33 results on '"Schihada, Hannes"'
Search Results
2. A molecular mechanism to diversify Ca2+ signaling downstream of Gs protein-coupled receptors.
- Author
-
Brands, Julian, Bravo, Sergi, Jürgenliemke, Lars, Grätz, Lukas, Schihada, Hannes, Frechen, Fabian, Alenfelder, Judith, Pfeil, Cy, Ohse, Paul Georg, Hiratsuka, Suzune, Kawakami, Kouki, Schmacke, Luna C., Heycke, Nina, Inoue, Asuka, König, Gabriele, Pfeifer, Alexander, Wachten, Dagmar, Schulte, Gunnar, Steinmetzer, Torsten, and Watts, Val J.
- Subjects
ADENYLATE cyclase ,GENOME editing ,ISOENZYMES ,PHOSPHOINOSITIDES ,CALCIUM ions ,G protein coupled receptors - Abstract
A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca
2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+ . By combining CRISPR/Cas9 genome editing to delete Gαs , the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs -dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs. Gs heterotrimers are considered to be poor providers of free Gβγ subunits. Here, the authors show that—despite this—Gs-derived Gβγ dimers are active transducers of GPCR-initiated Ca2+ signals involving phosphoinositide-based signaling routes. [ABSTRACT FROM AUTHOR]- Published
- 2024
- Full Text
- View/download PDF
3. Cryo-EM structure of constitutively active human Frizzled 7 in complex with heterotrimeric Gs
- Author
-
Xu, Lu, Chen, Bo, Schihada, Hannes, Wright, Shane C., Turku, Ainoleena, Wu, Yiran, Han, Gye-Won, Kowalski-Jahn, Maria, Kozielewicz, Pawel, Bowin, Carl-Fredrik, Zhang, Xianjun, Li, Chao, Bouvier, Michel, Schulte, Gunnar, and Xu, Fei
- Published
- 2021
- Full Text
- View/download PDF
4. A virtual library of small molecules mimicking dipeptides.
- Author
-
Lim, Victor Jun Yu, Gerber, Hans‐Dieter, Schihada, Hannes, Trinh, Van Tuan, Hilger, Daniel, Vázquez, Olalla, and Kolb, Peter
- Published
- 2024
- Full Text
- View/download PDF
5. Residue 6.43 defines receptor function in class F GPCRs
- Author
-
Turku, Ainoleena, Schihada, Hannes, Kozielewicz, Pawel, Bowin, Carl-Fredrik, and Schulte, Gunnar
- Published
- 2021
- Full Text
- View/download PDF
6. Fluorescent Tools for the Imaging of Dopamine D2‐Like Receptors.
- Author
-
Nagl, Martin, Mönnich, Denise, Rosier, Niklas, Schihada, Hannes, Sirbu, Alexei, Konar, Nergis, Reyes‐Resina, Irene, Navarro, Gemma, Franco, Rafael, Kolb, Peter, Annibale, Paolo, and Pockes, Steffen
- Published
- 2024
- Full Text
- View/download PDF
7. Shedding Light on the D1‐Like Receptors: A Fluorescence‐Based Toolbox for Visualization of the D1 and D5 Receptors.
- Author
-
Rosier, Niklas, Mönnich, Denise, Nagl, Martin, Schihada, Hannes, Sirbu, Alexei, Konar, Nergis, Reyes‐Resina, Irene, Navarro, Gemma, Franco, Rafael, Kolb, Peter, Annibale, Paolo, and Pockes, Steffen
- Published
- 2024
- Full Text
- View/download PDF
8. Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment.
