Conformational Basis of G Protein-Coupled Receptor Signaling Versatility

Abstract

G protein-coupled receptors (GPCRs) are privileged structural scaffolds in biology that have the versatility to regulate diverse physiological processes. Interestingly, many GPCR ligands exhibit significant 'bias' - the ability to preferentially activate subsets of the many cellular pathways downstream of these receptors. Recently, complementary information from structural and spectroscopic approaches has made significant inroads into understanding the mechanisms of these biased ligands. The consistently emerging theme is that GPCRs are highly dynamic proteins, and ligands with varying pharmacological properties differentially modulate the equilibrium among multiple conformations. Biased signaling and other recently appreciated complexities of GPCR signaling thus appear to be a natural consequence of the conformational heterogeneity of GPCRs and GPCR-transducer complexes.

Keywords: G protein-coupled receptor; arrestin; biased ligand; biophysics; cellular signaling; pharmacology.

Figure 1.. Diversity and conservation in GPCR structures.

(A) General structure of a GPCR (pink) bound to an agonist (purple). Structural conservation in the family is lowest in the extracellular-facing orthosteric site and highest in the intracellular transducer site. Eye indicates viewing angle in panel B. (B) Conserved conformational changes in the intracellular transducer site of GPCRs in moving from the canonical inactive conformation (gray) to the canonical active conformation (pink). Structures of the AT1R (inactive, PDB: 4YAY; active, PDB: 6OS0), the case study presented in this review, are used. Spheres highlight the movement of C_α atoms of residues at the intracellular ends of TMs 5, 6, and 7. Intracellular loops and helix 8 are not shown for clarity.

Figure 2.. Mechanism of AT1R activation by AngII and biased agonists.

(A) Overview of AT1R-AngII structure (PDB: 6OS0) and structure-activity relationships of biased AngII analogs. (B)-(D) Cartoon representations of the distinct states of the AT1R orthosteric site, receptor core, and transducer site—classified as “inactive” (gray), “partially activated” (blue), and “fully activated” (red)—observed by crystallography, spectroscopy, and MD simulations. (B) Compared to the inactive state (i), binding of either AngII or β-arrestin-biased ligands (lacking F8) causes contraction of the orthosteric site (ii). Certain AngII F8 conformations promote an additional rotation of TM3 (iii). (C) A hydrogen-bonding network involving TMs 2, 3, and 7 in the receptor core stabilizes the inactive state (i). In structures with β-arrestin-biased ligands (ii) and AngII (iii), the movement of TM7 breaks its connection to this network. In the AngII-bound structure, the additional rotation of TM3 removes the key residue from the network (iii). (D) Besides transducer site conformations consistent with the canonical inactive (i) and fully activated (iv) GPCR conformations, DEER spectroscopy delineated two partially activated, “occluded” conformations—one occluded by virtue of an inward TM6 (ii), and the other due to differences in the second intracellular loop (ICL2) and TM5 (iii). Stars represent nitroxide labels; positions of labels from the inactive state (i) are shown in gray in each panel for reference. (E) Model of AT1R biased ligand activation. β-Arrestin-biased ligands stabilize only partially activated conformations; AngII promotes both partially and fully activated conformations; and Gq-biased ligands stabilize fully activated conformations more strongly than AngII.

Figure I.

Ligand efficacy and bias. (A), (B) Dose response curves for five different ligands in assays measuring G protein (A) and β-arrestin (B) activation in a hypothetical scenario where the maximal signal is directly proportional to ligand efficacy. (C) Ligand bias is determined based on the relative efficacy of ligands in two (or more) assays. Points falling below the (dashed) line of unity represent G protein-biased ligands, points above the line of unity are β-arrestin-biased ligands, and points lying on the line are unbiased relative to the reference ligand. A reference ligand must be selected to define bias.