Approximately one-third of clinical drugs mediate their therapeutic effects through G protein-coupled receptors (GPCRs), highlighting their immense therapeutic relevance. Novel approaches to modulate GPCR activity have the potential to yield unique pharmacological profiles. Conventionally, the G protein and β-arrestin signaling pathways downstream of GPCRs have been viewed as mutually exclusive. Using the in-house developed survival pressure selection (SPS) method, a high-throughput platform for GPCR agonist discovery, we identified an allosteric ligand that stabilizes a GPCR-G protein-β-arrestin megacomplex, thereby mediating sustained receptor signaling following internalization. Remarkably, this compound, atazanavir, exhibits pan-receptor activation across multiple family A GPCRs, including GPR119, β1AR, and β2AR, demonstrating the broad applicability of this regulatory mechanism. This discovery uncovers a distinct mechanism of GPCR regulation, opening alternative avenues for the development of therapeutics targeting GPCRs.

Abstract Endogenous inverse agonists suppress constitutive G protein-coupled receptor (GPCR) signaling, but their mechanisms remain poorly understood. Here we report cryo-EM structures of melanocortin-1 receptor (MC1R) bound to its endogenous inverse agonist, agouti signaling protein (ASIP) and melanocortin-4 receptor (MC4R) bound to agouti-related protein (AgRP). Together with previously reported structures and those we determined using extracellular nanobodies developed here, these data delineate a conformational continuum underlying receptor activation and silencing. Both inverse agonists occlude the orthosteric pocket as molecular corks and drive a shared TM3-centered extracellular remodeling. Against this common mechanism, subtype selectivity is encoded not by the conserved orthosteric pocket but by the divergent extracellular receptor surface, engaged through an ASIP C-terminal loop-dependent clasp. These findings establish a mechanistic framework for endogenous inverse agonism and identify the receptor periphery as a tractable target for subtype-selective modulation.

Deciphering transducer selectivity in G protein-coupled receptors (GPCRs) is essential for developing next-generation therapeutics with improved safety profiles. Here, we identify (R)-141, a μ-opioid receptor (μOR) agonist with a distinct scaffold that exhibits exceptional G protein bias. To decode the underlying mechanism, we determined the cryo-EM structures of μOR bound to (R)-141 in complex with Gi and with GRK2. Our structural, functional and dynamics data together reveal that (R)-141 achieves this selectivity through a stepwise gating mechanism, in which the conformational dynamics of TM7 serves as a terminal checkpoint. This "conformational veto" by TM7 provides a mechanism to modulate β-arrestin recruitment at the final step. Collectively, our work provides a systemic vision of transducer selectivity and a framework for rational biased drug design.

Brain organic cation transporter 1 (BOCT1), also known as the solute carrier family 22 member 17 (SLC22A17) or the receptor for lipocalin-2 (LCN2), plays critical roles in health and disease. Its deficiency in mice results in early postnatal mortality and severe neurogenesis impairments. Despite its importance in physiology and pathophysiology, BOCT1’s structure and transport mechanism remain elusive. Here, we integrate cryo-electron microscopy (cryo-EM), functional assays, biochemical experiments, and molecular dynamics simulations to elucidate the structure, substrate recognition, and transport mechanism of mouse BOCT1 (mBOCT1). The high-resolution cryo-EM structure reveals a distinctive N-terminal domain with a unique folding pattern dominated by a transmembrane loop atop TM6 (TML6), diverging from both known structures of SLC22 transporters and AlphaFold predictions. Notably, mBOCT1 functions as a high-capacity, low-affinity iron transporter independent of LCN2 binding. Iron transport is facilitated by a substrate gating mechanism involving TML6. These findings establish a structural basis for BOCT1’s role as an independent iron transporter, enhancing our understanding of the transport mechanisms within major facilitator superfamily (MFS) transporters and providing new insights into brain iron homeostasis. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, Grants 32130048, 92157301 to L.C

