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Fully understanding the mechanisms of signaling proteins such as G protein-coupled receptors (GPCRs) will require the characterization of their conformational states and the pathways connecting those states. The recent crystal structures of the β 2 - and β 1 -adrenergic receptors in a nominally inactive state constituted a major advance toward this goal, but also raised new questions. Although earlier biochemical observations had suggested that these receptors possessed a set of contacts between helices 3 and 6, known as the ionic lock, which was believed to form a molecular switch for receptor activation, the crystal structures lacked these contacts. The unexpectedly broken ionic lock has raised questions about the true conformation(s) of the inactive state and the role of the ionic lock in receptor activation and signaling. To address these questions, we performed microsecond-timescale molecular dynamics simulations of the β 2 -adrenergic receptor (β 2 AR) in multiple wild-type and mutant forms. In wild-type simulations, the ionic lock formed reproducibly, bringing the intracellular ends of helices 3 and 6 together to adopt a conformation similar to that found in inactive rhodopsin. Our results suggest that inactive β 2 AR exists in equilibrium between conformations with the lock formed and the lock broken, whether or not the cocrystallized ligand is present. These findings, along with the formation of several secondary structural elements in the β 2 AR loops during our simulations, may provide a more comprehensive picture of the inactive state of the β-adrenergic receptors, reconciling the crystal structures with biochemical studies.

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), anothfer SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

The group of proton-sensing G-protein coupled receptors (GPCRs) consists of the four receptors GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132). These receptors are cellular sensors of acidification, a property that has been attributed to the presence of crucial histidine residues. However, the pH detection varies considerably among the group of proton-sensing GPCRs and ranges from pH of 5.5 to 7.8. While the proton-sensing GPCRs were initially considered to detect acidic cellular environments in the context of inflammation, recent observations have expanded our knowledge about their physiological and pathophysiological functions and many additional individual and unique features have been discovered that suggest a more differentiated role of these receptors in health and disease. It is known that all four receptors contribute to different aspects of tumor biology, cardiovascular physiology, and asthma. However, apart from their overlapping functions, they seem to have individual properties, and recent publications identify potential roles of individual GPCRs in mechanosensation, intestinal inflammation, oncoimmunological interactions, hematopoiesis, as well as inflammatory and neuropathic pain. Here, we put together the knowledge about the biological functions and structural features of the four proton-sensing GPCRs and discuss the biological role of each of the four receptors individually. We explore all currently known pharmacological modulators of the four receptors and highlight potential use. Finally, we point out knowledge gaps in the biological and pharmacological context of proton-sensing GPCRs that should be addressed by future studies.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existing literature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.Graphical Abstract

The mu-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOPR) are closely related yet functionally distinct modulators of rewards, motivation, and affect. Within the mesocorticolimbic system, including the prefrontal cortex (PFC), the ventral tegmental area (VTA) and the nucleus accumbens (NAc), these receptors exhibit divergent, cell type–specific expression patterns that drive opposing behavioral outcomes. For example, MOR activation enhances rewards processing and reinforcement by facilitating dopamine transmission, whereas NOPR signaling in the VTA can reduce dopamine cell activity. In addition, MOR and NOPR are positioned within cortical circuits to preferentially reduce GABA and glutamate transmission, respectively. This review synthesizes current knowledge on how MOR and NOPR coordinate motivational and affective states through distinct neuronal populations across the mesocorticolimbic circuit. We also discuss emerging evidence for functional interactions between these systems and the therapeutic implications of pharmacological strategies targeting both receptors, including dual-acting MOR/NOPR ligands that enhance analgesic efficacy with reduced abuse liability. By integrating behavioral, molecular, and circuit-level findings, this synthesis aims to clarify how MOR and NOPR signaling jointly shape rewards and stress pathways, and provide insight into the development of safer and more effective treatments for opioid use disorders.

Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished CaV1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function.

G-protein-coupled receptors (GPCRs) are the largest family of cell surface signaling receptors known to play a crucial role in various physiological functions, including tumor growth and metastasis. Various molecules such as hormones, lipids, peptides, and neurotransmitters activate GPCRs that enable the coupling of these receptors to highly specialized transducer proteins, called G-proteins, and initiate multiple signaling pathways. Integration of these intricate networks of signaling cascades leads to numerous biochemical responses involved in diverse pathophysiological activities, including cancer development. While several studies indicate the role of GPCRs in controlling various aspects of cancer progression such as tumor growth, invasion, migration, survival, and metastasis through its aberrant overexpression, mutations, or increased release of agonists, the explicit mechanisms of the involvement of GPCRs in cancer progression is still puzzling. This review provides an insight into the various responses mediated by GPCRs in the development of cancers, the molecular mechanisms involved and the novel pharmacological approaches currently preferred for the treatment of cancer. Thus, these findings extend the knowledge of GPCRs in cancer cells and help in the identification of therapeutics for cancer patients.

Aminergic G protein-coupled receptors (GPCRs) have been a major focus of pharmaceutical research for many years. Due partly to the lack of reliable receptor structures, drug discovery efforts have been largely ligand-based. The recently determined X-ray structure of the β 2 -adrenergic receptor offers an opportunity to investigate the advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR targets. Approximately 1 million commercially available, “lead-like” molecules were docked against the β 2 -adrenergic receptor structure. On testing of 25 high-ranking molecules, 6 were active with binding affinities <4 μM, with the best molecule binding with a K i of 9 nM (95% confidence interval 7–10 nM). Five of these molecules were inverse agonists. The high hit rate, the high affinity of the most potent molecule, the discovery of unprecedented chemotypes among the new inhibitors, and the apparent bias toward inverse agonists among the docking hits, have implications for structure-based approaches against GPCRs that recognize small organic molecules.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

G-Protein-Coupled Receptors (GPCRs) make up around 3–4% of the human genome and are the targets of one-third of FDA-approved drugs. GPCRs typically exist as monomers but also aggregate to form higher-order oligomers, including dimers. β2AR, a pharmacologically relevant GPCR, is known to be targeted for the treatment of asthma and cardiovascular diseases. The activation of β2AR at the dimer level remains under-explored. In the current study, molecular dynamics (MD) simulations have been performed to understand activation-related structural changes in β2AR at the dimer level. The transition from inactive to active and vice versa has been studied by starting the simulations in the apo, agonist-bound, and inverse agonist-bound β2AR dimers for PDB ID: 2RH1 and PDB ID: 3P0G, respectively. A cumulative total of around 21-μs simulations were performed. Residue-based distances, RMSD, and PCA calculations suggested that either of the one monomer attained activation-related features for the apo and agonist-bound β2AR dimers. The TM5 and TM6 helices within the two monomers were observed to be in significant variation in all the simulations. TM5 bulge and proximity of TM2 and TM7 helices may be contributing to one of the early events in activation. The dimeric interface between TM1 and helix 8 were observed to be well maintained in the apo and agonist-bound simulations. The presence of inverse agonists favored inactive features in both the monomers. These key features of activation known for monomers were observed to have an impact on β2AR dimers, thereby providing an insight into the oligomerization mechanism of GPCRs.