G protein-coupled receptor interactions with arrestins and GPCR kinases: The unresolved issue of signal bias

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins interact with agonist-bound GPCRs to promote receptor desensitization and downregulation. They also trigger signaling cascades distinct from those of heterotrimeric G proteins. Biased agonists for GPCRs that favor either heterotrimeric G protein or GRK/arrestin signaling are of profound pharmacological interest because they could usher in a new generation of drugs with greatly reduced side effects. One mechanism by which biased agonism might occur is by stabilizing receptor conformations that preferentially bind to GRKs and/or arrestins. In this review, we explore this idea by comparing structures of GPCRs bound to heterotrimeric G proteins with those of the same GPCRs in complex with arrestins and GRKs. The arrestin and GRK complexes all exhibit high conformational heterogeneity, which is likely a consequence of their unusual ability to adapt and bind to hundreds of different GPCRs. This dynamic behavior, along with the experimental tactics required to stabilize GPCR complexes for biophysical analysis, confounds these comparisons, but some possible molecular mechanisms of bias are beginning to emerge. We also examine if and how the recent structures advance our understanding of how arrestins parse the "phosphorylation barcodes" installed in the intracellular loops and tails of GPCRs by GRKs. In the future, structural analyses of arrestins in complex with intact receptors that have well-defined native phosphorylation barcodes, such as those installed by the two nonvisual subfamilies of GRKs, will be particularly illuminating.

Keywords: G protein-coupled receptor; GPCR kinase; GRK; allostery; arrestin; biased agonism; cryo-electron microscopy; desensitization; single particle reconstruction.

Figure 1

Arrestins contain conserved structural elements that serve as sensors for active, phosphorylated GPCRs and their surrounding anionic lipid environment.A, structure of Arr2 in its basal, inactive state (PDB entry 1G4M) (13). Details of two interdomain interactions stabilizing this state are highlighted in the insets. Named loops mentioned in the review are shown in purple with corresponding residue ranges. Structural elements serving as the phosphorylation and membrane sensors are highlighted in yellow and green, respectively. The activation sensor includes the finger loop and the groove it forms primarily with the C loop. B, sequence alignment of human arrestin phosphorylation, activation, and membrane sensors. Residue numbering is based on human Arr2. C, cartoon representation of the tail interaction, core interaction, and trimodal mode formed between activated GPCRs, arrestins, and the membrane. Arr2, arrestin-2; GPCR, G protein-coupled receptor.

Figure 2

Conformational heterogeneity in GPCR–arrestin interactions.A and B, superposition of the Rho–Arr1 (PDB entry 4ZWJ) (19) and NSTR₁–Arr2 structures (PDB entry 6PWC and 6UP7) (33, 34) with either (A) receptor or (B) Arr2 aligned. C and D, superposition of the Rho–Arr1 (PDB entry 4ZWJ) (19), β₁AR–Arr2 (PDB entry 6TKO) (36), and M₂R–Arr2 structures (PDB entry 6U1N) (35) with either (C) receptor or (D) Arr2 aligned. β₁AR, β1 adrenergic receptor; Arr1, arrestin-1; Arr2, arrestin-2; GPCR, G protein-coupled receptor; H8, helix 8; M₂R, M2 muscarinic receptor; NTSR₁, neurotensin receptor 1; Rho, rhodopsin; TM, transmembrane.

