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Nature Sep 2023Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets....
Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (G) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with G. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.
Topics: Humans; Arrestin; Binding Sites; GTP-Binding Protein alpha Subunits, Gs; Imidazolidines; Ligands; Peptides; Protein Conformation; Receptor, Parathyroid Hormone, Type 1; Receptors, G-Protein-Coupled; Signal Transduction; Spiro Compounds; Drug Design
PubMed: 37524305
DOI: 10.1038/s41586-023-06467-w -
Clinical Science (London, England :... Dec 2023Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently,...
Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the β-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated β-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine β-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated β-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the β-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the β-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated β-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.
Topics: Rats; Animals; Humans; Podocytes; TRPC6 Cation Channel; Calcium; beta-Arrestins; Angiotensin Receptor Antagonists; Rats, Inbred Dahl; Angiotensin-Converting Enzyme Inhibitors; Kidney Diseases; Hypertension; TRPC Cation Channels
PubMed: 38051199
DOI: 10.1042/CS20230313 -
Current Protocols Oct 2023G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in...
G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptors Basic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptors Alternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membrane Alternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membrane Support Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection.
Topics: Arrestin; Ligands; Arrestins; Research Design; Cell Membrane
PubMed: 37787634
DOI: 10.1002/cpz1.890 -
Molecular Cell Sep 2023Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body β-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for...
Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body β-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for inflammation-related diseases. HCAR2 mediates distinct pathophysiological events by activating G protein or β-arrestin effectors. Here, we characterize compound 9n as a G-biased allosteric modulator (BAM) of HCAR2 and exhibit anti-inflammatory efficacy in RAW264.7 macrophages via a specific HCAR2-G pathway. Furthermore, four structures of HCAR2-G complex bound to orthosteric agonists (niacin or monomethyl fumarate), compound 9n, and niacin together with compound 9n simultaneously reveal a common orthosteric site and a unique allosteric site. Combined with functional studies, we decipher the action framework of biased allosteric modulation of compound 9n on the orthosteric site. Moreover, co-administration of compound 9n with orthosteric agonists could enhance anti-inflammatory effects in the mouse model of colitis. Together, our study provides insight to understand the molecular pharmacology of the BAM and facilitates exploring the therapeutic potential of the BAM with orthosteric drugs.
Topics: Animals; Mice; Allosteric Regulation; Colitis; GTP-Binding Protein alpha Subunits, Gi-Go; Inflammation; Ketone Bodies; Niacin; Receptors, G-Protein-Coupled
PubMed: 37597514
DOI: 10.1016/j.molcel.2023.07.030 -
Cell Mar 2024Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular...
Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the β-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-G complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.
Topics: Apelin Receptors; Cryoelectron Microscopy; GTP-Binding Proteins; Receptors, G-Protein-Coupled; Signal Transduction; Humans; Cardiovascular Agents; Drug Design
PubMed: 38428423
DOI: 10.1016/j.cell.2024.02.004 -
Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.Science (New York, N.Y.) Jan 2024β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven...
β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues.
Topics: beta-Arrestins; Cryoelectron Microscopy; Receptors, G-Protein-Coupled; Signal Transduction; Protein Conformation, beta-Strand; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Humans
PubMed: 38175886
DOI: 10.1126/science.adj3347 -
Drugs Jun 2024Tegileridine () is a small molecule μ-opioid receptor biased agonist developed by Jiangsu Hengrui Pharmaceuticals Co., Ltd for the treatment of postoperative pain.... (Review)
Review
Tegileridine () is a small molecule μ-opioid receptor biased agonist developed by Jiangsu Hengrui Pharmaceuticals Co., Ltd for the treatment of postoperative pain. Tegileridine selectively activates the G-protein-coupled pathway, which mediates strong central analgesic effects and only weakly activates the β-arrestin-2 pathway implicated in adverse events like respiratory depression and gastrointestinal dysfunction. In January 2024, tegileridine received its first approval in China for the treatment of moderate to severe pain after abdominal surgery. This article summarizes the milestones in the development of tegileridine leading to this first approval for the treatment of moderate to severe pain after abdominal surgery.