- Author
-
Köck, Zoe, Schnelle, Kilian, Persechino, Margherita, Umbach, Simon, Schihada, Hannes, Januliene, Dovile, Parey, Kristian, Pockes, Steffen, Kolb, Peter, Dötsch, Volker, Möller, Arne, Hilger, Daniel, and Bernhard, Frank
- Abstract
Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H
2 R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2 R and the inactive and Gq -coupled active state of H1 R 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 H2 R, its agonist-dependent internalization and its interaction with endogenously synthesized H1 R and H2 R in HEK293 cells by applying a recently developed nanotransfer technique.The study describes the molecular structure of the human histamine 2 receptor in active conformation and in complex with Gs heterotrimer, synthesized in a cell-free system and co-translationally inserted into preformed nanodiscs. [ABSTRACT FROM AUTHOR]- Published
- 2024
- Full Text
- View/download PDF
9. Development of Fluorescent AF64394 Analogues Enables Real-Time Binding Studies for the Orphan Class A GPCR GPR3.
- Author
-
Bresinsky, Merlin, Shahraki, Aida, Kolb, Peter, Pockes, Steffen, and Schihada, Hannes
- Published
- 2023
- Full Text
- View/download PDF
10. A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics
- Author
-
Schihada, Hannes, Vandenabeele, Sylvie, Zabel, Ulrike, Frank, Monika, Lohse, Martin J., and Maiellaro, Isabella
- Published
- 2018
- Full Text
- View/download PDF
11. WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction.
- Author
-
Bowin, Carl-Fredrik, Kozielewicz, Pawel, Grätz, Lukas, Kowalski-Jahn, Maria, Schihada, Hannes, and Schulte, Gunnar
- Subjects
G protein coupled receptors ,FLUORESCENCE resonance energy transfer ,G proteins ,BIOLUMINESCENCE ,PROTEIN receptors ,CELLULAR signal transduction - Abstract
Frizzleds (FZDs) are G protein–coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD
5 -DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5 -DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5 -DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5 . The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs. WNT bends FZD-DVL complexes: The binding of WNTs to G protein–coupled receptors (GPCRs) of the FZD family drives the oligomerization of the intracellular protein DVL at the receptor complex. Although the activation of G proteins to GPCRs depends on ligand-induced conformational changes in the receptors, WNT-induced conformational changes in FZD have not been thought to contribute substantially to DVL-mediated signaling because FZD and DVL can interact in the absence of WNT. Using bioluminescence resonance energy transfer (BRET) assays, Bowin et al. confirmed that FZD5 associated with DVL2 or the isolated DEP domain of DVL2 in the absence of WNT and showed that WNT stimulation promoted changes in the conformation of FZD5 -DVL2 and FZD5 -DEP complexes. These findings suggest that ligand-induced conformational changes in FZD may influence whether downstream signaling proceeds through DVL or G proteins. —AMV [ABSTRACT FROM AUTHOR]- Published
- 2023
- Full Text
- View/download PDF
12. A guide to adhesion GPCR research.
- Author
-
Liebscher, Ines, Cevheroğlu, Orkun, Hsiao, Cheng‐Chih, Maia, André F., Schihada, Hannes, Scholz, Nicole, Soave, Mark, Spiess, Katja, Trajković, Katarina, Kosloff, Mickey, and Prömel, Simone
- Subjects
CELL receptors ,G protein coupled receptors ,APPLIED sciences - Abstract
Adhesion G protein‐coupled receptors (aGPCRs) are a class of structurally and functionally highly intriguing cell surface receptors with essential functions in health and disease. Thus, they display a vastly unexploited pharmacological potential. Our current understanding of the physiological functions and signaling mechanisms of aGPCRs form the basis for elucidating further molecular aspects. Combining these with novel tools and methodologies from different fields tailored for studying these unusual receptors yields a powerful potential for pushing aGPCR research from singular approaches toward building up an in‐depth knowledge that will facilitate its translation to applied science. In this review, we summarize the state‐of‐the‐art knowledge on aGPCRs in respect to structure–function relations, physiology, and clinical aspects, as well as the latest advances in the field. We highlight the upcoming most pressing topics in aGPCR research and identify strategies to tackle them. Furthermore, we discuss approaches how to promote, stimulate, and translate research on aGPCRs 'from bench to bedside' in the future. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
13. Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias.
