Structures of the entire human opioid receptor family.

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (µOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including ß-endorphin- and endomorphin-bound µOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

GPR35 promotes neutrophil recruitment in response to serotonin metabolite 5-HIAA.

Rapid neutrophil recruitment to sites of inflammation is crucial for innate immune responses. Here, we reveal that the G-protein-coupled receptor GPR35 is upregulated in activated neutrophils, and it promotes their migration. GPR35-deficient neutrophils are less recruited from blood vessels into inflamed tissue, and the mice are less efficient in clearing peritoneal bacteria. Using a bioassay, we find that serum and activated platelet supernatant stimulate GPR35, and we identify the platelet-derived serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) as a GPR35 ligand. GPR35 function in neutrophil recruitment is strongly dependent on platelets, with the receptor promoting transmigration across platelet-coated endothelium. Mast cells also attract GPR35+ cells via 5-HIAA. Mice deficient in 5-HIAA show a loss of GPR35-mediated neutrophil recruitment to inflamed tissue. These findings identify 5-HIAA as a GPR35 ligand and neutrophil chemoattractant and establish a role for platelet- and mast cell-produced 5-HIAA in cell recruitment to the sites of inflammation and bacterial clearance.

What Makes a Discovery Successful? The Story of Linda Buck and the Olfactory Receptors.

In 1991, Buck and Axel published a landmark study in Cell for work that was awarded the 2004 Nobel Prize. The identification of the olfactory receptors as the largest family of GPCRs catapulted olfaction into mainstream neurobiology. This BenchMark revisits Buck's experimental innovation and its surprising success at the time.

Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of ß-arrestin recruitment to MC4R, rather than canonical Gαs-mediated cyclic adenosine-monophosphate production, explained 88% of the variance in the association of MC4R variants with BMI. While most MC4R variants caused loss of function, a subset caused gain of function; these variants were associated with significantly lower BMI and lower odds of obesity, type 2 diabetes, and coronary artery disease. Protective associations were driven by MC4R variants exhibiting signaling bias toward ß-arrestin recruitment and increased mitogen-activated protein kinase pathway activation. Harnessing ß-arrestin-biased MC4R signaling may represent an effective strategy for weight loss and the treatment of obesity-related cardiometabolic diseases.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones.

Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone.

Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.

Oxytocin and vasopressin signaling in health and disease.

Neurohypophysial peptides are ancient and evolutionarily highly conserved neuropeptides that regulate many crucial physiological functions in vertebrates and invertebrates. The human neurohypophysial oxytocin/vasopressin (OT/VP) signaling system with its four receptors has become an attractive drug target for a variety of diseases, including cancer, pain, cardiovascular indications, and neurological disorders. Despite its promise, drug development faces hurdles, including signaling complexity, selectivity and off-target concerns, translational interspecies differences, and inefficient drug delivery. In this review we dive into the complexity of the OT/VP signaling system in health and disease, provide an overview of relevant pharmacological probes, and discuss the latest trends in therapeutic lead discovery and drug development.

G Protein-Coupled Receptor Signaling: New Insights Define Cellular Nanodomains.

G protein-coupled receptors are the largest and pharmacologically most important receptor family and are involved in the regulation of most cell functions. Most of them reside exclusively at the cell surface, from where they signal via heterotrimeric G proteins to control the production of second messengers such as cAMP and IP3 as well as the activity of several ion channels. However, they may also internalize upon agonist stimulation or constitutively reside in various intracellular locations. Recent evidence indicates that their function differs depending on their precise cellular localization. This is because the signals they produce, notably cAMP and Ca2+, are mostly bound to cell proteins that significantly reduce their mobility, allowing the generation of steep concentration gradients. As a result, signals generated by the receptors remain confined to nanometer-sized domains. We propose that such nanometer-sized domains represent the basic signaling units in a cell and a new type of target for drug development.

International Union of Basic and Clinical Pharmacology. CXV: The Class F of G Protein-Coupled Receptors.

The class F of G protein-coupled receptors (GPCRs) consists of ten Frizzleds (FZD1-10) and Smoothened (SMO). FZDs bind and are activated by secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family and SMO is indirectly activated by the Hedgehog (Hh) family of morphogens acting on the transmembrane protein Patched (PTCH). The advance of our understanding of FZDs and SMO as dynamic transmembrane receptors and molecular machines, which emerged during the past 14 years since the first class F GPCR IUPHAR nomenclature report, justifies an update. This article focuses on the advances in molecular pharmacology and structural biology providing new mechanistic insight into ligand recognition, receptor activation mechanisms, signal initiation and signal specification. Furthermore, class F GPCRs continue to develop as drug targets, and novel technologies and tools such as genetically encoded biosensors and CRISP/Cas9 edited cell systems have contributed to refined functional analysis of these receptors. Also, advances in crystal structure analysis and cryogenic electron microscopy contribute to a rapid development of our knowledge about structure-function relationships providing a great starting point for drug development. Despite the progress questions and challenges remain to fully understand the complexity of the WNT/FZD and Hh/SMO signaling systems. Significance Statement The recent years of research have brought about substantial functional and structural insight into mechanisms of activation of Frizzleds and Smoothened. While the advance furthers our mechanistic understanding of ligand recognition, receptor activation, signal specification and initiation, broader opportunities emerge that allow targeting class F GPCRs for therapy and regenerative medicine employing both biologics and small molecule compounds.