Topics: Humans; Drug Approval; Pain, Postoperative; Receptors, Opioid, mu; Analgesics, Opioid; China; Thiophenes; Spiro Compounds
PubMed: 38771484
DOI: 10.1007/s40265-024-02033-4 -
Handbook of Experimental Pharmacology Jul 2023The first structure of an adrenoceptor (AR), the human β-adrenoceptor (hβAR) was published in 2007 and since then a total of 78 structures (up to June 2022) have been...
The first structure of an adrenoceptor (AR), the human β-adrenoceptor (hβAR) was published in 2007 and since then a total of 78 structures (up to June 2022) have been determined by X-ray crystallography and electron cryo-microscopy (cryo-EM) of all three βARs (β, β and β) and four out of six αARs (α, α, α, α). The structures are in a number of different conformational states, including the inactive state bound to an antagonist, an intermediate state bound to agonist and active states bound to agonist and an intracellular transducer (G protein or arrestin) or transducer mimetic (nanobody). The structures identify molecular details of how ligands bind in the orthosteric binding pocket (OBP; 19 antagonists, 18 agonists) and also how three different small molecule allosteric modulators bind. The structures have been used to define the molecular details of receptor activation and also the molecular determinants for transducer coupling. This chapter will give a brief overview of the structures, receptor activation, a comparison across the different subfamilies and commonalities of ligand-receptor interactions.
PubMed: 37460660
DOI: 10.1007/164_2023_674 -
Trends in Biochemical Sciences Jun 2024G protein-coupled receptors (GPCRs) located at the cell surface bind extracellular ligands and convey intracellular signals via activation of heterotrimeric G proteins.... (Review)
Review
G protein-coupled receptors (GPCRs) located at the cell surface bind extracellular ligands and convey intracellular signals via activation of heterotrimeric G proteins. Traditionally, G protein signaling was viewed to occur exclusively at this subcellular region followed by rapid desensitization facilitated by β-arrestin (βarr)-mediated G protein uncoupling and receptor internalization. However, emerging evidence over the past 15 years suggests that these βarr-mediated events do not necessarily terminate receptor signaling and that some GPCRs continue to activate G proteins after having been internalized into endosomes. Here, we review the recently elucidated mechanistic basis underlying endosomal GPCR signaling and discuss physiological implications and pharmacological targeting of this newly appreciated signaling mode.
Topics: Endosomes; Signal Transduction; Receptors, G-Protein-Coupled; Humans; Animals; beta-Arrestins
PubMed: 38643023
DOI: 10.1016/j.tibs.2024.03.006 -
Pharmacology & Therapeutics Mar 2024The original paradigm of classical - also referred to as canonical - cellular signal transduction of heterotrimeric G proteins (G protein) is defined by a hierarchical,... (Review)
Review
The original paradigm of classical - also referred to as canonical - cellular signal transduction of heterotrimeric G proteins (G protein) is defined by a hierarchical, orthograde interaction of three players: the agonist-activated G protein-coupled receptor (GPCR), which activates the transducing G protein, that in turn regulates its intracellular effectors. This receptor-transducer-effector concept was extended by the identification of regulators and adapters such as the regulators of G protein signaling (RGS), receptor kinases like βARK, or GPCR-interacting arrestin adapters that are integrated into this canonical signaling process at different levels to enable fine-tuning. Finally, the identification of atypical signaling mechanisms of classical regulators, together with the discovery of novel modulators, added a new and fascinating dimension to the cellular G protein signal transduction. This heterogeneous group of accessory G protein modulators was coined "activators of G protein signaling" (AGS) proteins and plays distinct roles in canonical and non-canonical G protein signaling pathways. AGS proteins contribute to the control of essential cellular functions such as cell development and division, intracellular transport processes, secretion, autophagy or cell movements. As such, they are involved in numerous biological processes that are crucial for diseases, like diabetes mellitus, cancer, and stroke, which represent major health burdens. Although the identification of a large number of non-canonical G protein signaling pathways has broadened the spectrum of this cellular communication system, their underlying mechanisms, functions, and biological effects are poorly understood. In this review, we highlight and discuss atypical G protein-dependent signaling mechanisms with a focus on inhibitory G proteins (G) involved in canonical and non-canonical signal transduction, review recent developments and open questions, address the potential of new approaches for targeted pharmacological interventions.
Topics: Humans; Signal Transduction; Heterotrimeric GTP-Binding Proteins; Receptors, G-Protein-Coupled
PubMed: 38295906
DOI: 10.1016/j.pharmthera.2024.108589