Approximately 1/3 of all clinical drugs exert their therapeutic effects by modulating the activity of G protein-coupled receptors (GPCRs). Thus, there is a constant interest in finding novel ways to modulate GPCR activity. In this work, through a newly established Survival Pressure Selection (SPS) method for high-throughput screening of GPCR agonists, we discover that atazanavir functions as an agonist for GPR119. Further studies suggest that atazanavir is capable of activating a number of Family A GPCRs, including the β1 adrenergic receptor (β1AR), the β2 adrenergic receptor (β2AR) and the μ opoioid receptor (μOR). Cryo-EM structures reveal that atazanavir binds to an allosteric pocket near TM6/7 in both GPR119 and the β1AR. Pharmacological studies suggest that atazanavir mediates non-canonical signaling of Family A GPCRs. During this process, G protein and β-arrestin are spatially close, but β-arrestin forms a complex with the receptor and G protein instead of desensitizing G protein signaling, thus resulting in sustained G protein signaling. The work expands the signal transduction modes and pharmacological properties of allosteric modulators for GPCRs and provides a general starting point for developing therapeutics targeting this allosteric site. ### Competing Interest Statement The authors have declared no competing interest.

There is growing interest in peptide or small protein based drugs targeting G protein-coupled receptors (GPCRs) for improved subtype selectivity over small molecules. Naturally occurring toxins represent rich sources of such ligands. AdTx1 (ρ-Da1a), a three-finger toxin (3FTx) from the green Mamba Snake, selectively binds and antagonizes α-adrenoceptors. Here, we present the cryo-electron microscopy structure of α1A-adrenoceptor in complex with AdTx1. The structure reveals the molecular mechanism of the subtype selectivity and antagonist activity of AdTx1 for α1A-adrenoceptor, which is different from those revealed by the only 3FTx-GPCR structure reported so far, the Muscarinic toxins 7 (MT7) - Muscarinic acetylcholine receptor 1 (M1AChR) structure. Based on the structural information, we further engineered the AdTx1 and enhanced its antagonist activity by introducing three mutations. The results highlight the potential of developing potent toxin drugs towards GPCRs based on the 3FTx scaffold and structural information.

GPCRs signal through both G protein pathways and β–arrestin pathways. Previous work suggests that β–arrestins bind to GPCRs through different modes, including core engagement and tail engagement. Core engagement competes with G proteins and terminates G protein signaling, while tail engagement can coexist with G proteins, mediating sustained intracellular activation of the receptor – a process dependent on the high affinity between β–arrestin and the phosphorylated C-terminus of the receptor. In this study, we determined the structure of a GPCR - G protein - β-arrestin-1 complex stabilized by an allosteric modulator. The compound, atazanavir, acts like molecular glue to anchor β-arrestin-1 to the receptor′s TM6 and TM7 regions. This ′pendulum′binding mode is structurally compatible with simultaneous G protein binding. We further demonstrate that the atazanavir–mediated β–arrestin recruitment does not require the receptor′s C-terminal region. This work illustrates a novel paradigm of GPCR–G protein–β–arrestin1 megacomplex assembly and opens up new avenues for modulating GPCR function. ### Competing Interest Statement The authors have declared no competing interest.

Purinergic P2Y2 receptor (P2Y2R) represents a typically extracellular ATP and UTP sensor for mediating purinergic signaling. Despite its importance as a pharmacological target, the molecular mechanisms underlying ligand recognition and G-protein coupling have remained elusive due to lack of structural information. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of the apo P2Y2R in complex with Gq, ATP-bound P2Y2R in complex with Gq or Go, and UTP-bound P2Y4R in complex with Gq. These structures reveal the similarities and distinctions of ligand recognition within the P2Y receptor family. Furthermore, a comprehensive analysis of G-protein coupling reveals that P2Y2R exhibits promiscuity in coupling with both Gq and Go proteins. Combining molecular dynamics simulations and signaling assays, we elucidate the molecular mechanisms by which P2Y2R differentiates pathway-specific Gq or Go coupling through distinct structural components on the intracellular side. Strikingly, we identify a helix-like segment within the N-terminus that occupies the orthosteric ligand-binding pocket of P2Y2R, accounting for its self-activation. Taken together, these findings provide a molecular framework for understanding the activation mechanism of P2Y2R, encompassing ligand recognition, G-protein coupling, and a novel N-terminus-mediated self-activation mechanism.