GPR35 and mediators from platelets and mast cells in neutrophil migration and inflammation.

Neutrophil recruitment from circulation to sites of inflammation is guided by multiple chemoattractant cues emanating from tissue cells, immune cells, and platelets. Here, we focus on the function of one G-protein coupled receptor, GPR35, in neutrophil recruitment. GPR35 has been challenging to study due the description of multiple ligands and G-protein couplings. Recently, we found that GPR35-expressing hematopoietic cells respond to the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). We discuss distinct response profiles of GPR35 to 5-HIAA compared to other ligands. To place the functions of 5-HIAA in context, we summarize the actions of serotonin in vascular biology and leukocyte recruitment. Important sources of serotonin and 5-HIAA are platelets and mast cells. We discuss the dynamics of cell migration into inflamed tissues and how multiple platelet and mast cell-derived mediators, including 5-HIAA, cooperate to promote neutrophil recruitment. Additional actions of GPR35 in tissue physiology are reviewed. Finally, we discuss how clinically approved drugs that modulate serotonin uptake and metabolism may influence 5-HIAA-GPR35 function, and we speculate about broader influences of the GPR35 ligand-receptor system in immunity and disease.

Cryo-EM structure of the human chemerin receptor 1-Gi protein complex bound to the C-terminal nonapeptide of chemerin.

Chemerin is a processed protein that acts on G protein-coupled receptors (GPCRs) for its chemotactic and adipokine activities. The biologically active chemerin (chemerin 21-157) results from proteolytic cleavage of prochemerin and uses its C-terminal peptide containing the sequence YFPGQFAFS for receptor activation. Here we report a high-resolution cryo-electron microscopy (cryo-EM) structure of human chemerin receptor 1 (CMKLR1) bound to the C-terminal nonapeptide of chemokine (C9) in complex with Gi proteins. C9 inserts its C terminus into the binding pocket and is stabilized through hydrophobic interactions involving its Y1, F2, F6, and F8, as well as polar interactions between G4, S9, and several amino acids lining the binding pocket of CMKLR1. Microsecond scale molecular dynamics simulations support a balanced force distribution across the whole ligand-receptor interface that enhances thermodynamic stability of the captured binding pose of C9. The C9 interaction with CMKLR1 is drastically different from chemokine recognition by chemokine receptors, which follow a two-site two-step model. In contrast, C9 takes an "S"-shaped pose in the binding pocket of CMKLR1 much like angiotensin II in the AT1 receptor. Our mutagenesis and functional analyses confirmed the cryo-EM structure and key residues in the binding pocket for these interactions. Our findings provide a structural basis for chemerin recognition by CMKLR1 for the established chemotactic and adipokine activities.

Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses.

Latrophilin-1 (Lphn1, aka CIRL1 and CL1; gene symbol Adgrl1) is an adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knock-in/knock-out mice for Lphn1 that contain an extracellular myc epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but is required for some inhibitory synaptic connections.

Bayesian network models identify cooperative GPCR:G protein interactions that contribute to G protein coupling.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

G protein-coupled receptor-mediated signaling of immunomodulation in tumor progression.

G protein-coupled receptors (GPCRs) are essential contributors to tumor growth and metastasis due to their roles in immune cell regulation. Therefore, GPCRs are potential targets for cancer immunotherapy. Here, we discuss the current understanding of the roles of GPCRs and their signaling pathways in tumor progression from an immunocellular perspective. Additionally, we focus on the roles of GPCRs in regulating immune checkpoint proteins involved in immune evasion. Finally, we review the progress of clinical trials of GPCR-targeted drugs for cancer treatment, which may be combined with immunotherapy to improve treatment efficacy. This expanded understanding of the role of GPCRs may shed light on the mechanisms underlying tumor progression and provide a novel perspective on cancer immunotherapy.

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions.

