A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes

Abstract

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z’ factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations

“…Recently, we have developed a dynamic split-luciferase based mini-G protein recruitment assay for the histamine receptor family [ 52 ] meeting the requirements of a proximal readout as well as a simple, robust, non-radioactive and homogenous performance [ 75 ]. In this assay, HEK293T cells express HR subtypes that are C-terminally fused to the small fragment of the NanoLuc (NlucC) and mini-G proteins N-terminally attached to the large fragment (NlucN).…”

“…The cells were transiently transfected with a total amount of 3 µg plasmid DNA using linear polyethyleneimine (PEI, 1 mg/mL in PBS, transfection ratio 1:5 (3 µg DNA + 15 µL PEI)). Therefore, combinations of the following plasmids, the construction of which has been described previously [ 52 ], were used: pcDNA3.1 H 2 R–NlucC, pcDNA3.1 H 4 R–NlucC, pIRESpuro3 NlucN–mGs, pIRESpuro3 NlucN–mGsi, and pIRESpuro3 NlucN–mGsq. The cells were incubated for 48 h to allow for an adequate protein expression.…”

“…A similar approach has been used in the development of genetically engineered mini-G (mG) proteins consisting of the GTPase domain of Gαs and the α5 helices of G proteins of the four major G protein families Gs, Gi/o, Gq/11, and G12/13 [ 45 , 46 , 47 ]. Combined with BRET or split-luciferase complementation techniques, the mini-G proteins are suitable for ligand characterization in cell-based assays [ 47 , 48 , 49 , 50 , 51 , 52 ]. Moreover, these proteins were originally designed to stabilize GPCRs in their active state and thus to enable the elucidation of GPCR–G protein complexes by X-ray and cryo-electron microscopy (cryo-EM), which has been achieved for the adenosine A 2A (A 2A R) [ 53 ], the dopamine D 1 (D 1 R) [ 54 ], the GPR52 [ 55 ], the serotonin 5-HT 1B (5-HT 1B R) [ 56 ], and 5-HT 2A (5-HT 2A R) [ 57 ] receptors so far.…”

“…Three types of engineered G proteins, mGs, mGsi, and mGsq, were used to characterize the coupling profiles of each receptor. We have explored the functional responses of both receptors upon activation by the endogenous ligand histamine using a recently published mini-G protein recruitment assay [ 52 ]. Furthermore, we performed all-atom GaMD simulations on the H 2 R and H 4 R in complex with mGs, mGsi, and mGsq in explicit lipids and solvent.…”

Optical sensors of heterotrimeric G protein signaling

“…Moreover, the GPCR-binding specificity of mini G proteins can be easily altered by modifying their C-terminal sequence in the same way as for Ga subunits. Mini G proteins have recently been used in BRET [114] and luciferase complementation studies [115], as well as in live-cell imaging [54] of GPCR-G protein interactions. The stable binding of mini G proteins to active GPCRs also makes mini G proteins suitable for studies of localisation and dynamics of endosomal signaling [116].…”