Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors

Abstract

β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo–electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues.

A genetically-encoded nanobody sensor reveals conformational diversity in β-arrestins orchestrated by distinct seven transmembrane receptors

“…For example, most of these receptors can be categorized as class A vs. B receptors based on stability of their interaction 30 (class A: CXCR1, CXCR2, CXCR3, CXCR4, GPR109A; class B: C5aR1, AT1R, B2R), and they contain potential phosphorylation sites primarily in their carboxyl-terminus. We also used the muscarinic receptor subtype 2 (M2R) that interacts with βarrs primarily through the 3 rd intracellular loop instead of the carboxyl-terminus (M2R) 13 , and three β-arrestin-biased receptors namely the complement receptor C5aR2, decoy D6 receptor, and chemokine receptor CXCR7, which lack functional G-protein-coupling but robustly interact with βarrs 31,32 . While we observed a response for multiple GPCRs, the signal was most prominent for the B2R and AT1R (Figure 2a, Supplementary Figure 3a-d).…”

“…12800-017) at 37 °C under 5% CO 2 . The expression constructs for the receptors, βarrs, NanoBiT-CAXX, and Ib30 have been described previously 13,20,27,31,32,45–48 . Ib32 constructs in NanoBiT format as described in Figure 1b were generated by sub-cloning the Ib32 coding region in pCAGGS vector as described previously for Ib30 27 .…”

“…12800-017) at 37 °C under 5% CO2. The expression constructs for the receptors, βarrs, NanoBiT-CAXX, and Ib30 have been described previously 13,20,27,31,32,[45][46][47][48] .…”

“…NanoBiT assay to monitor GPCR-βarr interaction and Ib32/Ib30 reactivity was carried out following the previously described protocols 13,20,27,31,[45][46][47]51 . For the Ib32 reactivity assay described here for the first time, various combinations of LgBiT and SmBiT tagged at the Nterminus and C-terminus of ꞵarr1/2 and Ib32 were screened to obtain the optimal combination.…”

“…Agonist-induced activation and phosphorylation of GPCRs leads to binding of β-arrestins (βarrs), which is a critical step in regulating receptor signaling and trafficking [4][5][6][7] . Interaction with GPCRs imparts an active conformation on βarrs that is manifested in the form of a significant inter-domain rotation between the N-and the Cdomains, and the rearrangement of multiple loops in βarrs [8][9][10][11][12][13][14] . As the paradigm of agonistinduced βarr recruitment is typically conserved across nearly the entire repertoire of GPCRs, monitoring βarr activation may serve as a readout of receptor activation and ensuing downstream signaling.…”