Development of subtype-selective drugs for G protein-coupled receptors poses a significant challenge due to high similarity between subtypes, as exemplified by the three β-adrenergic receptors (βARs). The β 3 AR agonists show promise for treating the overactive bladder or preterm birth, but their potential is hindered by off-target activation of β 1 AR and β 2 AR. Interestingly, several β-blockers, which are antagonists of the β 1 ARs and β 2 ARs, have been reported to exhibit agonist activity at the β 3 AR. However, the molecular mechanism remains elusive. Understanding the underlying mechanism should facilitate the development of β 3 AR agonists with improved selectivity and reduced off-target effects. In this work, we determined the structures of human β 3 AR in complex with the endogenous agonist epinephrine or with a synthetic β 3 AR agonist carazolol, which is also a high-affinity β-blocker. Structure comparison, mutagenesis studies and molecular dynamics simulations revealed that the differences on the flexibility of D 3.32 directly contribute to carazolol's distinct activities as an antagonist for the β 2 AR and an agonist for the β 3 AR. The process is also indirectly influenced by the extracellular loops (ECL), especially ECL1. Taken together, these results provide key guidance for development of selective β 3 AR agonists, paving the way for new therapeutic opportunities.

Adiponectin is an important adipokine involved in glucose and lipid metabolism, but its secretion and potential role in regulating glucose utilization during ovarian development remains unclear. This study aims to investigate the mechanism and effects of follicle-stimulating hormones (FSHs) on adiponectin secretion and its following impact on glucose transport in the granulosa cells of rat ovaries. A range of experimental techniques were utilized to test our research, including immunoblotting, immunohistochemistry, immunofluorescence, ELISA, histological staining, real-time quantitative PCR, and transcriptome analysis. The immunohistochemistry results indicated that adiponectin was primarily located in the granulosa cells of rat ovaries. In primary granulosa cells cultured in vitro, both Western blot and immunofluorescence assays demonstrated that FSH significantly induced adiponectin secretion within 2 h of incubation, primarily via the PKA signaling pathway rather than the PI3K/AKT pathway. Concurrently, the addition of the AdipoR1/AdipoR2 dual agonist AdipoRon to the culture medium significantly stimulated the protein expression of GLUT1 in rat granulosa cells, resulting in enhanced glucose absorption. Consistent with these in vitro findings, rats injected with eCG (which shares structural and functional similarities with FSH) exhibited significantly increased adiponectin levels in both the ovaries and blood. Moreover, there was a notable elevation in mRNA and protein levels of AdipoRs and GLUTs following eCG administration. Transcriptomic analysis further revealed a positive correlation between the expression of the intraovarian adiponectin system and glucose transporter. The present study represents a novel investigation, demonstrating that FSH stimulates adiponectin secretion in ovarian granulosa cells through the PKA signaling pathway. This mechanism potentially influences glucose transport (GLUT1) and utilization within the ovaries.

