Chapter Five - Fluorescent bioorthogonal labeling of class B GPCRs in live cells

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Abstract

In this method paper, we describe the protocols for selective labeling of GCGR, a member of the class B GPCR family regulating glucose homeostasis, in live cells. A two-step procedure is presented in which a strained alkene chemical reporter is inserted into any desired location within the GPCR in the first step, followed by a robust bioorthogonal ligation reaction with a fluorophore-conjugated tetrazine or tetrazole reagent in the second step. The amber codon suppression strategy was adopted for site-specific incorporation of the strained alkene reporter, either spirohexene or trans-cyclooctene, in HEK293T cells. Subsequently, the inverse electron-demand Diels–Alder reaction with an AF647-conjugated 3,6-di (2-pyridyl)-S-tetrazine (DpTz) was performed with the alkene-encoded GCGR on live-cell surface. Alternatively, a photo-induced cycloaddition with a Cy5-conjugated, sterically shielded tetrazole was carried out, giving rise to faster fluorescent labeling along with excellent selectivity. Owing to their robust reaction kinetics and excellent chemoselectivity, the bioorthogonal labeling protocols described here could be readily adapted to labeling any accessible protein targets, e.g., membrane proteins, in live cells.

Section snippets

Introduction to fluorescent labeling of GPCR

The class B G protein-coupled receptors (GPCR) are composed of 15 peptide-binding receptors in humans, including those for secretin, glucagon, glucagon-like peptide 1 (GLP-1), corticotropin-releasing factor (CRF), parathyroid hormone (PTH), and the calcitonin gene-related peptide (CGRP), and are validated drug targets for diabetes, depression, and osteoporosis (Bortolato et al., 2014). A recent surge of structural data of full-length class B GPCRs have shed new light into the structural basis

Site-specific incorporation of alkene reporters into the class B GPCR

Given the vast array of functional groups present in cellular environment, stringent criteria need to be met for chemical reporters and the cognate bioorthogonal probes to be useful for bioorthogonal labeling (Tian & Lin, 2019). For site-specific incorporation of a chemical reporter into class B GPCRs, we focused on the genetically encodable strained alkenes because of their small size and outstanding reactivity in the strain-promoted cycloaddition reactions such as the tetrazole-based

Bioorthogonal labeling of GCGR in live cells using tetrazine ligation

The second step in our bioorthogonal labeling strategy involves the introduction of a fluorophore into class B GPCR in live cells using a bioorthogonal reaction. Among various bioorthogonal reactions reported in the literature (Ramil & Lin, 2013), we chose the inverse electron demand Diels − Alder reaction between Sph and DpTz (Fig. 3A) because its fast reaction kinetics permits labeling of low-abundance proteins such as class B GPCRs. In the live-cell labeling experiments, we compared the

Bioorthogonal labeling of GCGR in live cells using photo-click chemistry

The use of light to generate highly reactive species in situ from stable precursors has attracted substantial interest in the bioorthogonal chemistry field (Herner & Lin, 2016). For example, the photo-induced cycloaddition reaction between the tetrazoles and the electron-deficient or strained alkenes has shown broad utility in labeling proteins in biological systems (Lim & Lin, 2011; Ramil & Lin, 2014; Song, Wang, Qu, Madden, & Lin, 2008; Yu, Lim, & Lin, 2010), particularly where the

Precursor techniques

The chemical reporters used in these experiments, SphK and TCOK, were prepared according to the published procedure (Ramil et al., 2017). The corresponding tetrazine reagent, DpTz-AF647, was synthesized by following the literature procedure (Ramil et al., 2017). The water-soluble tetrazole reagent, Tet-Cy5, used in bioorthogonal labeling of GCGR, was prepared according to the published procedure (An et al., 2018).

Discussions and critical parameters

In constructing the fluorescent-labeled GPCR biosensors, these mutant receptors must retain their functions. To this end, functional assays were carried out during each step of the bioorthogonal labeling. In the first step, the suitable positions within GCGR that can tolerate UAA mutagenesis need to be identified. In our studies, we employed the cAMP response assay to measure the activities of the GCGR mutants encoding BocK at the various loop positions. We found that the ECL3 mutants displayed

Summary and future directions

As a first step toward the construction of fluorescence-based GPCR biosensors, we present the protocols for successful bioorthogonal labeling of the extracellular loop 3 (ECL3) region of GCGR, a member of the class B GPCR family, in live cells. The site-specific incorporation of spirohexene as a robust chemical reporter and the subsequent fluorescent labeling based on either tetrazine ligation or photo-click chemistry underline the two critical steps involved in the implementation of this

Acknowledgment

We gratefully acknowledge the National Institutes of Health (R35GM130307) for supporting our work on bioorthogonal chemistry.

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