Background

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface receptors and modulate a variety of cell functions under physiological and pathological conditions (Hauser et al., 2017; Hilger et al., 2018; Weinberg and Puthenveedu, 2019). The precise functions of individual GPCRs at the cell surface are initiated by their binding to specific ligands which in turn activates cognate heterotrimeric G proteins or other signaling molecules, leading to the activation of intracellular signal transduction cascades. Although GPCR-mediated signaling and functioning are sophisticated processes which are coordinated by many factors, the net amount of receptor surface expression is undoubtedly a crucial element regulating the magnitude and duration (Zhang and Wu, 2019).

The quantity of GPCR expression at the cell surface is a balance of highly regulated, dynamic intracellular trafficking, including maturation, internalization, recycling, and degradation. Over the past decades, numerous methods have been well established to quantify the surface GPCRs, such as ELISA, flow cytometry, biotinylation, radioligand binding and imaging (Dunham et al., 2009; Qin et al., 2011; Zhu et al., 2015; Shiwarski et al., 2017 and 2019). Here, we describe an intact live-cell ligand binding assay to quantify their surface expression at steady state by using cell membrane-nonpermeable radioligands. In this assay, live cells will be incubated with individual receptor-specific antagonists or agonists labelled with radioactive isotopes at a saturating concentration. Since these radioligands are not able to penetrate the plasma membrane, the radioactivity of ligands bound to the cells will reflect the quantity of receptor expression at the cell surface. As the ligands used are radiolabeled and stoichiometrically bind to specific GPCRs, this method provides a very sensitive and highly specific approach to accurately quantify the surface functional GPCRs which are able to bind to their ligands in intact live cells and is particularly useful for endogenous, low-abundant GPCRs whose quantification by other methods is extremely difficult.

We have used this method to measure the surface expression of a number of family A GPCRs in different cell types at the endogenous levels or after overexpression (Filipeanu et al., 2004; Dong and Wu, 2006 and 2007; Dong et al., 2008; Duvernay et al., 2009a and 2009b; Dong et al., 2010a and 2010b; Duvernay et al., 2011; Zhang et al., 2011; Dong et al., 2012; Fan et al., 2012; Li et al., 2012; Zhang et al., 2016a and 2016b; Li et al., 2017; Wei et al., 2019; Zhang et al., 2019). Here, we first describe the procedures to measure the surface expression of α_2B-adrenergic receptor (AR), which has long been used as a model GPCR in our studies, in NG108-15 neuroblastoma-glioma and MCF-7 breast cancer cells which express α_2B-AR endogenously, and in human embryonic kidney 293 (HEK293) cells in which α_2B-AR was overexpressed by transient transfection or inducible systems. We will then briefly discuss the quantification of surface expression of other GPCRs by using radioligand binding of intact live cells.