Kise, R. | bioRxiv Science

bioRxiv2021

Membrane phosphoinositides stabilize GPCR-arrestin complexes and offer temporal control of complex assembly and dynamics

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized some GPCRs may complex with arrestin, while others interact transiently; this difference affects receptor signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one requiring PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a receptor core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.

biochemistry↗

bioRxiv2021

Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and {beta}-arrestins ({beta}arrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with {beta}arrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to {beta}arrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that {beta}arrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.

biochemistry↗