Biology subjects

Inoue, A.

Publications and source records attributed to Inoue, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Comprehensive dissection of GPCR signaling using a NanoBiT-based platform

G-protein-coupled receptors (GPCRs) signal through multiple heterotrimeric G proteins, {beta}-arrestins, GPCR kinases (GRKs), and downstream effectors, whose combinatorial interactions shape cellular responses. These events are typically measured with separate assay formats that each capture only part of the network, making comparison across signaling layers difficult. Here, we consolidate a broad set of previously reported GPCR signaling interactions and assay concepts, together with newly designed sensors, into a single NanoBiT split-luciferase framework, allowing multiple layers of signal transduction to be examined side by side in living cells. We show that rational sensor engineering, in particular the positioning of NanoBiT fragments and targeted modification of the tagged proteins, is essential for detecting transient protein-protein interactions. The framework implements assays for G-protein dissociation, {beta}-arrestin recruitment, conformational activation and trafficking, and GRK recruitment, and extends to G-protein-effector and inter-effector interactions across diverse G, G{beta}, G{gamma}, {beta}-arrestin, GRK, adenylyl cyclase, PLC{beta}, and RhoGEF subtypes. It also enables real-time monitoring of the difficult-to-access G12/13-RhoGEF-RhoA pathway. Together, these assays provide a unified NanoBiT readout for systematic, side-by-side dissection of GPCR signaling.

pharmacology and toxicology↗

bioRxiv2026

Minimally invasive measurement of maternal transcripts enables predicting the developmental potential of mammalian zygotes

Maternal transcripts are stored in the oocyte cytoplasm during oogenesis and play a pivotal role in early embryonic development after fertilization. However, specific maternal transcripts that reflect the developmental potential of embryos have not been systematically identified, and the use of maternal transcript levels as an indicator of successful development has not been explored. Here, we link the maternal transcriptome to the zygotes developmental potential by examining transcripts in a single polar body. The transcriptome of a zygote or an oocyte was highly similar to that of its accompanying polar body in mouse, cow, and human. We have identified a set of maternal transcripts whose expression levels fluctuate between poor- and good-quality zygotes. Specifically, Sipa1and Zmym6 were identified as marker transcripts that accurately reflect the developmental potential of zygotes. Using these marker genes, combined with machine learning, the development of zygotes to the blastocyst stage was successfully predicted with more than 80% specificity as early as 12 hours after fertilization. Furthermore, our prediction platform significantly improved implantation rates and live births to term. Thus, we have demonstrated a minimally invasive method for identifying maternal transcripts associated with zygote developmental potential. Our developed prediction system provides a generalizable conceptual framework for human infertility treatment to reduce the risk of implantation failure by excluding embryos with low developmental potential, especially when early embryos are transferred, and for livestock propagation to assess selected expressed maternal trait-associated variants before embryo transfer.

developmental biology↗

bioRxiv2026

Active repression of muscle fate preserves neural lineage identity during cerebellum development

Cell fate commitment is commonly thought to entail progressive restriction of developmental potential, enforced by passive, heterochromatin-based silencing of alternative lineage programs. Here we show that maintenance of neural identity during cerebellum development instead requires active repression of a starkly divergent fate by the TEAD-INSM1 transcriptional complex. Loss of TEAD1/2 or INSM1 activates the myogenic master regulator Myod1, resulting in neural cells acquiring transcriptional, structural, and metabolic features of skeletal muscle cells. Deletion of Myod1 fully suppresses neural-to-muscle conversion while partially rescuing neural developmental defects. Our results uncover a latent alternative lineage during neurodevelopment and a surprising role for sequence-specific transcription factors in enforcing lineage boundaries, including those previously thought essentially unbreachable, with implications for understanding aberrant differentiation in disease contexts and cell-type evolution.

developmental biology↗

bioRxiv2026

A surrogate marker of protection confirms the efficacy of an AddaS03-adjuvanted West Nile virus subunit vaccine

