Emerging structural insights into GPCR-β-arrestin interaction and functional outcomes

Abstract

Agonist-induced recruitment of β-arrestins (βarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR signaling. Several recent studies have provided novel structural and functional insights into our understanding of GPCR-βarr interaction, subsequent βarr activation and resulting functional outcomes. In this review, we discuss these recent advances with a particular emphasis on recognition of receptor-bound phosphates by βarrs, the emerging concept of spatial positioning of key phosphorylation sites, the conformational transition in βarrs during partial to full-engagement, and structural differences driving functional outcomes of βarr isoforms. We also highlight the key directions that require further investigation going forward to fully understand the structural mechanisms driving βarr activation and functional responses.

Figure 1. GPCR-βarr interaction and decisive contribution of single phosphorylation site in V₂R-βarr interaction.

(a). Agonist-induced activation of GPCRs results in receptor phosphorylation by GRKs followed by βarr binding and activation in a biphasic manner. Distinct patterns of receptor phosphorylation drive functionally-specific βarr conformations. (b). Superimposition of the crystal structures of βarr1 in complex with V₂Rpp^WT and V₂Rpp^T360A reveals repositioning of the phosphopeptides, especially in the proximal segment. The structural snapshots were designed based on PDB IDs 4JQI and 7DFA, respectively. (c). Mutation of Thr³⁶⁰ results in the disruption of a key interaction with the Lys²⁹⁴ in the lariat loop of βarr1, which in turn leads to a dramatic alteration in agonist-induced βarr1 trafficking pattern.

Figure 2. Structural differences in barr1 between partially-engaged and fully-engaged complexes.

(a) Schematic representation of the partially-and fully-engaged β₂V₂R–βarr1 complexes reconstituted in nanodisc and studies using methyl-TROSY NMR spectroscopy. The lower panel shows the position of fifteen residues in βarr1 used in this study and the three isoleucine residues exhibiting significant differences between partially-and fully-engaged conformations are highlighted in blue. (b) Differences in the chemical shift for selected isoleucine (Ile) residues in basal, partially-engaged and fully-engaged βarr1 conformations as measured by NMR spectroscopy are shown. The position of the corresponding isoleucine residues in βarr1 are indicated in the table. These data are adapted from a previous publication [22].

Figure 3. Structural differences between phosphopeptides-bound βbarr isoforms.

(a) Superimposition of V₂Rpp-bound βarr1 and CXCR7pp-bound βarr2. The structural snapshots are generated in ChimeraX [44] using previously determined crystal structures (PDB ID 4JQI and 6K3F) [25,45]. (b) Surface representation of V₂Rpp-bound βarr1 and CXCR7-bound βarr2 to depict the difference in the inter-domain rotation between βarr1 and 2. (c)V₂Rpp and CXCR7pp are differently positioned in the corresponding crystal structures and an additional phosphate-binding site is identified in βarr2 that is not apparent in V₂Rpp-bound βarr1 structure.

Figure 4. Schematic depiction of key knowledge gaps and emerging directions.

(a) While most GPCRs harbor phosphorylation sites in their carboxylterminus, several others have a very short carboxyl-terminus and phosphorylatable Ser/Thr residues are predominantly located in the 3rd intracellular loop. The commonality and differences in the binding modes of barrs for these two types of GPCRs remains to be explored in detail. (b) Arrestin-coupled receptors (ACRs) robustly recruit βarrs despite lacking an inherent ability to functionally couple to G-proteins. While βarr conformations for ACRs vs. GPCRs are qualitatively distinct from each other, structural details at high-resolution remains to be determined. (c) The structural and mechanistic basis of catalytic activation of βarrs upon their transient interaction with selected GPCRs, despite rapid dissociation of the receptor from their complexes, remains to be explored.