![]() ![]() These two high- and low-affinity systems can therefore be configured to investigate either transient or stable protein interactions. The split NLuc system (NanoBiT) comprises a small 11-amino acid peptide engineered to interact with an 18-kDa polypeptide of NLuc (LgBiT) with either high (∼700 pM) or low (∼190 μM) affinity ( Dixon et al., 2016). This approach allowed for quantification of protein expression by changes in luminescence following luciferase complementation ( Oh-Hashi et al., 2017, Schwinn et al., 2018) as well as post-translational modifications of endogenous proteins to be investigated by NanoBRET (with addition of an exogenous fluorescent probe) ( Schwinn et al., 2018). In addition, reports have also demonstrated the use of CRISPR/Cas9 genome editing to insert small self-complementing fragments of NLuc into the endogenous genome ( Oh-Hashi et al., 2017, Schwinn et al., 2018). This results in NLuc fusion proteins being expressed under endogenous promotion and has been used to investigate ligand binding to adenosine A 2B receptors ( White et al., 2019), as well as CXCR4 receptor trafficking and β-arrestin2 recruitment to GPCRs ( White et al., 2017) by monitoring changes in resonance energy transfer between the NLuc luminescent donor and a fluorescent acceptor. To overcome the need for exogenous expression of a luciferase-tagged protein of interest in BRET assays, we and others have used CRISPR/Cas9 genome engineering to insert the 19-kDa nanoluciferase (NanoLuc, NLuc) into endogenous mammalian loci via homology-directed recombination ( White et al., 2017, White et al., 2019, Oh-Hashi et al., 2016). However, the use of these biosensors in cellular systems is typically accomplished by exogenous expression of the tagged protein(s) of interest that can perturb the normal cellular context and stoichiometry of the cellular interactome, particularly where the level of exogenous expression is high. Indeed, luciferase-based assays have been developed to investigate GPCR-ligand binding ( Stoddart et al., 2015), G protein activation, and protein-protein interactions ( Lohse et al., 2012), as well as receptor internalization and trafficking ( Lan et al., 2012, Tiulpakov et al., 2016) by monitoring changes in bioluminescence resonance energy transfer (BRET) or luciferase complementation. Many of these processes can be studied using genetically encoded luminescent and/or fluorescent fusion proteins that allow for investigation of receptor or protein function by sensitive microscopic or biophysical techniques such as resonance energy transfer. The response elicited by a given GPCR is dependent on the cellular context, i.e., the cellular proteome and a cascade of factors including receptor compartmentalization ( Ellisdon and Halls, 2016, Tsvetanova et al., 2015), association with interacting proteins ( Bockaert et al., 2004), binding of a specific ligand and subsequent conformational rearrangement resulting in activation ( Wang et al., 2018), coupling to specific intercellular effectors (e.g., G proteins) ( Wang et al., 2018, Rankovic et al., 2016), or scaffolding proteins (e.g., GPCR kinases and arrestins) ( Walther and Ferguson, 2015), as well as internalization, trafficking, and recycling of the receptor ( Magalhaes et al., 2012). G protein-coupled receptors (GPCRs) are a major class of membrane receptors that control numerous physiological responses via ligand-mediated signal transduction. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels. 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. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. ![]()
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