Solution NMR Analysis Reveals Synergistic β-Arrestin1 Activation by Chemically Synthesized Phosphopeptides of a C-Terminal Tail and ICL3 of NTSR1.

β-Arrestins are critical regulators of G-protein-coupled receptors (GPCRs), mediating desensitization, internalization, and activation of alternative downstream signal transduction pathways through selective binding to phosphorylated GPCRs. Although phosphorylation of C-terminal tails (C-tail) and intracellular loop 3 (ICL3) of GPCRs is essential for β-arrestin binding to GPCRs, cooperative interactions of the phosphorylated C-tail or ICL3 of GPCRs for β-arrestin recruitment remain elusive. Here, we chemically synthesized phosphorylated C-tail and ICL3 peptides of neurotensin receptor 1 (NTSR1) and investigated the conformational dynamics of β-arrestin1 during its interaction with the phosphopeptides. Two-dimensional ¹H-¹³C nuclear magnetic resonance (NMR) spectroscopy of ¹³C^εH3-methionine labeled β-arrestin1 revealed that the phosphorylated C-tail (C-tail-NC 6P), N-cluster of C-tail (C-tail-N 3P), or ICL3 4P triggered conformational changes of β-arrestin1, whereas the C-cluster of the C-tail (C-tail-C 3P) exhibited negligible influence. Additionally, analysis of successive binding of C-tail-NC 6P and ICL3 4P of NTSR1 to β-arrestin1 implied noncompetitive binding of the two segments and displayed allosteric modulation of C-tail or ICL3 in β-arrestin1. These 2D ¹³C-methyl-Met NMR data provide direct evidence for interactions between β-arrestin1 and phosphorylated segments of GPCRs, offering a framework to decode the details of β-arrestin signaling.