West Nile virus (WNV) is the causative agent of fatal West Nile encephalitis. To date, no human vaccine against WNV has been approved. Adjuvants are important for developing effective and affordable vaccines that enhance the immunogenicity and decrease the required antigen doses. In this study, we assessed the efficacy of AddaS03, a synthetic adjuvant analogous to AS03, in a WNV subunit vaccine composed of soluble recombinant envelope protein (sEnv). Using a passive immunization mouse model, we defined the neutralizing antibody titer threshold required for protection against lethal WNV infections and applied this threshold as a surrogate marker to evaluate adjuvant efficacy. AddaS03-adjuvanted formulations elicited markedly higher neutralizing antibody titers compared to Alhydrogel adjuvant 2% (Alhydrogel), even at suboptimal antigen doses, and consistently exceeded the defined protective threshold titer. Moreover, in a sequential challenge mouse model, AddaS03-adjuvanted vaccines completely protected mice from symptomatic WNV infections, whereas Alhydrogel-adjuvanted vaccines failed to confer full protection. Collectively, these findings demonstrate that AddaS03 is a promising adjuvant for WNV subunit vaccine development and highlights the utility of a passive immunization model for defining protective antibody thresholds as a surrogate marker for vaccine evaluation.

microbiology↗

bioRxiv2026

Efficient generation of hematopoietic progenitor cells from human pluripotent stem cells by robotic automation

Differentiation of human pluripotent stem cells (hPSCs) into therapeutic cell types requires stringent control of developmental signals, but conventional manual operations introduce substantial intra- and inter-operator variability. While automation can reduce such variability, fixed systems often lack compatibility with the dynamic, stage-specific, and format-diverse manipulations required for hPSC culture and differentiation. Here, we establish a flexible robotic platform to standardize a stroma- and xeno-free embryoid body (EB)-based hematopoietic progenitor cell (HPC) differentiation process and integrate machine learning (ML) to optimize key process parameters including the concentrations of multiple signaling molecules. Despite intrinsic biological stochasticity and technical complexity associated with EB formation, the robotic platform substantially reduced experimental variation. ML-guided, unbiased exploration not only identified combinations of signaling inputs that markedly improved the efficiency and reproducibility of HPC differentiation and subsequent natural killer cell generation, but also illuminated key signaling logic underlying early human hematopoietic development. Together, these results demonstrate that data-driven robotic automation can uncover optimal culture conditions that are difficult to identify through conventional human-driven experimentation.

developmental biology↗

bioRxiv2026

Integrated in vivo and transcriptomic analyses of lethal Oropouche virus infection reveal suppression of pathogenic host responses by antiviral therapy

Oropouche virus (OROV) is an emerging arbovirus responsible for large outbreaks of febrile illness in Central and South America, with increasing reports of severe neurological disease and fatal outcomes. Despite its growing public health impact, no approved antiviral therapies or vaccines are currently available. Here, we show that favipiravir, a broad-spectrum nucleoside analogue, robustly suppresses OROV replication and disease in vivo. In a lethal Syrian hamster model, favipiravir treatment provided complete protection against OROV infection, preventing viral dissemination to peripheral organs and the central nervous system, and remained highly effective when administration was initiated after infection. In contrast, insufficient antiviral control resulted in viral neuroinvasion and fatality. To define host responses associated with OROV pathogenesis and their modulation by antiviral therapy, we performed transcriptomic profiling of liver and brain tissues. OROV infection induced interferon-driven inflammatory programs accompanied by marked disruption of metabolic and tissue homeostatic pathways, whereas these transcriptional signatures were largely abrogated by favipiravir treatment. Together, our findings identify favipiravir as a potent antiviral candidate against OROV and provide the first in vivo, tissue-resolved transcriptomic framework of OROV infection, linking effective viral suppression with the prevention of neuroinvasion and pathogenic host responses. These results highlight antiviral intervention as a viable strategy to mitigate OROV-associated disease and mortality.

