The Molecular Basis of G Protein-Coupled Receptor Activation

Abstract

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

Keywords: 7-TM receptors; G protein–coupled receptor; GPCR; allostery; energy landscape; β2-adrenergic receptor.

Figure 1

(a) General architecture of a family A G protein–coupled receptor (GPCR). The seven transmembrane (TM) helices, the connecting intracellular loops (ICLs) and extracellular loops (ECLs), and a conserved disulfide bond are indicated. (b) Outline of GPCR activity upon binding of an agonist to a receptor (R). Top, the classical G protein pathway. Exchange of GDP for GTP in the G protein α subunit leads to dissociation and interaction with downstream effectors such as Gαs stimulation of adenylyl cyclase and Gβγ activation of ion channels. Bottom, activated GPCRs can also signal through arrestins. Phosphorylation of the receptor C-terminal tail by a G protein–coupled receptor kinase (GRK) promotes arrestin (Arr) recruitment and activation, including endocytosis through interactions with the clathrin adaptor protein 2 (AP2) complex and activation of extracellular signal-regulated kinase (ERK). (c) Efficacy of ligands. (d) Biased agonists stimulate one pathway preferentially over another. Figure modified with permission from Reference .

Figure 2

Overall GPCR structures. (a) Inactive state of β₂AR, bound to carazolol (PDB 2RH1). (b) Active state of β₂AR bound to adrenaline (left and center, PDB 3SN6, with adrenaline coordinates from PDB 4LDO; right, PDB 4LDO). Ligands are shown in space-filling representation, with carbon atoms of carazolol in purple and of epinephrine in yellow. The conserved prolines P211^5.50, P288^6.50, and P323^7.50 are shown as sticks. The center panels show the receptor with its extracellular face up and cytoplasmic face down. The left and right panels show views from the intracellular and extracellular surfaces, respectively. The labeling sites used for NMR probes (C265) and DEER and FRET probes (N148C and L266C) are shown in the left panel, and the opening of the G protein pocket upon receptor activation is indicated by the change in distance between the α carbons at N148 and L266. Abbreviations: β₂AR, β₂-adrenergic receptor; DEER, double electron-electron resonance; FRET, fluorescence resonance energy transfer; GPCR, G protein–coupled receptor; PDB, Protein Data Bank.

Figure 3

The conserved D(E)RY motif in the G protein–binding site. (a) The ionic lock in dark rhodopsin formed by R135^3.50 and E247^6.30. (b) The ionic lock is not present in the crystal structure of carazolol-bound β₂AR. The packing of R^3.50 with L272^6.34 and L275^6.37 is shown with space-filling models of these side chains. (c) Two conformations of the ionic lock region are observed in inverse-agonist bound structures of the β₁AR. Bending near the cytoplasmic end of TM6 results in an electrostatic interaction between R139^3.50 and E285^6.30 ( gray; PDB 2YCX). The alternative straight conformation of TM6 (salmon; PDB 2VT4) moves these two residues apart. (d) Movements of TM6 and TM7 in the β₂AR active state prevent ionic lock formation, and the intrahelical salt bridge between D130^3.49 and R131^3.50 is broken. (e) Packing of β₂AR ( green, with side chains in space-filling representation) and G_sα (orange, shown as a transparent surface). ( f) Interactions of β₂AR with the C-terminal region of bound G_sα. G_sα is shown in orange. Polar interactions are shown with dashed lines. Abbreviations: β₂AR, β₂-adrenergic receptor; G _sα, G_sβ, stimulatory heterotrimeric G protein α and β subunits; PDB, Protein Data Bank; TM, transmembrane.

Figure 4

The NP^7.50xxY motif. (a) The NPxxY motif in the inactive state of β₂AR. (b) Superposition of the NPxxY region in the inactive (gray; PDB 2RH1) and active ( green; PDB 3SN6) β₂AR structures. Abbreviations: β₂AR, β₂-adrenergic receptor; PDB, Protein Data Bank.

Figure 5

Water networks linking TM2, TM5, TM6, and TM7. Polar side chains conserved in the family A receptors are shown in stick representation and are labeled with their Ballesteros–Weinstein numbers. (a,b) Comparison of the inactive δ-OR (PDB 4N6H) and active μ-OR (PDB 5C1M) structures. These two receptors are highly homologous, and these structures are at sufficiently high resolution (1.8 A and 2.1 A, respectively) to visualize extensive water networks. (c) Overlay of inactive β₂AR (PDB 2RH1) and the muscarinic M2R structures (PDB 3UON) reveals similar polar side chain and water positions as those in the δ-OR. Abbreviations: β₂AR, β₂-adrenergic receptor; M2R, M2 muscarinic receptor; OR, opioid receptor; PDB, Protein Data Bank; TM, transmembrane.

Figure 6

Changes in the orthosteric site and connector of the β₂AR upon activation. (a,b) Top (a) and side (b) views of superimposed carazolol-bound inactive ( gray) and super adrenaline-bound active ( green) structures, highlighting changes in the orthosteric site. Carazolol is shown in blue sticks, and adrenaline in yellow sticks. In panel b, key hydrogen bonds formed with adrenaline are shown with dashed lines. (c) The connector region in the inactive (❶) and active (❷) states. The conserved nonpolar residues P211^5.50, I121^3.40, and F282 ^6.44 are shown in space-filling representation to highlight changes in their packing. The Cε methyl group of M82^2.53 is indicated with a dotted surface. The inward bulge of TM5 near P211^5.50 in the active state activation and the outward movement of TM6 are indicated by the black arrows in state ❷. Abbreviations: β₂AR, β₂-adrenergic receptor; TM, transmembrane.

Figure 7

Likely activation intermediate observed in the A_2aR. (a) Overlay of A_2aR ( yellow; PDB 3PWH) and β₂AR inactive ( gray; PDB 2RH1) states. (b) A_2aR bound to agonist (orange; PDB 2YDV) superimposed on the antagonist-bound structure (PDB 3PWH) and the active structure bound to the mini-G_sα (magenta; PDB 5G53). (c) Overlay of A_2aR (PDB 5G53) and β₂AR ( green; PDB 3SN6) active states. Abbreviations: A_2aR, adenosine 2a receptor; β₂AR, β₂-adrenergic receptor; G_sα, heterotrimeric G protein α subunit; PDB, Protein Data Bank.

Figure 8

Schematic energy landscape of β₂AR. The gray lines (solid and dashed) indicate the energy landscape of the ligand-free (basal) state. The solid black lines indicate the effect of the indicated ligand and G protein on the energy landscape. The two inactive states detected by spectroscopy are denoted S1 (intact ionic lock) and S2 (broken ionic lock). S3 is the intermediate detected in the presence of an agonist without G protein, and S4 states are active states in the presence of an agonist and a G protein. Single-molecule FRET analysis provides evidence for distinct active states in the presence of an agonist that are dependent on the nucleotide state of the G protein. Abbreviations: β₂AR, β₂-adrenergic receptor; FRET, fluorescence resonance energy transfer; G_s, stimulatory heterotrimeric G protein.