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G-protein signaling

Heterotrimeric G protein complexes are composed of 3 subunits, including an α-subunit which acts as the main signal mediator and whose active state is regulated via GTP (guanosine-5′-triphosphate) recruitment and hydrolysis. There is also a dimer of β- and γ-subunits, which form an inseparable complex with distinct signaling abilities.

Depending on the Gα subunit involved in the complex, the most well-known G-proteins are qualified as Gi, Gs, or Gq. They signal through different pathways. Gq proteins rely on enzymes of the phospholipase C family (PLC), while Gs and Gi proteins respectively stimulate and inhibit adenylate cyclase (AC) and thus act upon the amount of cytosolic cAMP.

G-protein Signaling – Gs, Gi, and Gq are types of G-protein alpha subunits​
GPCR signaling pathways activated by a canonical ligand and a biased ligand​

This webpage is intended to help you understand the G-protein signaling universe and navigate the differences and special features of Gs & Gi-coupled receptors and Gq-coupled receptors. We have developed a ready-to-use test panel to monitor GPCR signaling pathways (second messenger, phosphoproteins, and transcription factors), to help you in your research.

PerkinElmer offer a range of HTRF® and LANCE® Ultra™ kits, ensuring a match for your research needs: cAMP Gi kit, cAMP Gs dynamic kit, cAMP Gs HiRange kit, LANCE® Ultra™ cAMP kit, IP-One Gq kit, and GTP Gi Binding assay.

Guides cAMP guides

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Guides GTP guides

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Gs- and Gi-coupled receptors

Cyclic AMP (cyclic adenosine 3’,5’-monophosphate, or simply cAMP) is one of the most important GPCR intracellular mediators. In many cell types, cAMP production often results from the regulation of adenylate cyclase by the Gα subunit of heterotrimeric G-proteins. Gαs-coupled GPCRs act to positively stimulate the activity of adenylate cyclase, resulting in an increase in cellular cAMP. Gαi-coupled GPCRs lead to a negative regulation of adenylate cyclase, and thus to a decrease in cAMP production.

How to run a cAMP HTRF assay

Watch this video to learn about using a streamlined, no-wash, add-and-read HTRF® cAMP assay kit to monitor GPCR activation.

            

We guide you toward success

You’ll never be lost with our highly regarded cAMP Guides. Cisbio scientists have worked for many years with numerous Gαi- and Gαs-coupled GPCRs to produce a detailed set of recommendations, summarized in the guide. Thousands of GPCR researchers have already downloaded their copy of this valuable reference!

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GTPγS – GTP Binding assay​

Guanosine 5′- (trihydrogen – diphosphate), or GTPγS, is a stable analog (hydrolyzable or non-hydrolyzable) of GTP and benefits from various physiological actions, such as the stimulation of guanine nucleotide-binding proteins.

The GTP Binding assay measures G protein activation level following agonist occupation of a GPCR by determining the binding of the non-hydrolyzable analog GTPγS to the G subunits α. Thus the assay measures a functional consequence of receptor occupancy at one of the first receptor-mediated events.​

This simple assay provides a better understanding of contemporary pharmacological issues, including the role of accessory proteins in signaling, GPCR receptor constitutive activity, and agonist-specific signaling.​

Activation of GPCR leads to GDP/GTP nucleotide exchange on the Gα protein subunit
Activation of GPCR leads to GDP/GTP nucleotide exchange on the Gα protein subunit and binding of the Eu-GTP analog. Detection is enabled by the addition of the d2 anti-Gi antibody. An antagonist addition competes with the agonist binding in the orthosteric site of GPCR, and thus inactivates the G protein. Eu-GTP dissociates, leading to FRET signal extinction.​​

Get these 2 guides to master the art of optimizing GTP binding assays!​

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Guide: optimize your GTP binding assays with these Gαi agonist and antagonist

Gq-coupled receptors

Gq-coupled GPCRs stimulate PLC-β, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3 and diacylglycerol (DAG). IP3 binds IP3 receptors on the endoplasmic reticulum (ER) and induces calcium release. PLC-β promotes the termination of Gq-GPCR signaling complexes by stimulating GTP hydrolysis of the GTP-bound Gq.

Calcium release has historically been used as a readout for Gq activity, in spite of its limitations. Being ubiquitously involved in cell metabolism, calcium is tied to multiple G protein-independent mechanisms which make it lack specificity (Ca2+ permeable proteins, Ca2+ pumps, and Ca2+ transporters). The highly regulated nature of calcium also makes it a transient analyte in cells, which prevents equilibrium assays and gives bias to some pharmacological studies, in particular the correct screening and characterization of ligand classes.

As a degradation product of Gq-dependent IP-3, IP-1 is a specific readout and enables true equilibrium assays that ensure both signal specificity and accurate pharmacological studies of all compound classes. It measures only IP1, produced solely by phospholipase enzyme (PLC) action and mediated by GPCRs.

PerkinElmer offers an innovative approach with IP-One, a homogeneous, non-radioactive accumulation of inositol-phosphate for signaling Gαq. This kit has been shown to offer superior advantages over traditional tests, making it the ideal tool for your research.

Gq-coupled receptor signaling pathways and their related calcium flux and Gq-unrelated calcium flux
Gq-coupled receptor signaling pathways and their related calcium flux and Gq-unrelated calcium flux.

IP-One, the best alternative to calcium flux assays

Cisbio’s platform includes IP-One, a very unique, non-isotopic assay and the best alternative to calcium flux assays. IP-One specifically measures IP-1 levels in cells. Over the past decade, IP-One has demonstrated its efficiency on numerous occasions for discovering and/or characterizing new compounds that target Gαq coupled GPCR. Discover the many benefits of IP-One by watching this short video.

            

Ready for more? All essential information on IP-One are now available in one convenient eBook. This document offers an in-depth focus on the very unique benefits of IP-One accumulation assay over Calcium Flux technologies.

Never be at a loss with our acclaimed IP-One Guide. For years Cisbio’s scientists have worked with many Gαq coupled GPCR to produce a set of recommendations summarized in the guide. You’ll find all the guidance and support you need to get your experiment right the first time.