HTRF® Total and Phospho-YAP (Ser127) Cellular Assays

Zindel, Diana; Vol, Claire; Lecha, Odile; Béquignon, Isabelle; Bilgic, Merve; Vereecke, Marion; Charrier‐Savournin, Fabienne; Romier, Maïté; Trinquet, Eric; Pin, Jean‐Philippe; Pannequin, Julie; Roux, Thomas; Dupuis, Elodie; Prézeau, Laurent

doi:10.1007/978-1-4939-8910-2_13

Cited by 3 publications

(8 citation statements)

References 22 publications

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“…These Phospho‐YAP (S127) HTRF ® and Total‐YAP kits are based on a relative quantification of both the S127 phosphorylated form of YAP (P‐YAP) and of total YAP (T‐YAP) using a pair of antibodies labeled with time‐resolved FRET (TR‐FRET) compatible fluorophores. While the T‐YAP assay needs the use of two anti‐YAP antibodies that recognize the protein whether or not phosphorylated, the P‐YAP assay is based on the use of an antibody recognizing specifically YAP phosphorylated on Ser 127, one of the major events related to YAP inhibition, and a second anti‐YAP antibody (Supplementary Figure S1A, M&M, and 35 ). The signal obtained with the S127‐P‐YAP assay can be normalized to the signal obtained with the T‐YAP assay to generate the ratio P‐YAP/T‐YAP (Supplementary Figure S1A), in order to eliminate well to well cell number variation.…”

Section: Resultsmentioning

confidence: 99%

“…In order to be able to screen for conditions and drugs, we developed innovative and efficient assays, based on the HTRF ® technology allowing the relative quantification of total YAP and S127 phosphorylated YAP protein 35 . Using a stable HEK293 cell line expressing the GHS‐R1a receptor, we show that two different GPCRs activate YAP through two distinct pathways.…”

Section: Discussionmentioning

confidence: 99%

“…The HTRF ® kits to detect either phosphorylation of YAP on the S127 residues or total YAP relative protein amount were developed by Cisbio ( 35 and https://fr.cisbio.eu/, Cisbio, Codolet, France) in collaboration with Laurent Prézeau and Jean‐Philippe Pin. Reagents were purchased from Tocris unless otherwise specified.…”

Section: Methodsmentioning

confidence: 99%

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G protein‐coupled receptors can control the Hippo/YAP pathway through Gq signaling

et al. 2021

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The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell‐cell contact, apical cell polarity, and mechanical signals. Miss‐regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein‐coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up‐and‐down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up‐ or down‐regulate YAP activity through a pure Gq pathway.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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G protein‐coupled receptors can control the Hippo/YAP pathway through Gq signaling

et al. 2021

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“…To overcome this bias, the analysis of the activity of various elements of GPCR signaling, such as IP1 concentration for the Gα q pathway or cAMP for the Gα s pathway, can be jointly performed on a large scale [352,353]. The activity of molecules on downstream G-protein signaling pathways can also be assessed using kinase assays from protein extracts or purified protein or by FRET [354][355][356]. These screens can be virtually performed with high efficiency if the receptor structure is known [357][358][359].…”

Section: Novel Structures and Drug Design Approachesmentioning

confidence: 99%

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma

Raymond

Aktary

Larue

et al. 2022

Cancers

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G-protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G-proteins, which induce cellular signaling through various pathways. Such signaling modulates numerous essential cellular processes that occur during melanomagenesis, including proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden melanoma treatment options in the future.

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“…To overcome this bias, the analysis of the activity of various elements of GPCR signaling, such as IP1 concentration for the Gq pathway or cAMP for the Gs pathway, can be jointly performed on a large scale [343,344]. The activity of molecules on downstream G-protein signaling pathways can also be assessed using kinase assays from protein extracts or purified protein or by FRET [345][346][347]. These screens can be virtually performed with high efficiency if the receptor structure is known [348][349][350].…”

Section: B Novel Structures and Drug Design Approachesmentioning

confidence: 99%

Targeting GPCRs & their Signaling as a Therapeutic Option in Melanoma

Raymond¹,

Aktary²,

Larue³

et al. 2022

Preprint

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G protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G proteins, which induce cellular signaling through various pathways. Such signaling modulates essential cellular processes of melanomagenesis, such as proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden treatment options in melanoma in the future.

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HTRF® Total and Phospho-YAP (Ser127) Cellular Assays

Cited by 3 publications

References 22 publications

G protein‐coupled receptors can control the Hippo/YAP pathway through Gq signaling

G protein‐coupled receptors can control the Hippo/YAP pathway through Gq signaling

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma

Targeting GPCRs & their Signaling as a Therapeutic Option in Melanoma

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