β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking

Abstract

A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation

“…Acute knockdown of ARRB1 (encoding β-arrestin-1) reduced ERK1/2 phosphorylation, whereas, paradoxically, knockdown of ARRB2 (encoding β-arrestin-2) increased ERK1/2 phosphorylation ( Figure 3 D). Opposing effects of β-arrestin-1 versus -2 have been described previously for other GPCRs ( Shintani et al., 2018 ). Cross-talk between G-protein-dependent and G-protein-independent signaling has been demonstrated for a subset of (but not all) GPCRs ( Grundmann et al., 2018 ; Luttrell et al., 2018 ).…”

Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H4 Receptor in β-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling

“…Hence, these data suggest that phosphorylation by GRK2 and/or GRK3 contributes in part to β-arrestin1/2-mediated H 4 R internalization, as previously observed for agonist-activated PAC1, dopamine D2, and μ-opioid receptor. 22 − 25 Knockdown of β-arrestin1 and 2 decreased the histamine-induced H 4 R internalization by 56 ± 13.2% in comparison to control siRNA-treated cells ( Figure 1 I), indicating that β-arrestins are indeed involved in H 4 R internalization. The observed internalization in the presence of β-arrestin-targeting siRNA is most likely due to the only partial knockdown of β-arrestins ( Figure S3 ).…”

Signalling, trafficking and glucoregulatory properties of glucagon‐like peptide‐1 receptor agonists exendin‐4 and lixisenatide

“…The plasmid for β‐arrestin‐2 fused at the N‐terminus to LgBiT was obtained from Promega (plasmid no. CS1603B118); this configuration was used due to previous success with another class B GPCR (Shintani et al, 2018). The SmBiT tag was cloned in frame at the C‐terminus of the GLP‐1 receptor by substitution of the Tango sequence on a N‐terminally FLAG‐tagged GLP‐1 receptor‐Tango expression vector (Kroeze et al, 2015), a gift from Dr Bryan Roth, University of North Carolina (Addgene plasmid # 66295).…”