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-ß/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and ß-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

Dietary Fiber and Microbiota Metabolite Receptors Enhance Cognition and Alleviate Disease in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder with poorly understood etiology. AD has several similarities with other "Western lifestyle" inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing GPCRs and their ligands, short-chain fatty acids (SCFAs), in protection of numerous Western diseases in mouse models, such as Type I diabetes and hypertension. Depletion of GPR43, GPR41, or GPR109A accelerates disease, whereas high SCFA yielding diets protect in mouse models. Here, we extended the concept that metabolite-sensing receptors and SCFAs may be a more common protective mechanism against Western diseases by studying their role in AD pathogenesis in the 5xFAD mouse model. Both male and female mice were included. Depletion of GPR41 and GPR43 accelerated cognitive decline and impaired adult hippocampal neurogenesis in 5xFAD and WT mice. Lack of fiber/SCFAs accelerated a memory deficit, whereas diets supplemented with high acetate and butyrate (HAMSAB) delayed cognitive decline in 5xFAD mice. Fiber intake impacted on microglial morphology in WT mice and microglial clustering phenotype in 5xFAD mice. Lack of fiber impaired adult hippocampal neurogenesis in both W and AD mice. Finally, maternal dietary fiber intake significantly affects offspring's cognitive functions in 5xFAD mice and microglial transcriptome in both WT and 5xFAD mice, suggesting that SCFAs may exert their effect during pregnancy and lactation. Together, metabolite-sensing GPCRs and SCFAs are essential for protection against AD, and reveal a new strategy for disease prevention.Significance Statement Alzheimer's disease (AD) is one of the most common neurodegenerative diseases; currently, there is no cure for AD. In our study, short-chain fatty acids and metabolite receptors play an important role in cognitive function and pathology in AD mouse model as well as in WT mice. SCFAs also impact on microglia transcriptome, and immune cell recruitment. Out study indicates the potential of specialized diets (supplemented with high acetate and butyrate) releasing high amounts of SCFAs to protect against disease.

Bivalent and bitopic ligands of the opioid receptors: The prospects of a dual approach.

Opioid receptors belonging to the class A G-protein coupled receptors (GPCRs) are the targets of choice in the treatment of acute and chronic pain. However, their on-target side effects such as respiratory depression, tolerance and addiction have led to the advent of the 'opioid crisis'. In the search for safer analgesics, bivalent and more recently, bitopic ligands have emerged as valuable tool compounds to probe these receptors. The activity of bivalent and bitopic ligands rely greatly on the allosteric nature of the GPCRs. Bivalent ligands consist of two pharmacophores, each binding to the individual orthosteric binding site (OBS) of the monomers within a dimer. Bitopic or dualsteric ligands bridge the gap between the OBS and the spatially distinct, less conserved allosteric binding site (ABS) through the simultaneous occupation of these two sites. Bivalent and bitopic ligands stabilize distinct conformations of the receptors which ultimately translates into unique signalling and pharmacological profiles. Some of the interesting properties shown by these ligands include improved affinity and/or efficacy, subtype and/or functional selectivity and reduced side effects. This review aims at providing an overview of some of the bivalent and bitopic ligands of the opioid receptors and, their pharmacology in the hope of inspiring the design and discovery of the next generation of opioid analgesics.

Get ready to sharpen your tools: A short guide to heterotrimeric G protein activity biosensors.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors encoded in the human genome, and they initiate cellular responses triggered by a plethora of extracellular stimuli ranging from neurotransmitters or hormones to photons. Upon stimulation, GPCRs activate heterotrimeric G proteins (Gαßγ) in the cytoplasm, which then convey signals to their effectors to elicit cellular responses. Given the broad biological and biomedical relevance of GPCRs and G proteins in physiology and disease, there is great interest in developing and optimizing approaches to measure their signaling activity with high accuracy and across experimental systems pertinent to their functions in cellular communication. This review provides a historical perspective on approaches to measure GPCR-G protein signaling, from quantification of second messengers and other indirect readouts of activity, to biosensors that directly detect the activity of G proteins. The latter is the focus of a more detailed overview of the evolution of design principles for various optical biosensors of G protein activity with different experimental capabilities. We will highlight advantages and limitations of biosensors that detect different G protein activation hallmarks, like dissociation of Gα and Gßγ or nucleotide exchange on Gα, as well as their suitability to detect signaling mediated by endogenous versus exogenous signaling components, or in physiologically-relevant systems like primary cells. Overall, this review intends to provide an assessment of the state-of-the-art for biosensors that directly measure G protein activity to allow readers to make informed decisions on the selection and implementation of currently available tools. Significance Statement G protein activity biosensors have become essential and widespread tools to assess GPCR signaling and pharmacology. Yet, investigators face the challenge of choosing from a growing list of G protein activity biosensors. This review provides an overview of the features and capabilities of different optical biosensor designs for the direct detection of G protein activity in cells, with the aim of facilitating the rational selection of systems that align with the specific scientific questions and needs of investigators.