Structural insight into Class F receptors – What have we learnt regarding agonist‐induced activation?

Schulte, Gunnar; Kozielewicz, Paweł

doi:10.1111/bcpt.13235

Cited by 20 publications

(15 citation statements)

References 87 publications

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“…Meanwhile, the Wnt/PCP and Wnt/Ca 2+ pathways induce cytoskeletal rearrangements, as well as cell polarity and movement by upregulating intracellular phosphorylation cascades. 5 Although the role of Fzds in cancer varies by specific Fzd receptor and cancer type, their signaling pathways have emerged as potentially important drug targets due to their critical role in Wnt/β-catenin signaling and cell mobility ( Schulte and Kozielewicz, 2020 ). For instance, compounds that bind to the transmembrane domain of Fzd7 and interfere with Wnt binding could reduce oncogenic signaling in many cancer types ( Kalhor et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Non-Coding RNA and Frizzled Receptors in Cancer

Smith

Sompel

Elango

et al. 2021

Front. Mol. Biosci.

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Frizzled receptors have been long recognized for their role in Wnt/β-catenin signaling, a pathway known for its tumorigenic effects. More recent studies of frizzled receptors include efforts to understand non-coding RNA (ncRNA) regulation of these receptors in cancer. It has become increasingly clear that ncRNA molecules are important for regulating the expression of both oncogenic and tumor-suppressive proteins. The three most commonly described ncRNA molecules are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Here, we review ncRNA molecules that directly or indirectly affect frizzled protein expression and downstream signaling. Exploring these interactions highlights the potential of incorporating ncRNA molecules into cancer prevention and therapy strategies that target frizzled receptors. Previous investigations of frizzled receptors and ncRNA have established strong promise for a role in cancer progression, but additional studies are needed to provide the substantial pre-clinical evidence required to translate findings to clinical applications.

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Section: Introductionmentioning

confidence: 99%

Non-Coding RNA and Frizzled Receptors in Cancer

Smith

Sompel

Elango

et al. 2021

Front. Mol. Biosci.

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“…On a structural level, this seven-transmembrane domain spanning receptor is characterized by a large, extracellular cysteine-rich domain (CRD) and a long C-terminal domain (Schulte, 2010). While SMO is essential for transmitting transcriptional responses via heterotrimeric G proteins and Glioma-associated oncogene (Gli) signaling induced by hedgehog proteins (three mammalian homologs: Desert, Indian, and Sonic hedgehog), the nature and mode of action of the endogenous ligand and the mechanisms of receptor activation are not fully understood (Byrne et al, 2016;Kong et al, 2019;Schulte and Kozielewicz, 2019). It is known that hedgehog proteins bind Patched, a cholesterol transporter, which in turn regulates SMO activation by postulated regulation of cholesterol levels (Zhang et al, 2018b).…”

Section: Introductionmentioning

confidence: 99%

A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine

et al. 2019

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Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligandreceptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.

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“…On a structural level, this seven transmembrane domain spanning receptor is characterized by a large, extracellular cysteine-rich domain (CRD) and a long C-terminal domain (Schulte, 2010). While SMO is essential for transmitting transcriptional responses via heterotrimeric G proteins and Gliomaassociated oncogene (Gli) signaling induced by hedgehog proteins (three mammalian homologues: Desert, Indian and Sonic hedgehog), the nature and mode of action of the endogenous ligand and the mechanisms of receptor activation are not fully understood (Byrne et al, 2016;Schulte & Kozielewicz, 2019). It is known that hedgehog proteins bind Patched, a cholesterol transporter, which in turn regulates SMO activation by postulated regulation of cholesterol levels .…”

Section: Introductionmentioning

confidence: 99%

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

Kozielewicz

Bowin

Schulte

2019

Preprint

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Background and Purpose: Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the Patched, which in turn regulates SMO activation.Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here, we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists.Experimental Approach: In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allows kinetic analysis of ligand-receptor binding in living HEK293 cells and competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine).Key Results: The NanoBRET binding assay for SMO is sensitive and superior to purely fluorescence-based binding assays. BODIPY-cyclopamine showed complex binding parameters suggesting separate binding sites.Conclusions and Implications: The NanoBRET ligand binding assay for SMO provides a fast, sensitive and reliable alternative to assess SMO ligand binding.Furthermore, this assay is sufficiently sensitive to dissect a SANT-1-sensitive and a SANT-1-insensitive cyclopamine binding site in the 7TM core, and will be important to further dissect and understand the molecular pharmacology of Class F receptors.

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Structural insight into Class F receptors – What have we learnt regarding agonist‐induced activation?

Cited by 20 publications

References 87 publications

Non-Coding RNA and Frizzled Receptors in Cancer

Non-Coding RNA and Frizzled Receptors in Cancer

A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

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