microbiology↗

bioRxiv2026

Computed atlas of the human GPCR-G protein signaling complexes

Experimental mapping of G protein-coupled receptors (GPCR)-G protein signaling coupling has illuminated hundreds of receptors, yet the coupling specificity of a large fraction of this large receptor family remains unknown, thereby preventing the development of new GPCR-targeting therapies. Here, we used AlphaFold3 (AF3) to predict the 3D structures of the human GPCRome in complex with heterotrimeric G proteins. We used experimental GPCR-G protein binding data to show that AF3 predictions significantly discriminate between positive and negative binders, and used 3D structural features to train a machine learning (ML) algorithm to predict coupling potency. Interpretation of the ML model helped discriminate universal features governing the strength of G protein coupling from those determining binding specificity. We computationally illuminated the coupling preferences of 180 non-olfactory GPCRs (non-OR) with previously unreported transduction mechanisms and experimentally validate the predicted couplings for multiple previously uncharacterized GPCRs, including QRFPR, GPR50, GPR37, GPR37L1 and GPRC5A. Our predictions established that Gi/o is the most prevalent coupling among non-OR GPCRs, which is often co-occurring with Gq/11 and, to a lesser extent, G12/13 signaling. Gs coupling is less common and restricted to specific clusters within the non-OR GPCRome phylogeny, likely due to stricter structural requirements for its binding. We also computed G protein complexes for over 400 ORs, establishing Gs as the most prevalent coupling. ORs are predicted to bind to Gs with a simpler interface compared to non-ORs, ultimately leading to energetically less stable complexes. Additionally, we predict recurrent bindings to Gq/11 and Gi/o proteins for ORs, suggesting potentially novel ORs signaling mechanisms. We exploited the GPCRome coupling atlas to interpret healthy and cancer expression data, revealing the coupling of most GPCR-G protein co-expressed pairs. This analysis highlights a richer coupling repertoire in healthy tissues compared to cancer, likely reflecting the high signaling requirements of specialized normal cell functions, which are lost in most cancer cells due to their de-differentiated state or under cancer selection processes. In summary, this study provides the first computational 3D atlas of the human GPCR-G protein transductome, thereby illuminating the signaling mechanisms of neglected GPCR classes and providing the basis for interpreting omics datasets from a myriad of pathological conditions, thus enabling the development of novel precision therapeutics.

bioinformatics↗

bioRxiv2026

Cytoplasmic mRNA granules regulate cell fate decisions during PINK1/Parkin mitophagy

Mitophagy is generally considered to promote cell survival by removing damaged mitochondria in response to mitochondrial stress, whereas apoptosis occurs during prolonged stress. However, the mechanisms that determine cell survival and cell death under these stress conditions remain poorly understood. Here, we showed that cytoplasmic mRNA granules, designated as mitophagy-induced mRNA granules (mitoRGs), were formed transiently and played an important role in cell fate decisions during PINK1/Parkin-dependent mitophagy. Although some components, such as G3BP1, were shared with stress granules (SGs), mitoRGs were distinct from SGs because mitoRG assembly required the mitochondrial protein phosphatase PGAM5. In response to mitochondrial stress, PGAM5 was released into the cytosol from mitochondria and incorporated into mitoRGs, but was then released back into the cytosol during mitoRG disassembly following prolonged mitochondrial stress, corresponding with the induction of apoptosis. Impairment of mitoRG assembly through G3BP1 depletion sensitized cells to apoptosis during mitophagy in a PGAM5-dependent manner. These results suggest that mitoRGs regulate cell fate decisions by spatiotemporally controlling PGAM5 and its pro-apoptotic activity during PINK1/Parkin mitophagy.

cell biology↗

bioRxiv2026

A lethal mouse model of Oz virus infection reveals hepatic involvement and enables evaluation of antiviral and vaccine efficacy

