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Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα o K46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα o K46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα o , without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells., (© 2024. The Author(s).)

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors in humans. Over 800 GPCRs regulate the (patho)biology of every organ, tissue, and cell type. Consequently, GPCRs are the most prominent therapeutic targets in medicine. Although over 30% of current U.S. Food and Drug Administration-approved drugs target GPCR signaling, most receptors remain understudied and therapeutically underutilized. Challenges include an incomplete understanding of GPCR signaling, pharmacology, structural biology, and the multiplicity of endogenous GPCR ligands, in addition to a scarcity of biological and pharmacological tools for elucidating GPCR-mediated cellular processes beyond initial signaling events. Various mammalian, insect, and yeast cell models currently address some of these needs. Here, we review recent advances in yeast synthetic biology that are helping to catalyze new and unexpected conceptual and technical breakthroughs in GPCR-based medicine and biotechnology., Competing Interests: Declaration of Competing Interest D.G.I and N.J.K. declare no interests. G.J.T. was supported in part by an appointment to the NRC Research Associateship Program at the National Institute of Standards and Technology, administered by the Fellowships Office of the National Academies of Sciences, Engineering, and Medicine. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification implies neither recommendation or endorsement by the National Institute of Standards and Technology nor that the materials or equipment identified are necessarily the best available for the purpose. The views expressed in this publication are those of the authors and do not necessarily represent the views of the U.S. Department of Commerce or the National Institute of Standards and Technology., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ligands for the serotonin 2B receptor (5-HT 2B ) have shown potential to treat pulmonary arterial hypertension in preclinical models but cannot be used in humans because of predicted off-target neurological effects. The aim of this study was to develop novel systemically restricted compounds targeting 5-HT 2B . Here, we show that mice treated with VU6047534 had decreased RVSP compared with control treatment in both the prevention and intervention studies using Sugen-hypoxia. VU6047534 is a novel 5-HT 2B partial agonist that is peripherally restricted and able to both prevent and treat Sugen-hypoxia-induced pulmonary arterial hypertension. We have synthesized and characterized a structurally novel series of 5-HT 2B ligands with high potency and selectivity for the 5-HT 2B receptor subtype. Next-generation 5-HT 2B ligands with similar characteristics, and predicted to be systemically restricted in humans, are currently advancing to investigational new drug-enabling studies., Competing Interests: This work was supported by the National Institutes of Health (HL135790, HL095797, and HL087738) and Vanderbilt University Discovery Grant. Research undertaken in the Vanderbilt Mouse Neurobehavior Core was supported by the EKS NICHD of the National Institutes of Health under Award P50HD103537. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

In this issue of Molecular Cell, Park et al. 1 comprehensively profile how neurons utilize the Gα inhibitory interacting protein, GINIP, to modulate neurotransmission at a systems level through bias of downstream G protein-coupled receptor (GPCR) signaling., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Engineered signaling proteins permit precise modulation of cell signaling networks and are valuable tools for basic and translational research. In this issue of Structure, Tichy and colleagues leverage high-resolution GPCR-G protein complex structures to rationally design improved light-activated chimeric GPCRs (termed OptoXRs) with increased sensitivity and tunable signaling features., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The evolutionary expansion of G protein-coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision-making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular sensors capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are coincidence detectors that couple proton (H + ) binding to GPCR signaling. Using a panel of 28 receptors covering 280 individual GPCR-Gα coupling combinations, we show that H + gating both positively and negatively modulates GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H + gating and report the discovery of an acid sensor, the adenosine A2a receptor, which can be activated solely by acidic pH. Together, these findings establish a paradigm for GPCR signaling, biology, and pharmacology applicable to acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature., Competing Interests: Competing interest statement: N.J.K., J.B.R., G.J.T., W.M.M., and D.G.I. have filed a patent application with the US Patent and Trademark Office related to this work.

A rich diversity of transmembrane G protein-coupled receptors (GPCRs) are used by eukaryotes to sense physical and chemical signals. In humans alone, 800 GPCRs comprise the largest and most therapeutically targeted receptor class. Recent advances in GPCR structural biology have produced hundreds of GPCR structures solved by X-ray diffraction and increasingly, cryo-electron microscopy (cryo-EM). Many of these structures are stabilized by site-specific cholesterol binding, but it is unclear whether these interactions are a product of recurring cholesterol-binding motifs and if observed patterns of cholesterol binding differ by experimental technique. Here, we comprehensively analyze the location and composition of cholesterol binding sites in the current set of 473 human GPCR structural chains. Our findings establish that cholesterol binds similarly in cryo-EM and X-ray structures and show that 92% of cholesterol molecules on GPCR surfaces reside in predictable locations that lack discernable cholesterol-binding motifs., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H + ). How these receptors have uniquely obtained this ability is unknown. Here, we show these receptors evolved the capability to sense H + signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed deep variant profiling, a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using deep variant profiling and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. In addition, we found that an allosteric modulator of many class A GPCRs, Na + , synergistically regulated pH sensing by maintaining the pK a values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H + and Na + . Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Rowe et al.)

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs., Competing Interests: The authors declare no competing interest.

The yeast Saccharomyces cerevisiae employs multiple pathways to coordinate sugar availability and metabolism. Glucose and other sugars are detected by a G protein-coupled receptor, Gpr1, as well as a pair of transporter-like proteins, Rgt2 and Snf3. When glucose is limiting, however, an ATP-driven proton pump (Pma1) is inactivated, leading to a marked decrease in cytoplasmic pH. Here we determine the relative contribution of the two sugar-sensing pathways to pH regulation. Whereas cytoplasmic pH is strongly dependent on glucose abundance and is regulated by both glucose-sensing pathways, ATP is largely unaffected and therefore cannot account for the changes in Pma1 activity. These data suggest that the pH is a second messenger of the glucose-sensing pathways. We show further that different sugars differ in their ability to control cellular acidification, in the manner of inverse agonists. We conclude that the sugar-sensing pathways act via Pma1 to invoke coordinated changes in cellular pH and metabolism. More broadly, our findings support the emerging view that cellular systems have evolved the use of pH signals as a means of adapting to environmental stresses such as those caused by hypoxia, ischemia, and diabetes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)