Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.

The beta(2) adrenergic receptor (beta(2)AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 angstrom) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The beta(2)AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free beta(2)AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in beta(2)AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of G alpha subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-G alpha(i) and Ric-8A-G alpha(q) complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of G alpha, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal cx5 helix of Goc is held away from the Ras-like domain through Ric-8A core domain interactions, which crit-ically depend on recognition of the G alpha C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal cx5 helix before al-lowing GTP-gated release of G alpha from Ric-8A.

Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways(1,2). Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human beta-arrestin 1 (beta arr1(Delta CT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with beta arr1(Delta CT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85 degrees relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions. A cryo-electron microscopy structure of the neurotensin receptor 1 in complex with beta-arrestin 1 is reported.

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric G(i1) protein, at a resolution of 3 A. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-G(i/o) complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with (CH3)-C-13-epsilon-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of (CH3)-C-13-epsilon-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

The beta(2) adrenergic receptor (beta(2)AR) signals through both G(s) and G(i) in cardiac myocytes, and the G(i) pathway counteracts the G(s) pathway. However, G(i) coupling is much less efficient than G(s) coupling in most cell-based and biochemical assays, making it difficult to study beta(2)AR-G(i) interactions. Here we investigate the role of phospholipid composition on G(s) and G(i) coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the beta(2)AR, we find that they impair coupling to G(i3) and facilitate coupling to G(s). Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates G(i3) coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of G(i3). Taken together, our observations suggest that local membrane charge modulates the interaction between beta(2)AR and competing G protein subtypes.

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

G protein-coupled receptors (GPCRs) have evolved to recognize incredibly diverse extracellular ligands while sharing a common architecture and structurally conserved intracellular signaling partners. It remains unclear how binding of diverse ligands brings about GPCR activation, the common structural change that enables intracellular signaling. Here, we identify highly conserved networks of water-mediated interactions that play a central role in activation. Using atomic-level simulations of diverse GPCRs, we show that most of the water molecules in GPCR crystal structures are highly mobile. Several water molecules near the G protein-coupling interface, however, are stable. These water molecules form two kinds of polar networks that are conserved across diverse GPCRs: (i) a network that is maintained across the inactive and the active states and (ii) a network that rearranges upon activation. Comparative analysis of GPCR crystal structures independently confirms the striking conservation of water-mediated interaction networks. These conserved water-mediated interactions near the G protein-coupling region, along with diverse water-mediated interactions with extracellular ligands, have direct implications for structure-based drug design and GPCR engineering.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.