Oz virus (OZV), a member of the genus Thogotovirus in the family Orthomyxoviridae, is an emerging tick-borne virus reported in Japan. A fatal human case and seroepidemiological evidence of widespread exposure among wild animals and humans suggest its potential public health significance. However, no animal models suitable for pathogenic studies or evaluation of countermeasures are available for OZV. Here, we have established a lethal mouse model of OZV infection using cell-adapted virus and mice lacking type I interferon signaling (B6 Ifnar1 KO mice). OZV infection resulted in 100% mortality and was characterized by robust viral replication in the liver and spleen, severe hepatitis, and acute liver injury. Using this model, we also demonstrated that oral administration of T-705, an antiviral drug widely used against RNA viruses, as well as immunization with an inactivated whole virus particle vaccine, protected B6 Ifnar1 KO mice from lethal OZV infection by mitigating the acute hepatitis. The present study provides critical insights into OZV pathogenesis and establishes a practical in vivo platform for the development of countermeasures against OZV infection. Significance statementEmerging tick-borne viruses pose a growing public health concern, yet progress in understanding their pathogenesis and in developing countermeasures is often limited by the lack of relevant animal models. Oz virus (OZV), a recently identified thogotovirus associated with a fatal human case, exemplifies this challenge. Here, we establish a lethal mouse model of OZV infection that reveals pronounced hepatic involvement as a central pathological feature. Using this model, we demonstrate effective protection by both an antiviral drug and an inactivated vaccine against lethal OZV challenge. This study provides a practical in vivo platform for investigating OZV pathogenesis and for accelerating the development of medical countermeasures against this emerging tick-borne virus.

microbiology↗

bioRxiv2026

Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation

Phospholipase C {beta} (PLC{beta}) enzymes are activated by heterotrimeric G protein subunits, increasing hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. All four human PLC{beta} isoforms (PLC{beta}1-4) are activated by Gq, while PLC{beta}1-3 are activated to varying extents by G{beta}{gamma}. The binding sites for Gq on PLC{beta} are well-established and much has been learned about its mechanism of activation, but comparatively little is known about G{beta}{gamma}-dependent activation. In this work, we used cryo-electron microscopy (cryo-EM) single particle analysis (SPA), functional assays, and bioluminescence resonance energy transfer (BRET) to investigate how G{beta}{gamma} interacts with PLC{beta}3 in concert with activated Gq to regulate phospholipase activity. G{beta}{gamma} heterodimers bind multiple surfaces of PLC{beta}3 to promote activation but alone do not recruit the enzyme to the plasma membrane. Instead, G{beta}{gamma} facilitates activation by Gq, most likely by reorienting the phospholipase catalytic site at the membrane to maximize PI(4,5)P2 hydrolysis and downstream Ca2+ release. Cell-based functional assays demonstrate that G{beta}{gamma} is required for maximal PLC{beta}3 activation even when Gq heterotrimers are the sole source of G{beta}{gamma}. Together, these findings demonstrate that G{beta}{gamma} acts as a critical positive allosteric modulator that regularly acts in concert with Gq to activate PLC{beta}3 at the plasma membrane.

biochemistry↗

bioRxiv2025

Single-molecule behavior and cell-growth regulation in human RTKs

Receptor tyrosine kinases (RTKs) are a major family of cell surface receptor proteins responsible for various cellular functions in animal cells, including fate decisions, metabolism, polarization and migration. Lateral mobility, dimerization, clustering, and oligomerization are crucial behaviors in the activation process of RTKs on the cell surface. However, relationships between these molecular behaviors and molecular function remain to be elucidated, except for a few RTK members. Here, using an automated live-cell single-molecule imaging and analysis system, we studied the behavior of 52 of the 58 human RTK species on living cells over time during stimulation with ligands. We extracted 72 single-molecule parameters for each RTK species to examine their relationship to function, structure, and evolution. We noticed that RTKs ability to inhibit or support cell growth, as observed in a large-scale loss-of-function experiment in the public domain, significantly relates to their behavior. Growth-inhibitory signaling was coupled with the immediate formation of immobile clusters, followed by the enlargement of immobile and slow-mobile domains. In contrast, growth-supportive signaling coupled with higher lateral diffusivity and delayed clustering of immobile molecules. The relationship between structure and function suggests that functional differences are related to partitioning into membrane rafts and changes in mobility associated with phosphatidylinositol turnover. In multiple linear regression models, molecular behavior explained half or more of the molecular function related to cell growth. This level of explainability is comparable to that of evolutionary grouping.

