Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ...ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.
G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to ...monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.
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•Active state structures of a μOR–nanobody complex determined to 2.3Å resolution.•The first GPCR–arrestin structure defines the arrestin–opsin interface.•Structure determined of A2AR ...coupled to an engineered G protein.•Inactive, intermediate-active and active structures have now been solved for A2AR.•G protein and arrestin complexes provide insight into GPCR coupling specificity.
G protein-coupled receptors (GPCRs) regulate cellular signalling through heterotrimeric G proteins and arrestins in response to an array of extracellular stimuli. Structure determination of GPCRs in an active conformation bound to intracellular signalling proteins has proved to be highly challenging. Nonetheless, three new structures of GPCRs in an active state have been published during the last year, namely the adenosine A2A receptor (A2AR) bound to an engineered G protein, opsin bound to visual arrestin and the μ opioid receptor (μOR) bound to a G protein-mimicking nanobody. These structures have provided novel insight into the sequence of events leading to GPCR activation, and have highlighted both similarities and differences in the structure of the interface between GPCRs and different signalling proteins.
Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. ...Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.
G protein-coupled receptors (GPCRs) regulate intracellular signalling pathways that contribute to virtually all aspects of cell function. Characterising GPCRs in each of their conformational states ...is key to understanding their mechanism of action, but structure determination of receptors in their active state, bound to a heterotrimeric G protein or b-arrestin, has proved challenging. A number of G protein surrogates have been developed to simplify this process, including G protein-derived peptides, nanobodies and, most recently, mini G proteins. The aim of these surrogates is to bind the receptor and stabilise its active conformation, whilst eliminating the problems inherent to native signalling proteins, namely their large size, instability and conformational dynamics. Mini G proteins are composed of a single domain from the G protein a-subunit that has been engineered to form a stable complex with GPCRs. They induce comparable pharmacological and structural changes in the receptor to those elicited by heterotrimeric G proteins, and retain their native receptor-coupling specificity. At least one member of each G protein family has been converted into a mini G protein, which means that they can be used to characterise a wide variety of GPCRs. Since their initial publication two years ago, mini G proteins have facilitated the structure determination of three different receptors in their active state and enabled the development of a methodology to thermostabilise GPCRs in their fully active conformation. They have also been used to develop a range of assays that can measure mini G protein coupling to receptors in vitro, and a sensitive cell-based assay that is capable of accurately reporting ligand efficacy and quantifying G protein coupling in vivo. This review presents an overview of the current applications of mini G proteins to study the structure and function of GPCRs.
Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's ...mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.
► Structure of the leptin binding domain of the human obesity receptor (ObR) ► Structure of a leptin blocking antibody was solved in both free and ObR-bound forms ► A model is proposed for leptin binding to the leptin-binding domain (LBD) of ObR ► 9F8 Fab blocks leptin binding to ObR through a small overlap in their epitopes
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and ...functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-G
, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A
receptor (A
R) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-G
.
The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy ...(cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.
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