The human organic cation transporter 1 (hOCT1), also known as SLC22A1, is integral to hepatic uptake of structurally diversified endogenous and exogenous organic cations, influencing both metabolism and drug pharmacokinetics. hOCT1 has been implicated in the therapeutic dynamics of many drugs, making interactions with hOCT1 a key consideration in novel drug development and drug–drug interactions. Notably, metformin, the frontline medication for type 2 diabetes, is a prominent hOCT1 substrate. Conversely, hOCT1 can be inhibited by agents such as spironolactone, a steroid analog inhibitor of the aldosterone receptor, necessitating a deep understanding of hOCT1–drug interactions in the development of new pharmacological treatments. Despite extensive study, specifics of hOCT1 transport and inhibition mechanisms remain elusive at the molecular level. Here, we present cryo-electron microscopy structures of the hOCT1-metformin complex in three distinct conformational states — outward open, outward occluded, and inward occluded as well as substrate-free hOCT1 in both partially and fully open states. We also present hOCT1 in complex with spironolactone in both outward and inward facing conformations. These structures provide atomic-level insights into the dynamic metformin transfer process via hOCT1 and the mechanism by which spironolactone inhibits it. Additionally, we identify a ‘YER’ motif critical for the conformational flexibility of hOCT1 and likely other SLC22 family transporters. Our findings significantly advance the understanding of hOCT1 molecular function and offer a foundational framework for the design of new therapeutic agents targeting this transporter.

Prostaglandins and their receptors regulate various physiological processes. Carboprost, an analog of prostaglandin F 2α and an agonist for the prostaglandin F2-alpha receptor (FP receptor), is clinically used to treat postpartum hemorrhage (PPH). However, off-target activation of closely related receptors such as the prostaglandin E receptor subtype EP3 (EP3 receptor) by carboprost results in side effects and limits the clinical application. Meanwhile, the FP receptor selective agonist latanoprost is not suitable to treat PPH due to its poor solubility and fast clearance. Here, we present two cryo-EM structures of the FP receptor bound to carboprost and latanoprost-FA (the free acid form of latanoprost) at 2.7 Å and 3.2 Å resolution, respectively. The structures reveal the molecular mechanism of FP receptor selectivity for both endogenous prostaglandins and clinical drugs, as well as the molecular mechanism of G protein coupling preference by the prostaglandin receptors. The structural information may guide the development of better prostaglandin drugs.

Abstract Background: Adiponectin, also known as Acrp30, GBP28, or apM1, plays a crucial role in regulating glucose levels, lipid metabolism and insulin sensitivity in various tissues and organs. Despite its broad physiological relevance in female reproduction, its role in ovarian adiponectin secretion and energy utilization remains unknown. Methods: In the present investigation, we employed a suite of sophisticated molecular and cellular techniques to elucidate the expression of lipocalin system components and glucose transporter proteins within the ovarian tissue of rats. Specifically, cellular immunofluorescence, RT-PCR, Western blotting, and immunohistochemistry, alongside ELISA, were meticulously applied for the identification of these proteins. Furthermore, granulosa cells, harvested from rat ovaries, were subjected to an in vitro culture protocol to establish a primary cell culture. Results: In our study, we utilized primary cultured rat granulosa cells to demonstrate for the first time that FSH quickly stimulated adiponectin protein expression in granulosa cells, particularly through the PKA signaling pathway. Consistently, further in vivo study indicated that eCG administration significantly increased adiponectin protein levels in rat ovaries. The subsequent ovarian transcriptome and RT-PCR analysis showed a positive correlation between the expression of intraovarian glucose transporters (Slc2a1, Slc2a3, and Slc2a4) and the adiponectin system (Adipoq, Adipor1, and Adipor2), as well as the protein expression patterns. Although eCG administration in rats notably elevated circulating adiponectin levels, it had no significant effects on blood glucose levels. The current findings proved that AdipoRon (an AdipoR agonist) significantly increased the expression of glucose transporters (GLUT1 and GLUT2) proteins levels, enhancing glucose uptake in granulosa cells. Conclusions: These findings indicate that FSH may modulate ovarian glucose transporter protein expression and glucose utilization through the autocrine actions of adiponectin, thereby influencing ovarian development and function.