biophysics↗

bioRxiv2025

Structural basis of stepwise G protein activation by a viral chemokine receptor

G protein-coupled receptors (GPCRs) govern diverse cellular responses and are crucial drug targets. However, the sequence of structural events from G protein recognition to GDP release has remained elusive. Here, we leveraged the viral chemokine GPCR US28 to capture multiple activation states of the US28-Gq complex. Using cryo-electron microscopy and an engineered chemokine superagonist, we determined three distinct complex structures, capturing a GDP-bound Encounter state, the nucleotide-free Canonical state, and a putative intermediate bridging the two states, the Transition-to-Canonical state. These structures, along with simulations and functional data, provide high-resolution snapshots of a plausible G protein activation trajectory and support a stepwise conformational model for G protein activation. This activation cascade closely parallels mechanisms proposed for human GPCRs, suggesting a conserved GPCR signaling mechanism.

biophysics↗

bioRxiv2025

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2025

Noradrenaline and microglia maintain plasticity-rigidity balance to safeguard rapid emotional learning in the prefrontal cortex

Synaptic plasticity is governed by rules that determine how patterns of neural activity are converted into changes in synaptic weights. While neocortical plasticity rules appear to shift from Hebbian to three-factor forms during development, the cellular mechanisms underlying this transition remain unclear. Here, we show that microglia mediate the developmental transition of plasticity rules at individual dendritic spines in the medial prefrontal cortex (mPFC) during juvenile-to-adolescent maturation. After maturation, microglia suppressed Hebbian activity-induced spine enlargement via a humoral factor. This suppression was relieved by noradrenaline through microglia-intrinsic cAMP signaling, thereby enabling a three-factor rule for gating plasticity. The adolescent mPFC required three-factor plasticity signaling for the acquisition of socially learned fear. Local microglial ablation enhanced learning efficiency but also altered pain sensitivity in a learning-dependent manner, suggesting that microglial suppression safeguards pre-existing emotional circuits. Together, these findings identify microglia as critical regulators of developmental plasticity-rule transitions in the mPFC.

neuroscience↗

bioRxiv2025

Membrane phosphoinositides allosterically tune β-arrestin dynamics to facilitate GPCR core engagement

Arrestin proteins bind active G protein-coupled receptors (GPCRs) through coordinated protein-protein, protein-phosphate, and protein-lipid interactions to attenuate G protein signaling and promote GPCR internalization and trafficking. While there are hundreds of diverse GPCRs, only two {beta}-arrestin isoforms ({beta}arrs) must recognize and engage this wide range of receptors with varied phosphorylation patterns. Traditional models suggest that {beta}arr activation requires displacement of its autoinhibitory C-tail by a phosphorylated GPCR C-terminus; however, this paradigm fails to explain how minimally phosphorylated GPCRs still complex with {beta}arrs. Using single-molecule Forster resonance energy transfer imaging and hydrogen-deuterium exchange mass spectrometry, we observe basal dynamics in which the {beta}arr1 C-tail spontaneously releases from the N-domain, transiently adopting an active conformation that can facilitate binding of the phosphorylated GPCR C-terminus. We further demonstrate the importance of an intermediate state of {beta}arr1 arising from spontaneous C-tail release stabilized by the membrane phosphoinositide PI(4,5)P2. Both PI(4,5)P2 and mutations in the proximal or middle regions of the C-tail shift {beta}arr1 towards a partially released state, revealing an allosteric connection that informs a refined model for {beta}arr activation. In this model, membrane engagement conformationally primes {beta}arrs prior to receptor binding, thereby explaining how {beta}arrs are recruited by diverse GPCRs, even those with limited C-terminal phosphorylation.

