Temporally gated molecular tools for tracking protein-protein interactions in live cells

Abstract

Protein-protein interactions (PPIs) are essential in most biological processes. Even though many methods were designed to detect PPIs, detecting PPIs in a large volume of cells with a temporal resolution remains challenging. Recent development of light gated transcriptional reporters, such as SPARK and iTANGO, enabled detection of PPI in a large population of cells with a temporal resolution on the order of minutes. In this chapter, we discussed in detail the application of SPARK to detect PPIs between the activated β-2 adrenergic receptor (B2AR) and both Gα mimic and β-arrestin2. Because SPARK is a multi-component system, the protein expression level is critical for its optimal performance. We also discussed the detailed protocols for using SPARK with either transfection or lentiviral infection in HEK296T/17 cells.

Introduction

Protein-protein interactions (PPI) play crucial roles in most biological processes. While a lot of proteins form stable and tight protein complexes, many PPIs are highly dynamic depending on the cellular context, its post-translational modifications, or its conformational state. For example, GPCRs (G-protein coupled receptors) interact with G-proteins transiently upon agonist activation, initiating downstream signaling (Jean-Charles, Kaur, & Shenoy, 2017). Arrestin could then interact with the activated GPCR leading to receptor desensitization or other downstream signaling such as the extracellular signaling kinases 1 and 2 (ERK) signaling (Jean-Charles et al., 2017).

Given the importance of PPIs, many methods were developed to study PPIs. Co-immunoprecipitation is commonly used to study stable PPI complexes but cannot be used to study dynamic PPIs. The existing methods for detecting dynamic PPIs can be generally divided into two classes. One class of methods provides a real-time readout of the PPIs, and these include FRET (Fluorescence Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy transfer). The other class of PPI detection methods integrate the PPI signals overtime and provide a permanent mark for analysis at a later time. They include the yeast two-hybrid system, TANGO (Inagaki et al., 2012) and most split protein systems, such as split luciferases (Luker et al., 2004), split ubiquitin (Iyer et al., 2005), split GFP (Hu & Kerppola, 2003), split TEV protease (Wehr et al., 2006), split Cre recombinase (O'Brien & Delisa, 2014), split DHFR (Remy, Campbell-Valois, & Michnick, 2007) and split galactosidase (Rossi, Charlton, & Blau, 1997). Real time detection methods are powerful, because they allow the tracking of the PPI dynamics with a temporal resolution on the order of milliseconds to seconds. However, real time detection methods are technically challenging and difficult for use in high-throughput detection and screening of PPIs. Integration methods are usually easy to perform and advantageous for high-throughput screening but lose the ability to resolve PPI dynamics. Recently, a few time-gated integration methods were reported that can integrate the PPI signals in a user-defined time window on the order of minutes, such as FLARE (Wang et al., 2017), SPARK (Kim et al., 2017), iTango (Lee et al., 2017) and Cal-Light (Lee, Hyun, Jung, Hannan, & Kwon, 2017). In this way, the PPI is recorded and the signal is integrated in this specific time window, thereby maintaining the advantages of integration methods but also resolving the PPI dynamics on the order of minutes. Further, these new time-gated integration methods also improve the signal-to-noise ratio of integration methods, as they reduce the background accumulation by shortening the time window of signal integration.

SPARK and iTANGO have very similar designs. In this chapter, we will describe in detail the application of SPARK to detect protein-protein interactions with a temporal control.