Biology subjects

Schihada, H.

Publications and source records attributed to Schihada, H..

10 recordsLinked to original sources

bioRxiv2026

Subcellular pharmacology of a novel cell-permeable fluorescent phosphodiesterase inhibitor using advanced fluorescence microscopy

Direct visualisation of drug-target engagement within living cells remains a major challenge. Here we develop a cell-permeable fluorogenic analogue of the phosphodiesterase 4 (PDE4) inhibitor rolipram (MAN193), generated by conjugation to fluorescein diacetate. Following intracellular activation, the probe functions as a near-neutral PDE4 antagonist and enables direct visualisation of endogenous PDE4 populations. Using advanced fluorescence spectroscopy imaging approaches, including molecular brightness analysis and fluorescence anisotropy imaging, we demonstrate rolipram-displaceable binding of the analogue to cytosolic PDE4 and resolve thesubcellular distribution of binding sites. We further show that the probe enables visualisation of untagged PDE4 at focal adhesion complexes in cardiomyocyte-like cells. Together these findings establish fluorogenic drug conjugation combined with quantitative spectroscopy imaging as a generalisable strategy to map intracellular drug-target engagement with subcellular resolution, providing an effective framework for interrogating the spatial pharmacology of small molecules in living systems.

biophysics↗

bioRxiv2026

Functional diversity of GPCR-Gustducin complexes controls signaling output and suppresses alternative pathways

Gustducin (Ggust), the G protein mediating taste receptor signaling, is expressed not only in sensory taste cells but throughout the human body alongside numerous non-taste G protein-coupled receptors (GPCRs). Whether and how these receptors engage Ggust, and the functional consequences of such interactions, remain poorly understood. Here, we developed two complementary biosensors that enable direct monitoring of GPCR-Ggust interaction and downstream signaling in living cells, allowing pharmacological characterization of Ggust activity by non-taste GPCRs. Unexpectedly, we find that several non-taste GPCRs do not activate Ggust but instead stabilize the G protein heterotrimer and suppress basal signaling by forming unproductive GPCR-G protein complexes, whereas others induce conventional Ggust activation and signal transduction. We further show that these unproductive GPCR-Ggust complexes suppress the activation of alternative signaling pathways by sequestering receptors from other G proteins. This previously unrecognized mode of interaction functionally partitions GPCRs into activators and inactivators of Ggust and represents a mechanism for balancing concurrent signaling pathways within a cell. HighlightsO_LIDevelopment of biosensors for gustducin activation and signal transduction C_LIO_LIAnalysis of single-cell co-expression data of non-tastant GPCRs and gustducin C_LIO_LIProfiling of non-tastant GPCR-induced gustducin activation and signal transduction C_LIO_LIProductive and unproductive GPCR-gustducin complexes revealed C_LIO_LIUnproductive GPCR-gustducin complexes suppress alternative signaling pathways C_LI

cell biology↗

bioRxiv2023

Cyclic peptide inhibitors stabilize Gq/11 heterotrimers

Heterotrimeric G proteins play a central role in cellular signaling, acting as switchable molecular regulators. Consequently, pharmacological agents to control G protein activity are of utmost importance to advance our understanding of this signal transduction system. The natural depsipeptides FR900359 (FR) and YM-254890 (YM) are two highly specific and widely used inhibitors of heterotrimeric Gq/11 proteins. These compounds have traditionally been understood to inhibit GDP dissociation by preventing the separation of the GTPase and -helical domains of the G subunit. In this work, we have determined the high-resolution crystal structures of FR and YM bound to heterotrimeric G11 and used them to explain the molecular basis underlying their efficient suppression of G protein signaling. Notably, our data show that FR and YM also function as stabilizers of the interface between the G and G{beta} subunits, acting as molecular adhesives that stabilize the entire heterotrimer. Our results reveal unrecognized mechanistic features that explain how FR and YM effectively blunt Gq/11 signaling in living cells.

biochemistry↗

bioRxiv2023

Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

molecular biology↗

bioRxiv2023

Fluorescent Tools for Imaging and Ligand Screening of Dopamine D2-Like Receptors

The family of dopamine D2-like receptors represent an interesting target for a variety of neurological diseases, e.g. Parkinsons disease (PD), addiction or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2-like receptors (pKi (D2longR) = 8.24, pKi (D3R) = 8.58, pKi (D4R) = 7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligands binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups.

molecular biology↗

bioRxiv2023

Cryo-EM structure of cell-free synthesized human histamine H2 receptor coupled to heterotrimeric Gs protein in lipid nanodisc environment

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 [A]. The complex was generated by cotranslational insertion into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. It is the first structure obtained by this detergent-free strategy and the first GPCR/Gs complex structure in lipid environment. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

molecular biology↗

bioRxiv2022

WNT stimulation induced conformational dynamics in the Frizzled-Dishevelled interaction

Frizzleds (FZD1-10) are G protein-coupled receptors containing an extracellular cysteine-rich-domain (CRD) that presents the orthosteric binding site of the 19 mammalian WNTs, the endogenous agonists of FZDs. FZDs signal via a diverse set of effector proteins, of which Dishevelled (DVL1-3) is the most well studied and which acts as a hub for several FZD-mediated signaling pathways. However, the mechanistic details of how FZD-DVL interaction mediate pathway initiation and provide pathway selectivity remain an enigma. Here, we use bioluminescence resonance energy transfer-based assays employing Venus-tagged DVL2 together with NanoLuciferase-tagged FZD5 to investigate the WNT-3A- and WNT-5A-induced dynamics of the FZD-DVL interaction. Our biophysical assessment suggests that the ligand-induced BRET changes over time originate from conformational dynamics in the FZD5-DVL2 complex rather than recruitment dynamics of DVL2 to FZD5. Thus, we suggest that extracellular agonist and intracellular transducers could cooperate with each other through allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs. One Sentence SummaryAnalysis of the interaction of FZD5 and DVL2 uncovers WNT-induced conformational dynamics of a WNT-FZD5-DVL2 complex.

pharmacology and toxicology↗

bioRxiv2021

Functional modulation of PTH1R activation and signalling by RAMP2

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered {beta}-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

pharmacology and toxicology↗

bioRxiv2021

Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/{beta}-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

cell biology↗

bioRxiv2021

Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors

Heterotrimeric G proteins constitute the primary transducers of G protein-coupled receptor (GPCR) signaling. Besides mediating ligand-induced GPCR activation, G proteins transduce basal levels of activity in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Several generations of optical biosensors were developed and optimized to monitor GPCR ligand-induced G protein activation, however, quantitative approaches to detect constitutively active GPCRs are not available. Here, we designed and validated a set of eight bioluminescence-resonance-energy-transfer (BRET)-based G protein sensors, covering all four major families of G proteins, and established a protocol to identify constitutive GPCR/G protein signaling in living cells. These sensors rely on the encoding of all three G protein subunits on a single plasmid, enabling their cellular expression at desired relative levels and resulting in reduced signal variability in mammalian cells. Based on this sensor platform, we further present here an experimental protocol to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will aid in the characterization of constitutively active GPCRs and the exploration of their role in health and disease. One Sentence SummaryThis Resource article describes the validation of a biophysical approach to directly assess the constitutive signaling activity of G protein-coupled receptors through heterotrimeric G proteins in living cells using optical biosensors.

cell biology↗