A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase

Abstract

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α 1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration

“…Heteromerization of GPCRs can alter the ligand binding affinity of their partner GPCRs via allosteric modulation [ 10 , 11 ]. To investigate the effect of LPA 1 on the binding of CXCL12 to CXCR4, we performed a BRET-based ligand binding assay utilizing Gluc [ 31 ] and the Alexa Fluor 488-conjugated CXCR4 antagonist TZ14011 (TZ14011-AF488). We expressed Gluc-tagged CXCR4 in HEK293A cells as a BRET donor and treated cells with TZ14011-AF488 as a BRET acceptor.…”

“…If necessary, a stop codon was introduced at the end of the coding sequence using site-directed mutagenesis. Gaussia luciferase (Gluc)-PM was a gift from Laszlo Hunyady (#164783, Addgene) [ 31 ]. Flag, HA, and Myc tags or Gluc were cloned at the N-terminus of each GPCR using one-step sequence- and ligation-independent cloning (SLIC) [ 32 ].…”

“…Ligand binding assay was performed as previously described [ 31 ]. HEK293A cells were transfected with Gluc-CXCR4 alone or in combination with Flag-LPA 1 .…”

Miniaturization of Bright Light-Emitting Luciferase ALuc: picALuc

“…A similar spectrum was also observed in the previous report for NanoLuc. 35 For the spectral aspects, further investigations will be required, especially for the wavelength shift by YFP fusion (observed in the case of picALuc).…”

PharmacoSTORM nanoscale pharmacology reveals cariprazine binding on Islands of Calleja granule cells

“…Functional pharmacological assays are convenient for the rapid screening of the best candidates, as we demonstrated via the development of a fluorescent ligand for the CB 1 cannabinoid receptor, the MAGL enzyme and a labeled analog of an approved drug (fluo-cariprazine). The exact effects of fluorophore tagging on equilibrium or kinetic ligand-binding parameters can certainly be obtained by further radioactivity- or fluorescence-based methods 55 , 56 . Moreover, the specificity of target binding can be analyzed by appropriate control PharmacoSTORM experiments using untransfected cells, displacement assays, point-mutant receptors for binding sites, or tissues obtained from knockout mice as we demonstrated all four control approaches in the case of fluo-cariprazine.…”