism*
  • beta-Arrestin 2 / metabolism*

Substances

  • ARRB1 protein, human
  • CALCRL protein, human
  • Calcitonin Receptor-Like Protein
  • Receptors, Adrenomedullin
  • Receptors, G-Protein-Coupled
  • beta-Arrestin 1
  • beta-Arrestin 2
  • Adrenomedullin