CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

© 2020 The Authors G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels

A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

© 2020 The Author(s) Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time

G protein-coupled receptor heteromers

G protein–coupled receptors (GPCRs) compose one of the largest families of membrane proteins involved in intracellular signaling. They are involved in numerous physiological and pathological processes and are prime candidates for drug development. Over the past decade, an increasing number of studies have reported heteromerization between GPCRs. Many investigations in heterologous systems have provided important indications of potential novel pharmacology; however, the physiological relevance of these findings has yet to be established with endogenous receptors in native tissues. In this review, we focus on family A GPCRs and describe the techniques and criteria to assess their heteromerization. We conclude that advances in approaches to study receptor complex functionality in heterologous systems, coupled with techniques that enable specific examination of native receptor heteromers in vivo, are likely to establish GPCR heteromers as novel therapeutic targets

Application of G Protein-Coupled Receptor-Heteromer Identification Technology to Monitor β-Arrestin Recruitment to G Protein-Coupled Receptor Heteromers

Understanding the role of G protein-coupled receptor (GPCR; also known as a 7 transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer function enables the so-called biochemical fingerprints of the receptor heteromer to be ascertained. This is the first step in establishing the physiological relevance of heteromerization, the goal of everyone in the field, as these fingerprints can then be utilized in future endeavors to elucidate heteromer function in native tissues. The simple, robust, ligand-dependent methodology described in this study utilizes a novel configuration of components of a proximity-based reporter system. This is exemplified by the use of bioluminescence resonance energy transfer due to the advantages of real-time live cell monitoring of proximity specifically between the heteromer complex and a protein that is recruited in a ligand-dependent manner, in this case, β-arrestin 2. Further, the demonstration of Z′-factor values in excess of 0.6 shows the potential of the method for screening compounds for heteromer-selective or biased activity. Three previously characterized GPCR heteromers, the chemokine receptor heteromers CCR2-CCR5 and CCR2-CXCR4, as well as the angiotensin II receptor type 1-bradykinin receptor type 2 heteromer, have been used to illustrate the profiling capability and specificity of the GPCR heteromer identification technology

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes

The type 1 angiotensin II (AngII) receptor (ATR) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the ATR was able to transactivate HER2, but not HER3. Mechanistically, ATR-mediated ERK1/2 activation was dependent on G and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of G and only partially dependent on EGFR tyrosine kinase activity. This G independence of EGFR transactivation was confirmed using ATR mutants and in CRISPR cell lines lacking G. EGFR transactivation was also apparently independent of β-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted ATR-EGFR heteromerization. In summary, we report an alternative approach to monitoring ATR-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the ATR and EGFR