- Author
-
Schihada, Hannes, Klompstra, Thomas M., Humphrys, Laura J., Cervenka, Igor, Dadvar, Shamim, Kolb, Peter, Ruas, Jorge L., and Schulte, Gunnar
- Subjects
- *
G protein coupled receptors , *INFLAMMATORY bowel diseases , *ALTERNATIVE RNA splicing , *G proteins , *BACTERIAL diseases , *GASTROINTESTINAL system - Abstract
Within the intestine, the human G protein--coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrest in recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor--β-arrest in interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G13 activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor-- β-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35--transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
14. Functional modulation of PTH1R activation and signaling by RAMP2.
- Author
-
Nemec, Katarina, Schihada, Hannes, Kleinau, Gunnar, Zabel, Ulrike, Grushevskyi, Eugene O., Scheerer, Patrick, Lohse, Martin J., and Maiellaro, Isabella
- Subjects
- *
G protein coupled receptors , *MEMBRANE proteins , *HORMONE receptors , *ADVANCED planning & scheduling , *BONE metabolism , *EXCHANGE - Abstract
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 signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonistdependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered ß-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
15. Novel optical methods to monitor G-protein-coupled receptor activation in microtiter plates
- Author
-
Schihada, Hannes
- Subjects
615 Pharmakologie, Therapeutik - Published
- 2021
- Full Text
- View/download PDF
16. Neue optische Methoden zur Messung der Aktivierung von G-Protein-gekoppelten Rezeptoren in Mikrotiter-Platten
- Author
-
Schihada, Hannes
- Subjects
ddc:615 ,G-Protein gekoppelte Rezeptoren ,Hochdurchsatz-Screening ,Förster Resonanz Energie Transfer - Abstract
G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function., Die Klasse der G-protein-gekoppelten Rezeptoren (GPCRs) stellt die größte Familie membranständiger Proteine dar. GPCRs regulieren eine Vielzahl diverser physiologischer Prozesse in eukaryotischen Zellen und kontrollieren so unterschiedliche Zellfunktionen im menschlichen Organismus. Sie stellen die Zelloberflächenrezeptoren für verschiedenartige extrazelluläre Stimuli, wie zum Beispiel Photonen, niedermolekulare chemische Verbindungen, Peptide und Lipide dar. Die Wechselwirkung mit diesen sogenannten Liganden stabilisiert spezifische GPCR-Konformationen. Diese dienen wiederum als Ausgangspunkt für nachgeschaltete intrazelluläre Signalkaskaden, die beispielweise über membranverankerte G-Proteine vermittelt werden können. Während endogene GPCR-Agonisten diese Signalweiterleitung verstärken, können andere Biomoleküle wie Lipide, Ionen oder andersartige Membranproteine die Funktion, und damit die Signalweiterleitung der GPCRs modulieren. Aufgrund ihrer Einbindung in eine Vielzahl physiologischer und pathophysiologischer Prozesse, wurden GPCRs schon früh als Angriffspunkte („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt. Heutzutage vermitteln etwa 30% aller zugelassenen Arzneistoffe ihre Wirkung über G-protein-gekoppelte Rezeptoren. Dennoch wird das große Potential dieser Rezeptorfamilie als Targets für medikamentöse Behandlungen noch nicht in vollem Umfang ausgeschöpft. Tatsächlich gibt es für mehr als 200 GPCRs, die nicht der olfaktorischen Wahrnehmung dienen, noch keine Arzneistoffe, da wenig über deren Pharmakologie und physiologische Bedeutung bekannt ist. Zudem wird die Entwicklung neuartiger GPCR-Liganden erheblich durch das eingeschränkte