Effects of some important structural parameters,i.e.slat pitch,and layout position,on dynamic forces acting on the baffles were examined in the fluidized bed of FCC particles operating under different super-ficial gas velocities.The experimental baffles were made of multiple inclined slats.We found that the forces acting on the baffles decreased significantly with reducing pitch between the slats.For the baf-fles with a small slat pitch,the forces acting on the baffles increased slightly and then decreased with increasing superficial gas velocity,which is very different from the measured results of a single slat or tube immersed in fluidized beds.The different results are greatly related to the appearance of the "gas cushion" beneath the baffles,whose height increases with increasing superficial gas velocity.On the other hand,a region with stronger particle circulation induced by the inclined slat array was observed in the experiments.The slat near the wall and located below the region of downward-flowing particles was found to be subjected to the severest forces.Therefore,the slats located in similar locations of industrial baffles are suggested to be reinforced to increase their structural strength.

Although there has been enormous progress in the last half-century in the drug discovery targeting obesity and associated co-morbidities, the clinical treatment of obesity remains tremendously challenging. GPR75 is an orphan receptor and is suggested to be a potential novel target for the control of obesity and related metabolic disorders. Inhibition of the GPR75 signaling pathway by small molecules, antibodies, or genetic manipulations may provide a therapeutic strategy for obesity. Here, we report the active-like Cryo-EM structure of human GPR75 with an intracellular nanobody, which reveals the receptor activation mechanism. The extensive interaction network required to achieve the active structure helps explain the allosteric coupling between the orthosteric pocket and the G-protein coupling domain. The well-defined orthosteric ligand binding pocket of human GPR75 provides a structural basis for anti-obesity drug discovery.

To satisfy the ever-increasing demands for high-performance ceramic foams that could be applied in catalysts loader, filtrations, and adsorptions, it is critical to develop technologies for ceramic foams with open-channel structure. In this work, we fabricated ceramic foams with a three-dimensional porous structure especially with open channels by combining the direct foaming method with adding a pore-forming agent method. There are two levels of length scale present in this hierarchically porous structure, that is, foam structure with spherical pores evolved from bubbles, and open pores on the cell wall derived from silica hollow spheres with a thin shell as the pore-former. Hierarchical ZrO2 based foams with a porosity of 86.5%-95.1% and compressive strength of 2.05–5.67 MPa and Al2O3 based foams with a porosity of 86.2%-91.0% and compressive strength of 6.8–13.2 MPa were fabricated. The prepared ceramic foams characterized by this open-channel structure are promising to perform outstandingly in the abovementioned fields due to their uniform pore size, low density, as well as high mechanical strength.

Forces acting on a horizontal slat immersed in a fluidized bed during its start-up stage were investigated systematically. Both stress and pressure signals were measured by using strain gauges and pressure transducers to describe the forces and their relationship with gas-solids hydrodynamics. The main influencing parameters are the increment of superficial gas velocity, installation height, inclination angle, static bed height, and installation method of the slat. The experimental results indicated that there is a dangerous upward impulse with a high peak and a long duration in the measured stress signal during starting up a fluidized bed. The effects of various operating and structural parameters on the forces acting on the test slat were elucidated in detail. Effective measures to alleviate the forces and critical conditions where the strength of internals needs to be re-enforced are proposed. The obtained knowledge is helpful to engineering design to ensure the safety of immersed internals in fluidized bed reactors.

We firstly fabricated CeO2 ceramic foams with tunable structure by using particle-stabilized bubbles as template, and designed their interconnected porous structure and even hierarchically porous structure, which endows them the penetration ability for gases or liquids. Hollow spheres with single-layer shell were innovatively selected as the pore-former, which allows for the formation of open pores on the cell wall. Moreover, 3D printing CeO2 particle-stabilized foams are realized with the aid of direct ink writing, which enables the production of CeO2 ceramic foams with complex shape. Highly porous CeO2 with relatively high compressive strength have been fabricated, the porosity of which varies from 81.0% to 92.0% while their excellent compressive strength ranges from 5.0 MPa to 20.0 MPa. Attributed to the hierarchical porous structure, uniform pore size distribution as well as densely assembled cell wall, 3D printing CeO2 ceramic foams possess superior mechanical performance at high porosity level.