Figure 3

Comparison of receptor-engaged phosphorylation and activation sensors.A–F, structures of arrestins bound to (presumably) phosphorylated C tails of GPCRs. When added, Fab30 interacts with the position analogous to pS362 in the V₂Rpp peptide, effectively stapling arrestin to the GPCR C tail (D–F). Density for the GPCR C tail in each structure (gray wire cages) in general has poor definition, as evidenced by poor stereochemistry in some cases (e.g., see colliding adjacent phosphates in panel E). The insets detail the interactions at the most consistently observed phosphosite (corresponding to residue Thr360 in V₂Rpp in panel A), which is coordinated by Arr2-Lys11, Arr2-Arg25, and presumably Arr2-Lys294 (because density is lacking). G, the Arr2 finger loop (76) shows many different conformations when bound to a GPCR, highlighting its ability to adapt to distinct cytoplasmic clefts and arrestin orientations relative to the receptor core. Shown is a superposition of Arr2 bound to β₁AR (PDB entry 6TKO) (36), M₂R (PDB entry 6U1N) (35), and NTSR₁ (PDB entry 6PWC and 6UP7) (33, 34). H and I, interactions of Arr2 finger loop within the cytoplasmic cleft of (H) NSTR₁ (PDB entry 6UP7) (34) and (I) β₁AR (PDB entry 6TKO) (36). J, interaction of the GRK1 αN helix with the cytoplasmic cleft of Rho (PDB entry 7MTA) (52). In (H–J), the side chains of residues contributing hydrophobic and hydrophilic interactions are shown with yellow and magenta carbons, respectively. β₁AR, β1 adrenergic receptor; Arr2, arrestin-2; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; H8, helix 8; M₂R, M2 muscarinic receptor; NTSR₁, neurotensin receptor 1; V₂Rpp, vasopressin 2 receptor–derived phosphopeptide; Rho, rhodopsin; TM, transmembrane.

Figure 4

GRKs also contain conserved structural elements that serve as sensors for active, phosphorylated GPCRs and their surrounding anionic lipid environment.A, activated structure of GRK1. The model was generated by merging the GRK1 αN and kinase domain from the structure of GRK1 in complex with Rho (PDB entry 7MTA) (52) and the RH domain from the crystal structure of GRK1 in its basal state (PDB entry 3C4W) (77). The RH domain was in fact disordered in the Rho–GRK1 complex. ATP is modeled in place of the adenosine analog sangivamycin used in the Rho complex. The GRK “membrane” and “phosphorylation” sensors, by loose analogy to those of arrestin, are highlighted with yellow and green side chains, respectively. The activation sensor is composed of the N-terminal half of αN and adjacent segments of the AST loop (purple). B, sequence alignment of the GRK phosphorylation, activation, and membrane sensors. Residue numbering is based on bovine GRK1. The N domain membrane sensor mainly interacts with negative phospholipids via electrostatic interactions and participating residues are shown in purple. Note that a significant role in membrane binding for the residues in this region has not been experimentally demonstrated in GRK2 and 3. C, cartoon representation of GRK activation, membrane, and phosphorylation sensors in basal (left) and activated, GPCR-bound (center and right) states. The GRK is speculated to partially dissociate from the receptor during the exchange of ATP, remaining tethered to the receptor either via its activation or phosphorylation sensors. AST, active site tether; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; Rho, rhodopsin; RH, regulator of G protein signaling-homology.

Figure 5

Conformational heterogeneity in GPCR–GRK1 interactions. Superposition of the Rho–GRK1 structure in the presence of Fab1 (PDB entry 7MTA) or Fab6 (PDB entry 7MTB) (52) with either (A) receptor or (B) GRK1 aligned. GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; H8, helix 8; Rho, rhodopsin; TM, transmembrane.

Figure 6

Comparison of GPCRs bound to G proteins, GRK1, and arrestins show subtle differences but somewhat more condensed cytoplasmic clefts.A, overlay of G protein mimic nanobody (NB80) (PDB entry 6IBL) (36), Gs (PDB entry 7JJO) (73), and Arr2 (PDB entry 6TKO) (36) bound to the β₁AR. B, overlay of G_i (PDB entry 6OSA and 6OS9) (78) and Arr2 (PDB entries 6PWC and 6UP7) (33, 34) bound to NTSR₁. C, overlay of G_o (PDB entry 6OIK) (79) and Arr2 (PDB entry 6U1N) (35) bound to the Μ₂R. D, overlay of GRK1 (PDB entry 7TMA) (52), G_t (PDB entry 6OYA) (71), and Arr1 (PDB entry 5W0P) (18) bound to Rho. β₁AR, β1 adrenergic receptor; Arr1, arrestin-1; Arr2, arrestin-2; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; H8, helix 8; ICL, intracelluar loop; NTSR₁, neurotensin receptor 1; Rho, rhodopsin; TM, transmembrane.