Methodenrepertoire beeinträchtigt. Alle derzeit etablierten Techniken zur Identifizierung neuer GPCR-Liganden erfassen entweder den Ligand-GPCR-Bindungsprozess, der keine Informationen über die tatsächliche Aktivität der Verbindung liefert, oder messen weit-nachgeschaltete Signale, wie Änderungen sogenannter „Second-Messenger“-Konzentrationen (meist cAMP oder Calcium) und Reporter-Gen-Expressionslevel. Aufgrund ihrer Entfernung vom eigentlichen Rezeptor-Aktivierungsprozess haben diese Methoden allerdings bedeutende Nachteile und produzieren so häufig Falsch-Positive und Falsch-Negative Ergebnisse. Seit den frühen 2000er wurden GPCR-Konformationssensoren auf Basis von Fluoreszenz-Resonanz-Energie-Transfer (FRET) zur Messung der Ligand-induzierten Rezeptordynamik genutzt. Jedoch wies keiner der bisher entwickelten FRET- oder BRET- (Biolumineszenz-Resonanz-Energie-Transfer) Sensoren ausreichende Signalstärke auf, um im Hochdurchsatz-Screening (HTS) angewendet werden zu können. Die vorliegende Studie beschreibt das erste GPCR-Sensordesign, das aufgrund seiner exzellenten Signalstärke im Hochdurchsatz-Verfahren verwendet werden kann. Wir haben 21 unterschiedliche FRET- und BRET-Sensoren des α2A-adrenergen Rezeptors (α2AAR) getestet und dabei die Kombination der kleinen und hellen Luziferase NanoLuciferase (Nluc) mit dem rot-fluoreszierenden HaloTag-Farbstoff 618 als sensitivstes RET-Paar identifiziert. Der α2AARNluc/Halo(618) Biosensor ermöglicht die Messung der Aktivität und Wirkstärke von α2AAR-Liganden im Mikrotiterplattenformat. Um die universelle Anwendbarkeit dieses Sensordesigns zu prüfen, wurden fünf weitere Nluc/Halo(618)-basierende Sensoren für GPCRs unterschiedlicher Unterfamilien entwickelt. Zudem konnten wir zeigen, dass diese GPCRNluc/Halo(618)-Fusionsproteine weiterhin ihre natürlichen Signalkaskaden in Gang setzen können und damit die biologische Funktionalität dieser Rezeptoren erhalten ist. Außerdem belegt die vorlegende Arbeit, dass diese neue Sensor-Generation zur Messung Ligand-vermittelter Rezeptordynamiken im Hochdurchsatz-Format und zur Untersuchung der GPCR-Regulation durch endogene Modulatoren genutzt werden kann. Zusammenfassend kann gesagt werden, dass wir den ersten HTS-kompatiblen Assay zur Messung der GPCR-Konformationsänderungen entwickelt haben. Diese Biosensoren erlauben die Charakterisierung neuartiger GPCR-Liganden direkt auf der Rezeptorebene und funktionieren damit unabhängig von nachgeschalteter Signalamplifikation oder Überlagerung verschiedener Signalwege, welche die Aussagekraft traditioneller GPCR-Screening-Verfahren häufig beeinträchtigen. Diese Technik kann zur Entdeckung neuartiger GPCR-Arzneistoffe genutzt werden, zu einem besseren Verständnis bisher kaum erforschter Rezeptoren beitragen und der Identifizierung und Charakterisierung potentieller GPCR-Modulatoren dienen.
- Published
- 2021
17. Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.
- Author
-
Kowalski-Jahn, Maria, Schihada, Hannes, Turku, Ainoleena, Huber, Thomas, Sakmar, Thomas P., and Schulte, Gunnar
- Subjects
- *
CATENINS , *TRANSFER RNA , *AMINO acids , *DETECTORS , *CELL receptors , *MEMBRANE proteins - Abstract
The article focuses on Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain. Topics include Frizzleds are G protein–coupled receptors containing an extracellular cysteine-rich domain (CRD) binding Wingless/Int-1 lipoglycoproteins (WNTs), and the role of WNT/FZD signaling in health and disease, our understanding of how WNT binding is translated into receptor activation and transmembrane signaling remains limited.