biophysics↗

bioRxiv2025

Antimuscarinic drugs exert β-arrestin-biased agonism at the muscarinic acetylcholine type 1 receptor

Previous studies indicate that both pirenzepine (PZ), a selective orthosteric muscarinic acetylcholine type 1 receptor (M1R) antagonist, and muscarinic toxin 7 (MT7), a negative M1R allosteric modulator (NAM), act via M1R to promote neuritogenesis in cultured adult rodent primary dorsal root ganglia (DRG) sensory neurons, in part, through {beta}-arrestin-dependent activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Furthermore, these antagonists reverse nerve degeneration in a variety of rodent models of peripheral neuropathy through multiple complementary pathways. To understand the therapeutic effects and mechanism of M1R antagonist-induced ERK1/2 phosphorylation, we tested the hypothesis that PZ and MT7 possess {beta}-arrestin-biased agonism at M1R to drive activation of ERK and enhance neurite outgrowth. Treatment for up to 30 min with PZ and MT7 dose-dependently recruited {beta}-arrestin2 to M1R (analyzed using nano-BRET) and increased ERK phosphorylation in both HEK293 cells and DRG neurons. DRG neurons of different sub-types express M1R, and ERK activation by MT7 was only observed in M1R-positive neurons. These novel pharmacological effects occurred in the absence of activation of G protein signaling or receptor internalization. PZ phosphorylated M1R at six specific serine/threonine residues (T230, S251, T254, S321, T354, S356) of intracellular loop 3 (ICL3) and deletion mutation of these sites suppressed PZ and MT7 induction of {beta}-arrestin binding to M1R and inhibited ERK activation. With regard to PZ signaling, alanine substitution at S251 and T254 was sufficient to impede {beta}-arrestin binding and ERK activation. {beta}-arrestin-biased activity of PZ and MT7 involved the mobilization of casein kinase 2 (CK2) and this occurred in the absence of Gq or G protein receptor kinase (GRK) activity. Pharmacological or siRNA-based inhibition of CK2 blocked PZ-induction of {beta}-arrestin association, ERK activation and neurite outgrowth in DRG neurons. In conclusion, PZ/MT7 activated M1R toward the {beta}-arrestin signaling pathway in both HEK293 cells and DRG neurons to augment ERK activation and neurite outgrowth via engagement of CK2. One-sentence summaryAntimuscarinic drugs act as {beta}-arrestin-biased agonists via casein kinase 2 activation to promote ERK1/2 phosphorylation and neurite outgrowth O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/649213v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1bb8da7org.highwire.dtl.DTLVardef@510218org.highwire.dtl.DTLVardef@608d69org.highwire.dtl.DTLVardef@e40d9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Schematic presentation of the effect of muscarinic ligands at M1R associated signaling pathway. (A) Muscarine/carbachol acts as a balanced ligand by engaging both Gq and -arrestin signaling pathways and treatment with pirenzepine/MT7 blocks these effects. (B) Pirenzepine/MT7 acts as a -arrestin biased ligand by 1) phosphorylating of ICL3 region of M1R via CK2 (but not GRKs), 2) no activation of G protein signaling, 3) recruitment of -arrestin 2 and 4) ERK1/2 activation leading to neurite outgrowth in DRG sensory neurons. This figure was generated by BioRender under license number EK285MUOPQ. C_FIG

pharmacology and toxicology↗

bioRxiv2025

FAST RESENSITIZATION OF G PROTEIN-COUPLED RECEPTORS REQUIRESTHEIR PI(4,5)P2-DEPENDENT SORTING INTO AN AP2 POSITIVE COMPARTMENT

