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

Area of Science:

• Pharmacology
• Molecular Biology
• Cell Biology

Background:

• Adrenomedullin (AM) is a peptide hormone with protective effects mediated by the AM type 1 receptor (AM1 receptor), composed of CLR and RAMP2.
• Beta-arrestins (β-arrs) are crucial for receptor internalization and resensitization, with β-arr-biased agonists in clinical trials for pain and heart failure.
• Understanding β-arr involvement in AM1 receptor trafficking is key for therapeutic development.

Purpose of the Study:

• To investigate the role of β-arrestin isoforms (β-arr-1 and β-arr-2) in the internalization of the human AM1 receptor.
• To determine if the C-terminal tail (C-tail) of CLR influences β-arr-mediated AM1 receptor internalization.

Main Methods:

• Constructed a chimeric AM1 receptor with the CLR C-tail replaced by that of the β2-adrenergic receptor (β2-AR).
• Transfected HEK-293 cells expressing RAMP2 with wild-type or chimeric receptors and β-arrs (or dominant-negative β-arrs).
• Quantified receptor internalization and cell-surface expression using flow cytometry; assessed AM binding and cAMP signaling.

Main Results:

• Co-expression of β-arr-1 or β-arr-2 significantly decreased AM-induced AM1 receptor internalization without affecting basal AM binding or signaling.
• Dominant-negative β-arrs also reduced AM1 receptor internalization, confirming a negative regulatory role.
• Internalization of the chimeric receptor was enhanced by β-arrs and reduced by dominant-negative β-arrs, mirroring β2-AR behavior.

Conclusions:

• Both β-arr-1 and β-arr-2 negatively regulate AM1 receptor internalization.
• The C-tail of CLR is critical for this β-arr-mediated negative control.
• Findings reveal a novel mechanism of AM1 receptor trafficking distinct from other G protein-coupled receptors.