- Published
- 2021
- Full Text
- View/download PDF
18. Quantitative assessment of constitutive G protein–coupled receptor activity with BRET-based G protein biosensors.
- Author
-
Schihada, Hannes, Shekhani, Rawan, and Schulte, Gunnar
- Subjects
G proteins ,FLUORESCENCE resonance energy transfer ,G protein coupled receptors ,BIOSENSORS ,CELLULAR signal transduction - Abstract
Better biosensors for constitutive activity: G protein activation is a common downstream signal transduction event upon agonist binding to the GPCR family of receptors. Genetically encoded BRET sensors are often used to measure the activity of G proteins but are limited to those stimulated by wild-type receptors. Schihada et al. developed BRET sensors capable of measuring the activity of GPCRs with disease-associated mutations that confer constitutive activity. By relying on plasmids that encoded all of the necessary components, these BRET sensors also showed better sensitivity than currently available BRET sensors that require cotransfection of cells with three separate plasmids. This set of BRET biosensors, which can detect the activity of the four major G protein families, will help to delineate the pathophysiological roles and effects of constitutively active GPCRs. Heterotrimeric G proteins constitute the primary transducers of G protein–coupled receptor (GPCR) signaling. In addition to mediating ligand-induced GPCR activation, G proteins transduce basal signaling in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Optical biosensors have been developed and optimized to monitor GPCR ligand–induced activation of G proteins, but these biosensors cannot be used to detect constitutively active GPCRs. Here, we designed and validated eight bioluminescence resonance energy transfer (BRET)–based G protein sensors that can measure the activity of all four major families of G proteins. We also established a protocol to identify constitutive GPCR or G protein signaling in live cells. These G protein–based, tricistronic activity sensors (G-CASE) rely on the encoding of all three G protein subunits by a single plasmid, enabling their expression at the desired relative amounts and resulting in reduced signal variability in mammalian cells. We also present an experimental protocol to use the G-CASE sensor toolbox to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will help to characterize constitutively active GPCRs and their role in health and disease. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
19. Development of a Conformational Histamine H3 Receptor Biosensor for the Synchronous Screening of Agonists and Inverse Agonists.
- Author
-
Schihada,, Hannes, Xiaoyuan Ma, Zabel, Ulrike, Vischer, Henry F., Schulte, Gunnar, Leurs, Rob, Pockes, Steffen, and Lohse, Martin J.
- Published
- 2020
- Full Text
- View/download PDF
20. Deconvolution of WNT-induced Frizzled conformational dynamics with fluorescent biosensors.
- Author
-
Schihada, Hannes, Kowalski-Jahn, Maria, Turku, Ainoleena, and Schulte, Gunnar
- Subjects
- *
BIOSENSORS , *G protein coupled receptors - Abstract
The G protein-coupled receptors Frizzled 1-10 (FZD 1-10) act as molecular checkpoints mediating intracellular signaling induced by 19 mammalian, secreted Wingless/Int-1 lipoglycoproteins (WNTs). Despite the vital roles of these signaling components in health and disease, our knowledge about WNT/FZD selectivity, and the mechanisms of receptor activation and intracellular signal propagation by individual ligand/receptor pairs is limited due to the current lack of suitable biophysical techniques. Here, we developed fluorescence-based biosensors that detect WNT-induced FZD conformational changes in living cells in order to assess WNT action via FZDs at the most proximal level, i.e. the receptor conformation. By testing a panel of recombinant ligands on conformational biosensors representing all four homology clusters of FZDs, we discover yet unappreciated selectivities of WNTs to their receptors and, surprisingly, identify distinct ligand-induced receptor conformations. Furthermore, we demonstrate that FZDs can undergo conformational changes upon WNT binding without being dependent on the WNT co-receptors LRP5/6. This sensor toolbox provides an advanced platform for a thorough investigation of the 190 possible WNT/FZD pairings and for future screening campaigns targeting synthetic FZD ligands. Furthermore, our findings shed new light on the complexity of the WNT/FZD signaling system and have substantial implications for our understanding of fundamental biological processes including embryonal development and tumorigenesis. Image 1 • Development and validation of conformational class F GPCR biosensors. • FZD-cpGFP sensors uncover WNT-induced conformational dynamics. • Investigation of WNT/FZD selectivity. • FZD-cpGFP sensors can serve as new tools to characterize FZD-targeting compounds. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
