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Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. Calcium ions regulate many processes in the human body. The calcium-sensing receptor, called CaSR, is responsible for maintaining a stable level of calcium ions in the blood. This receptor can detect small changes in the concentration of calcium ions, and activates signalling events within the cell to restore the level of calcium ions back to normal. Abnormal activity of this receptor is associated with severe diseases in humans CaSR is found in the surface membrane of cells and belongs to a family of proteins called G-protein coupled receptors. Much of the protein extends out of the cell and interacts with calcium ions, phosphate ions and certain other molecules such as amino acids. However, it was not well understood how these small molecules bind to CaSR and how this activates the receptor. Geng et al. have now used a technique called X-ray crystallography to view the three-dimensional structure of the exterior domain of CaSR in its resting state and active state. These structures revealed that, contrary to expectations, calcium ions are not the main activator of the receptor. Instead, Geng et al. found that CaSR adopts an inactive state in the absence or presence of calcium ions, while the active state only forms when an amino acid is bound. Furthermore investigation showed that calcium ions are needed to stabilise the active form, while phosphate ions keep the inactive form stable. Geng et al. also identified the shape changes that must occur as CaSR transitions from its inactive to its active state. In particular, an amino acid binding to the exterior domain causes it to close like a venus flytrap, which is a crucial step in activating the receptor. Taken together, the findings show that the amino acids and calcium ions act jointly to fully activate CaSR. The next steps are to determine the structure of the entire receptor with and without its small molecule partners and to use these structures to design drugs that can alter CaSR’s activity in order to treat human diseases.

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies 1 , 2 . CAR T cells have been less effective against solid tumours 3 – 5 , in part because they enter a hyporesponsive (‘exhausted’ or ‘dysfunctional’) state 6 – 9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19 + tumour-bearing mice. CD8 + CAR + tumour-infiltrating lymphocytes and CD8 + endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells) 10 – 12 . CD8 + T cells from humans with cancer or chronic viral infections 13 – 15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors ( Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8 + effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy. Transfer of NR4A-deficient T cells expressing chimeric antigen receptors is shown to reduce tumour burden and increase survival by shifting T cell transcriptional programs away from exhaustion and towards increased effector function.

Rationale Accumulating evidence indicates that the mixed serotonin and dopamine receptor agonist lysergic acid diethylamide (LSD) induces an altered state of consciousness that resembles dreaming. Objectives This study aimed to test the hypotheses that LSD produces dreamlike waking imagery and that this imagery depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods Twenty-five healthy subjects performed an audiorecorded guided mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). Cognitive bizarreness of guided mental imagery reports was quantified as a standardised formal measure of dream mentation. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) questionnaire. Results LSD, compared with placebo, significantly increased cognitive bizarreness ( p  < 0.001). The LSD-induced increase in cognitive bizarreness was positively correlated with the LSD-induced loss of self-boundaries and cognitive control ( p  < 0.05). Both LSD-induced increases in cognitive bizarreness and changes in state of consciousness were fully blocked by ketanserin. Conclusions LSD produced mental imagery similar to dreaming, primarily via activation of the 5-HT2A receptor and in relation to loss of self-boundaries and cognitive control. Future psychopharmacological studies should assess the differential contribution of the D2/D1 and 5-HT1A receptors to cognitive bizarreness.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immunemediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

The MRGPRX family of receptors (MRGPRX1–4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently 1 . Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions 2 – 5 . MRGPRX2 couples to both G i and G q in mast cells 6 . Here we describe agonist-stabilized structures of MRGPRX2 coupled to G i1 and G q in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G q . Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity. Structural studies of the itch receptors MRGPRX2 and MRGPRX4 in complex with endogenous and synthetic ligands provide a basis for the development of therapeutic compounds for pain, itch and mast cell-mediated hypersensitivity.

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT 1 receptor) — a prototypical class A GPCR—activation. Our findings suggest a synergistic transition mechanism for AT 1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and β-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT 1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT 1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry. G protein-coupled receptors (GPCRs) are a critical target in modern drug development across a wide range of indications. Here the authors provide a comprehensive characterization of a typical GPCR, the angiotensin II (AngII) type 1 receptor (AT1R), and provide insight into its activation mechanism that suggest avenues for the design of allosteric GPCR modulators.

The majority of insect olfactory receptors belong to two distinct protein families, the ionotropic receptors (IRs), which are related to the ionotropic glutamate receptor family, and the odorant receptors (ORs), which evolved from the gustatory receptor family. Both receptor types assemble to heteromeric ligand-gated cation channels composed of odor-specific receptor proteins and co-receptor proteins. We here present in short the current view on evolution, function, and regulation of IRs and ORs. Special attention is given on how their functional properties can meet the environmental and ecological challenges an insect has to face.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.A suite of generally applicable methods and tools, developed to enable single-molecule FRET-based studies of transmembrane proteins diffusing in the cell membrane of living cells, was used to study the oligomerization and dynamics of GPCRs.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome 1 – 3 . How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense. Through the use of cryo-electron microscopy and molecular dynamics stimulations, mechanistic insight into the binding of an odorant to the human odorant receptor OR51E2 is provided.

Receptors of insulin and insulin-like growth factors (IGFs) are receptor tyrosine kinases whose signalling controls multiple aspects of animal physiology throughout life. In addition to regulating metabolism and growth, insulin–IGF receptor signalling has recently been linked to a variety of new, cell type-specific functions. In the last century, key questions have focused on how structural differences of insulin and IGFs affect receptor activation, and how insulin–IGF receptor signalling translates into pleiotropic biological functions. Technological advances such as cryo-electron microscopy have provided a detailed understanding of how native and engineered ligands activate insulin–IGF receptors. In this Review, we highlight recent structural and functional insights into the activation of insulin–IGF receptors, and summarize new agonists and antagonists developed for intervening in the activation of insulin–IGF receptor signalling. Furthermore, we discuss recently identified regulatory mechanisms beyond ligand–receptor interactions and functions of insulin–IGF receptor signalling in diseases. Insulin and insulin-like growth factor (IGF) signalling regulates metabolism, cell growth and proliferation and diverse cell-specific processes. This Review discusses recent structural insights into insulin–receptor and IGF–receptor binding and activation, regulation of insulin and IGF signalling and new agonists with therapeutic potential.