GTP Gi binding assay HTRF®

The GTP Gi binding assay uses a cryptate-labelled GTP analog (donor) and  a d2-labelled anti-Gi antibody (acceptor). The GTP analog specifically binds into the GTP-binding pocket of active G proteins, while the d2-labelled antibody is specific to the Gαi subunit of G proteins. GPCR activation in presence of the two detection reagents brings the donor and acceptor into proximity, which triggers a FRET signal with an intensity proportional to the concentration of active Gi protein in the sample. GTP gamma S, a non-hydrolysable GTP analog, is used to displace the donor from the GTP-binding pocket and extinguish FRET to determine the assay non-specific signal an ensure its accuracy.

The GTP Gi binding assay is performed in a miniaturized format using 20 µl final volume, and features a streamlined 4-step reagent addition protocol where stimulation/compounds, agonist, detection reagents, and membranes are added successively. Plates are read after 3H or ON incubation at RT, depending on the biological model. No washing steps are required.

The assay was performed using the previously optimized conditions for this membrane model: stimulation buffer #3 was supplemented with 0.5 µM of GDP,  50 mM of MgCl2. The assay was performed using 5 µg of membranes / well. The SNC-162 dose-response curve fit shows an EC50 of 6.5 nM, which is in accordance with published values.

A case study in CHO-DOR membrane model with and without optimization of the MgCl2 and GDP concentrations in stimulation buffer. 5 µg of membrane were incubated in presence or absence of the SNC-162 agonist (1 µM). The plate was read after ON incubation at RT.

Optimal membrane  quantity/well may change from one membrane model to another. An additional optimization step featuring three conditions (2.5, 5, and 10 µg/well) enables optimal membrane quantity determination. Two case studies are presented here:

The graphs show the signal obtained in absence (basal) or presence of agonist (stimulated). S/B is presented for each condition. As demonstrated here, membrane quantity/well is a key parameter for pharmacological S/B optimization. For these two models, 5 µg of membrane/well is suitable. This optimization step can be a deciding factor in enhancing assay S/B, or in  selecting assay conditions using a lower quantity of membrane (2.5 µg/well for DOR).

In their inactive state, heterotrimeric G proteins bind GDP in a binding pocket of their Ga sub-unit. Upon stimulation of the GPCR by an agonist, the receptor-coupled G protein undergoes a conformational change that allows the Ga sub-unit to release its bound GDP and replace it with GTP. This nucleotide exchange fully activates the Ga sub-unit, which dissociates from the Gb/Gg dimer and performs inhibitory or stimulating functions on adenylate cyclase (AC) and phospholipase C (PLC), depending on its sub-type (Gai, Gas, Gaq, etc).

The following signal transduction steps are GPCR-specific and mobilize different phospho-proteins and pathways. Over time, the Ga-bound GTP undergoes hydrolysis, which returns the Ga protein to an inactive state.