21. ERNEST COST action overview on the (patho)physiology of GPCRs and orphan GPCRs in the nervous system.
- Author
-
Birgül Iyison, Necla, Abboud, Clauda, Abboud, Dayana, Abdulrahman, Abdulrasheed O., Bondar, Ana‐Nicoleta, Dam, Julie, Georgoussi, Zafiroula, Giraldo, Jesús, Horvat, Anemari, Karoussiotis, Christos, Paz‐Castro, Alba, Scarpa, Miriam, Schihada, Hannes, Scholz, Nicole, Güvenc Tuna, Bilge, and Vardjan, Nina
- Abstract
G protein‐coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group ‘Biological roles of signal transduction’. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
22. The dark sides of the GPCR tree ‐ research progress on understudied GPCRs.
- Author
-
Scharf, Magdalena M., Humphrys, Laura J., Berndt, Sandra, Di Pizio, Antonella, Lehmann, Juliane, Liebscher, Ines, Nicoli, Alessandro, Niv, Masha Y., Peri, Lior, Schihada, Hannes, and Schulte, Gunnar
- Abstract
A large portion of the human GPCRome is still in the dark and understudied, consisting even of entire subfamilies of GPCRs such as odorant receptors, class A and C orphans, adhesion GPCRs, Frizzleds and taste receptors. However, it is undeniable that these GPCRs bring an untapped therapeutic potential that should be explored further. Open questions on these GPCRs span diverse topics such as deorphanisation, the development of tool compounds and tools for studying these GPCRs, as well as understanding basic signalling mechanisms. This review gives an overview of the current state of knowledge for each of the diverse subfamilies of understudied receptors regarding their physiological relevance, molecular mechanisms, endogenous ligands and pharmacological tools. Furthermore, it identifies some of the largest knowledge gaps that should be addressed in the foreseeable future and lists some general strategies that might be helpful in this process. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
23. eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled.
- Author
-
Wesslowski, Janine, Kozielewicz, Pawel, Xianxian Wang, Haijun Cui, Schihada, Hannes, Kranz, Dominique, M., Pradhipa Karuna, Levkin, Pavel, Gross, Julia Christina, Boutros, Michael, Schulte, Gunnar, and Davidson, Gary
- Subjects
- *
WNT signal transduction , *TRAFFIC signs & signals , *WNT proteins , *FUNCTIONAL analysis , *BINDING site assay , *CHROMOSOME duplication , *CANCER cell culture - Abstract
The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalianWNTs to their receptors of the class Frizzled (FZD1-10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/b-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated lowdensity lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRETbased ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
24. A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics
- Author
-
Hannes Schihada, Isabella Maiellaro, Martin J. Lohse, Monika Frank, Ulrike Zabel, Sylvie P. Vandenabeele, Schihada, Hannes, Vandenabeele, Sylvie, Zabel, Ulrike, Frank, Monika, Lohse, Martin J., and Maiellaro, Isabella
- Subjects
0301 basic medicine ,Chemistry ,High-throughput screening ,technology, industry, and agriculture ,Medicine (miscellaneous) ,macromolecular substances ,Resonance (particle physics) ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Microtiter plate ,030104 developmental biology ,0302 clinical medicine ,Förster resonance energy transfer ,lcsh:Biology (General) ,Cardiovascular and Metabolic Diseases ,ddc:570 ,Biophysics ,Bioluminescence ,Sensitivity (control systems) ,ddc:610 ,General Agricultural and Biological Sciences ,lcsh:QH301-705.5 ,Biosensor ,030217 neurology & neurosurgery ,G protein-coupled receptor - Abstract
G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α2A-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β2-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.