G protein coupled receptors (GPCRs) are the target of about 35% of FDA-approved drugs, which underlines their importance in physiology and disease. Many GPCRs undergo rapid desensitization upon agonist activation, mediated by {beta}-arrestin binding to the phosphorylated receptors, which prevents their G protein coupling. How such receptors regain their G protein signaling competence is poorly understood. Using the AT1 angiotensin II receptor (AT1R), we show that its rapid re-sensitization requires the sorting of the receptors into an AP2-positive plasma membrane (PM) compartment without receptors having to be internalized. This process requires the scaffolding protein, EFR3 and a dedicated PI(4,5)P2 pool specifically produced by PIP5KA. While {beta}-arrestin 1 and -2 both can carry the receptors to the AP2 compartment, weaker binding of {beta}-arrestin 1 to the receptor allows some of the receptors to re-sensitize, while strong {beta}-arrestin 2 binding elicits stronger desensitization and directs the receptors for internalization. These results suggest that the rapid phase of re-sensitization of GPCRs occurs without their endocytosis and primarily takes place at the PM at specific steps during clathrin-coated pit maturation. Together with differences observed between the two {beta}-arrestins to interact with CCPs and the receptors, our data suggest that specific PI(4,5)P2 pools controlled by EFR3A and PIP5K1A determine the balance between {beta}-arrestin1 and -2 receptor interaction and delivery to the AP2 positive compartment, ultimately determining what fraction of the receptors regain their G protein signaling competence.

biochemistry↗

bioRxiv2025

Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and the most common pharmaceutical drug target. Through allosteric coupling, GPCRs transduce extracellular stimuli into physiologically-relevant intracellular signaling cascades via G proteins and {beta}-arrestin ({beta}arr). The growth hormone secretagogue receptor (GHSR) is a rhodopsin-like, peptide hormone GPCR considered a promising target for both metabolic and neurological diseases. Here, by characterizing an ultra-rare coding variant in intracellular loop-2 (ICL2) of the GHSR, GHSRL149P (L149P), we establish a unique role of ICL2 in GPCR biased signaling, lipid modulation, and intracellular trafficking. Using an array of bioluminescence resonance energy transfer (BRET)-based assays, we show that the natural L149P mutant exhibits (i) a constitutive plasma membrane [PM] expression bias, (ii) preferential partitioning away from cholesterol-enriched PM microdomains, (iii) enhanced agonist-directed endocytosis, and (iv) dramatic signaling bias towards {beta}arr1/2 over Gq, Gi/o, and G12/13. Using a combination of pharmacological and genetic tools, we demonstrate that {beta}arr1/2 recruitment to L149P requires G protein-coupled receptor kinase-2/3 (GRK2/3)-mediated phosphorylation, but it does not utilize protein kinase C (PKC), G{beta}{gamma}-dependent GRK2/3 translocation, or Gi/o, supporting a G protein-independent mechanism. Lastly, we found that {beta}arr1/2 recruitment to L149P requires both GRK2/3 and GRK5/6, while the wild-type GHSR relies exclusively on GRK2/3, consistent with increased GRK6 pre-coupling to the L149P mutant. Collectively, our findings using a rare, natural variant reveal novel mechanisms of GPCR regulation that could be leveraged to improve personalized medicine and facilitate the rational design/discovery of GPCRICL2-directed drugs. SIGNIFICANCE STATEMENTG protein-coupled receptors (GPCRs) are the largest class of membrane proteins and the most prevalent drug target class in medicine. However, the structural and mechanistic determinants of GPCR signaling and membrane compartmentalization remains incompletely understood. Here, we characterize an ultra-rare natural variant of the growth hormone secretagogue receptor (GHSR), L149P, that uncovers an unanticipated role for intracellular loop 2 (ICL2) in receptor biased signaling, intracellular trafficking, and membrane lipid modulation. The variant exhibits a strong bias for {beta}-arrestin signaling independent of canonical G protein pathways, mediated instead by unique GPCR kinase interactions and a distinct subcellular distribution. These findings reveal previously unrecognized regulatory layers in GPCR structure/function and identify ICL2 as a promising target for designing precision pharmacotherapeutics.

biochemistry↗