Background

GPCRs mediate cellular communications, both locally and over long distances, especially in the endocrine system. For instance, the cellular response to hormones such as adrenaline is mediated through adrenergic receptors, of which the β₂ adrenergic receptor (β₂AR) is a prominent member. β₂AR is expressed throughout the human body and is especially important in pulmonary, cardiac and immunological systems. Pharmacologically, agonists targeting β₂AR are medically proven to alleviate acute asthma attacks, since activation of β₂AR relaxes smooth muscle lining in the respiratory tracts. At the molecular level, β₂AR binds extracellular ligands and transmits signals across the cell membrane to intracellular effectors, such as G proteins or β arrestin. A variety of β₂AR ligands are known, and these are classified as agonists or inverse agonists, depending on whether they stimulate signaling activity or reduce the activity below the basal level, respectively (Baker, 2010). Crystallographic studies have revealed the three-dimensional structures of β₂AR in both inactive (Cherezov et al., 2007) and active (Rasmussen et al., 2011) conformations. However, less is known about how the receptor converts from the inactive to active conformation during signaling and how these transitions are linked to ligand binding.

Observation of single receptor molecules can reveal spontaneous conformational transitions and the influence of ligands on conformational switching, providing a unique perspective on receptor activation (Lamichhane et al., 2015). Development of a single-molecule assay requires labeling the receptor with a fluorescent reporter group at an informative position and a method for reconstituting individual receptor molecules in a membrane-like environment. A Cy3 fluorophore attached to the cytoplasmic end of trans-membrane helix 6 is a suitable reporter of conformational transitions, since the fluorescence quantum yield is sensitive to the local protein environment, a phenomenon referred to as protein-induced fluorescence enhancement (Hwang et al., 2011; Stennett et al., 2015). Moreover, phospholipid nanodiscs provide an ideal system for reconstitution and observation of single receptor molecules (Bayburt and Sligar, 2010). Individual labeled receptors in nanodiscs can be monitored over extended time periods by total internal reflection fluorescence (TIRF) microscopy, directly revealing transitions between inactive and active-like receptor conformations. The concept of the assay is illustrated in Figure 1. Statistical analysis of a collection of receptor molecules provides the rate constants for conformational exchange and the equilibrium distribution of the two conformational states, information that is difficult to obtain otherwise. These analyses can be readily performed in the absence or presence of various β₂AR ligands, providing detailed mechanistic information on the linkage of receptor activation to ligand binding. Such information should be useful in the design of improved GPCR-targeting drugs with finely tuned pharmacological efficacies and reduced side effects. Here we describe the protocols to label β₂AR with a Cy3 fluorophore, to reconstitute the labeled receptor in nanodiscs, to attach receptor-nanodisc complexes to a microscope slide and the procedures used to record and analyze single-molecule TIRF microscopy data. Although we describe these methods in the context of β₂AR, they are equally applicable to a variety of GPCRs.


Figure 1. Experimental system to monitor conformational transitions of β₂AR at the single-molecule level (reproduced from Lamichhane et al., 2015). A. An individual receptor molecule (black) labeled with Cy3 (red sphere) incorporated in a phospholipid nanodisc is tethered to a quartz surface coated with polyethylene glycol (wavy lines) via biotin (orange circles) and streptavidin (dark blue rectangles). The labeled receptor is illuminated in the evanescent field of a totally internally reflected 532 nm laser beam (green). The cartoon of the receptor-nanodisc complex is adapted from Bayburt and Sligar (2010). B. Expanded view of a single receptor-nanodisc complex, showing the receptor exchanging between inactive (blue) and active (red) conformations, with corresponding changes in the local environment of the Cy3 probe attached to Cys265 (light or dark red spheres, respectively). The transparent cylinder represents an abstraction of the lipid bilayer.