- Published
- 2018
25. A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors.
- Author
-
Brands J, Bravo S, Jürgenliemke L, Grätz L, Schihada H, Frechen F, Alenfelder J, Pfeil C, Ohse PG, Hiratsuka S, Kawakami K, Schmacke LC, Heycke N, Inoue A, König G, Pfeifer A, Wachten D, Schulte G, Steinmetzer T, Watts VJ, Gomeza J, Simon K, and Kostenis E
- Subjects
- Humans, HEK293 Cells, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, CRISPR-Cas Systems, GTP-Binding Protein alpha Subunits, Gs metabolism, GTP-Binding Protein alpha Subunits, Gs genetics, Cyclic AMP metabolism, Animals, Gene Editing, Cytosol metabolism, GTP-Binding Protein beta Subunits metabolism, GTP-Binding Protein beta Subunits genetics, Adenylyl Cyclases metabolism, Adenylyl Cyclases genetics, Calcium Signaling, Phospholipase C beta metabolism, Phospholipase C beta genetics, Receptors, G-Protein-Coupled metabolism, Receptors, G-Protein-Coupled genetics, Calcium metabolism
- Abstract
A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca
2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+ . By combining CRISPR/Cas9 genome editing to delete Gαs , the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs -dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
26. Cryo-EM structure of cell-free synthesized human histamine 2 receptor/G s complex in nanodisc environment.
- Author
-
Köck Z, Schnelle K, Persechino M, Umbach S, Schihada H, Januliene D, Parey K, Pockes S, Kolb P, Dötsch V, Möller A, Hilger D, and Bernhard F
- Subjects
- Humans, Cryoelectron Microscopy, HEK293 Cells, Receptors, Histamine H2 metabolism, Histamine metabolism, Escherichia coli metabolism
- Abstract
Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H
2 R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2 R and the inactive and Gq -coupled active state of H1 R 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 H2 R, its agonist-dependent internalization and its interaction with endogenously synthesized H1 R and H2 R in HEK293 cells by applying a recently developed nanotransfer technique., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
27. Shedding Light on the D 1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D 1 and D 5 Receptors.
- Author
-
Rosier N, Mönnich D, Nagl M, Schihada H, Sirbu A, Konar N, Reyes-Resina I, Navarro G, Franco R, Kolb P, Annibale P, and Pockes S
- Subjects
- Ligands, Fluorescence, Receptors, Dopamine D1 metabolism, Fluorescent Dyes
- Abstract
Dopamine D
1 -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 D1 R 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 (D1 R)=8.34, pKi (D5 R)=7.62) with excellent selectivity towards D2 -like receptors. Compound 25 proved to be a neutral antagonist at the D1 R and D5 R 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 D1 R 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., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)- Published
- 2024
- Full Text
- View/download PDF
28. Fluorescent Tools for the Imaging of Dopamine D 2 -Like Receptors.
- Author
-
Nagl M, Mönnich D, Rosier N, Schihada H, Sirbu A, Konar N, Reyes-Resina I, Navarro G, Franco R, Kolb P, Annibale P, and Pockes S
- Subjects
- Dopamine Antagonists pharmacology, Ligands, Radioligand Assay, Coloring Agents, Receptors, Dopamine D2 metabolism, Dopamine
- Abstract
The family of dopamine D
2 -like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's 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 (D2long R)=8.24, pKi (D3 R)=8.58, pKi (D4 R)=7.78) with decent selectivity towards D1 -like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2long R, D3 R, and D4 R, 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 D2long R in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's 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., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)- Published
- 2024
- Full Text
- View/download PDF
29. Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids.
- Author
-
Kowalski-Jahn M, Schihada H, and Schulte G
- Subjects
- Ligands, Protein Conformation, Cell Membrane, Energy Transfer, Amino Acids
- Abstract
Here we describe the application of genetic code expansion and site-specific incorporation of noncanonical amino acids that serve as anchor points for fluorescent labeling to generate bioluminescence resonance energy transfer (BRET)-based conformational sensors. Using a receptor with an N-terminal NanoLuciferase (Nluc) and a fluorescently labeled noncanonical amino acid in the receptor's extracellular part allows to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells. These BRET sensors can be used to investigate ligand-induced intramolecular (cysteine-rich domain [CRD] dynamics), but also intermolecular (dimer dynamics) receptor rearrangements. With the design of BRET conformational sensors based on the minimally invasive bioorthogonal labeling procedure, we describe a method that can be used in a microtiter plate format and can be easily adopted to investigate ligand-induced dynamics in various membrane receptors., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
