Structure and dynamics of GPCR signaling complexes

Abstract

G-protein-coupled receptors (GPCRs) relay numerous extracellular signals by triggering intracellular signaling through coupling with G proteins and arrestins. Recent breakthroughs in the structural determination of GPCRs and GPCR-transducer complexes represent important steps toward deciphering GPCR signal transduction at a molecular level. A full understanding of the molecular basis of GPCR-mediated signaling requires elucidation of the dynamics of receptors and their transducer complexes as well as their energy landscapes and conformational transition rates. Here, we summarize current insights into the structural plasticity of GPCR-G-protein and GPCR-arrestin complexes that underlies the regulation of the receptor's intracellular signaling profile.

Fig. 1. G-protein-coupled receptor signal transduction.

Receptors regulate multiple intracellular signaling cascades including G-protein-dependent and G-protein-independent pathways. Agonist binding activates the receptor by inducing conformational changes that involve an outward shift of the transmembrane domain (TM6, blue). The activated receptor can bind to a diverse set of intracellular signaling proteins including G proteins (orange), GRKs (red) and arrestins (green, inactive arrestin; blue-green, active arrestin). Coupling of heterotrimeric G proteins to the receptor triggers nucleotide exchange followed by dissociation of the G protein into the Gα and Gβγ subunits. Both subunits can regulate different downstream effector proteins. GTP-bound Gα subunits can modulate the activity of adenylyl cyclase (AC, yellow), while Gβγ can interact with G-protein-coupled inwardly rectifying potassium channels (GIRK, cylindrical TM representation, gray). G-protein-mediated signaling is terminated by hydrolysis of GTP and reassociation of Gα with Gβγ to form the inactive heterotrimer. Activation of the receptor can also lead to phosphorylation by GRKs and subsequent coupling to arrestin. Arrestin coupling to the receptor leads to desensitization and arrestin-mediated activation of downstream effector proteins like mitogen-activated protein kinases (MAPKs) or SRC kinases. Arrestin activation also promotes the internalization of the receptor into endosomes followed by degradation or recycling of the receptor to the plasma membrane. NL, N lobe of arrestin; CL, C lobe of arrestin.

Fig. 2. Receptor-mediated conformational changes in Gα.

a, Structural comparison of nucleotide-free Gαs (red) coupled to β2AR (gray, PDB 3SN6) and GTPγS-bound Gαs (orange, PDB 1AZT). GTPγS is shown as spheres. Receptor coupling of Gs induces an outward movement of the α-helical domain of Gαs (GαsAHD) relative to its position in the GTPγS-bound state. Gβ (cyan) and Gγ (magenta) have been made transparent for clarity. b, The α5 helix undergoes a rotational translation into an intracellular cavity of the receptor that is formed by outward movement of transmembrane helices TM5 and TM6 upon receptor activation. Displacement of α5 leads to perturbation of the β6–α5 loop and the hydrophobic core interaction between α5, β2 and β3 and α1, which results in a rearrangement of the β6–α5 loop and destabilization of α1, which are important for the binding of the purine ring and the phosphates of the nucleotide, respectively. Interaction between the intracellular loop 2 (ICL2) of the receptor and the αN–β1 hinge region of Gαs may also lead to conformational changes in β1 and the adjacent P loop that forms part of the phosphate-binding site in the GTPγS-bound Gαs structure.

Fig. 3. Conformational changes in arrestin-2 upon activation.

a, Overlay of arrestin-2 in its inactive (wheat, PDB 1G4M) and active (green, PDB 4JQI) states. In the inactive conformation, the arrestin C tail (dashed lines represent unresolved residues) docks onto the arrestin N lobe. The active state was obtained by crystallizing arrestin in the presence of a peptide corresponding to the fully phosphorylated vasopressin receptor 2 C terminus (V2Rpp, dark blue) and an active-state-stabilizing antibody fragment (not shown). Activation induces major conformational changes (indicated by purple arrows): rearrangements of the loops at the N–C-domain interface, displacement of the arrestin C tail and an ∼20° interdomain rotation. Two major interaction networks maintain arrestin in its basal, inactive conformation. b, The three-element interaction is mediated by bulky hydrophobic residues (shown in stick representation) between the C tail, the β-strand I and the α-helix I. c, The polar core is a conserved network of charged residues (shown in stick representation) forming ionic interactions (dashed gray lines) between the N-terminal β-strands III and X, the gate loop and the C tail.

Fig. 4. Structure and interaction interface of the rhodopsin–arrestin-1 complex.

a, Overview of the rhodopsin–arrestin-1 structure (PDB 5W0P), obtained by fusing a preactivated arrestin mutant (shown in green, a triple alanine mutation disrupting the three-element interaction) to a constitutively active rhodopsin mutant (gray). Two of the three known phosphorylation sites (red) on the rhodopsin C tail (orange) are resolved in the structure. b,c, Front view (b) and side view (c) of the rhodopsin–arrestin interface, with the structural elements of arrestin that interact with the receptor highlighted in dark green and the interacting receptor residues colored according to the arrestin loops that they are interacting with (blue, finger loop (FL); magenta, middle loop (ML); yellow, C loop (CL); orange, back loop (BL)).