- Full Text
- View/download PDF
30. A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H 3 Receptor.
- Author
-
Rosier N, Grätz L, Schihada H, Möller J, Işbilir A, Humphrys LJ, Nagl M, Seibel U, Lohse MJ, and Pockes S
- Subjects
- Binding Sites drug effects, Dose-Response Relationship, Drug, Fluorescent Dyes chemical synthesis, Fluorescent Dyes chemistry, HEK293 Cells, Histamine H3 Antagonists chemical synthesis, Histamine H3 Antagonists chemistry, Humans, Ligands, Molecular Structure, Structure-Activity Relationship, Bioluminescence Resonance Energy Transfer Techniques, Fluorescent Dyes pharmacology, Histamine H3 Antagonists pharmacology, Receptors, Histamine H3 metabolism, Single Molecule Imaging
- Abstract
The histamine H
3 receptor (H3 R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3 R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3 R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3 R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3 R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3 R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.- Published
- 2021
- Full Text
- View/download PDF
31. Employing Genetically Encoded, Biophysical Sensors to Understand WNT/Frizzled Interaction and Receptor Complex Activation.
- Author
-
Kozielewicz P, Schihada H, and Schulte G
- Subjects
- Animals, Cell Membrane, Humans, Ligands, Wnt Signaling Pathway, Frizzled Receptors genetics, Wnt Proteins genetics
- Abstract
The Frizzled (FZD) family of WNT receptors consists of ten paralogues in mammals. They belong to the superfamily of G protein-coupled receptors and regulate crucial processes during embryonic development. Dysregulated FZD signaling leads to disease, most prominently to diverse forms of cancer, which renders these receptors attractive for drug discovery. Recent advances in assay development and the design of genetically encoded biosensors monitoring ligand-receptor interaction, conformational dynamics, and protein-protein interaction have allowed for a better pharmacological understanding of WNT/FZD signal transduction and open novel avenues for mechanism-based drug discovery and screening. In this chapter, we summarize the recent progress in the molecular dissection of FZD activation based on advanced biosensors., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)
- Published
- 2021
- Full Text
- View/download PDF
32. Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology.
- Author
-
Schihada H, Nemec K, Lohse MJ, and Maiellaro I
- Subjects
- HEK293 Cells, Humans, Protein Conformation, Receptors, G-Protein-Coupled chemistry, Bioluminescence Resonance Energy Transfer Techniques methods, Drug Evaluation, Preclinical methods, Fluorescent Dyes chemistry, High-Throughput Screening Assays methods, Luciferases metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
Here we describe the stepwise application of bioluminescence resonance energy transfer (BRET)-based conformational receptor biosensors to study GPCR activation in intact cells. This technology can be easily adopted to various plate reader devices and microtiter plate formats. Due to the high sensitivity of these BRET-based receptor biosensors and their ability to quantify simultaneously receptor activation/de-activation kinetics as well as compound efficacy and potency, these optical tools provide the most direct and unbiased approach to monitor GPCR activity in a high-throughput-compatible assay format, representing a novel promising tool for the discovery of potential GPCR therapeutics.
- Published
- 2021
- Full Text
- View/download PDF
33. Development of a Conformational Histamine H 3 Receptor Biosensor for the Synchronous Screening of Agonists and Inverse Agonists.
- Author
-
Schihada H, Ma X, Zabel U, Vischer HF, Schulte G, Leurs R, Pockes S, and Lohse MJ
- Subjects
- Histamine, Ligands, Protein Binding, Biosensing Techniques, Receptors, Histamine H3 metabolism
- Abstract
The histamine H
3 receptor (H3 R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H3 R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H3 R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H3 R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H3 R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H3 receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H3 receptor ligand possesses even stronger inverse agonistic activity than reference H3 R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H3 R conformational biosensor that will aid in the discovery of novel H3 R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target.- Published
- 2020
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.