Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1

Background

The activities of proteins in cells can be regulated by various mechanisms including intramolecular interaction (Miernyk and Thelen, 2008). Several protein-protein interaction assays have been adopted for studying intramolecular interaction such as co-immunoprecipitation and protein complementation assays (PCAs). Despite being widely employed, there are known limitations of these conventional assays. For instance, co-immunoprecipitation is not ideal for detecting low-affinity interaction. Even though PCAs provide good sensitivity, the reporter fragments may hinder the interaction of interest (Ohad et al., 2007; Miernyk and Thelen, 2008). Proximity ligation assay (PLA) is an innovative technology that allows in situ detection of protein-protein interactions. The protein targets are first labeled by their specific primary antibodies from different species, followed by the recognition of species-specific secondary antibodies conjugated to oligonucleotides (PLA probes). Proximity ligation of the oligonucleotides generates the templates for single-stranded rolling circle amplification. The resultant products are detected with fluorescence-labeled complementary oligonucleotide detection probes. PLA offers exceptional specificity and sensitivity as target-specific antibodies and single-stranded rolling circle amplification are involved. The resultant products are detected with fluorescence-labeled complementary oligonucleotide detection probes, and PLA signals can be visualized and quantified by using fluorescence microscopy (Weibrecht et al., 2010). Although the technology has been available for over a decade, the possibility of using PLA for investigating protein intramolecular interaction has not been explored.

Engulfment and cell motility 1 (ELMO1) is the regulatory subunit of the bipartite ELMO1-dedicator of cytokinesis 180 (DOCK180) guanine nucleotide exchange factor (GEF) for the activation of the small GTPase Rac1. ELMO1 possesses several distinct domains: a Ras binding domain (RBD), ELMO inhibitory domain (EID), ELMO domain, pleckstrin homology (PH) domain, ELMO autoregulatory domain (EAD) and a PXXP motif (Figure 1). Similar to many other GEFs, the activity of ELMO1 is modulated by the intramolecular interaction between its EID and EAD which allows ELMO1 to adopt a closed autoinhibitory conformation at the basal state (Patel et al., 2010) (Figure 1). The relief of ELMO1 autoinhibition is required for the targeting of the complex of ELMO1-DOCK180 to the plasma membrane where it stimulates Rac1 (Patel et al., 2011). However, the mechanism that alleviates the autoinhibition of ELMO1 remains elusive. We have recently reported that the neuronal adaptor protein FE65 interacts with ELMO1 EAD and to disrupt the interaction of ELMO1^1-315 (comprises of ELMO1 amino acid residues 1-315) and ELMO1^315-727 (comprises of ELMO1 amino acid residues 315-727) fragments, which possess the EID and EAD respectively, by using both classical protein binding assay and PLA (Li et al., 2018). Therefore, we demonstrate for the first time that PLA can be applied for examining protein intramolecular interaction.

Here, we describe how to investigate the intramolecular interaction of ELMO1 EID and EAD by using PLA. The method may also be adopted for the investigation of other protein intramolecular interactions.


Figure 1. Schematic diagram shows the subdomains and the intramolecular interaction of ELMO1. At basal stage, the EAD and EID interact intramolecularly (indicated with double headed arrow) to form an autoinhibitory conformation. The disruption of the intramolecular interaction alleviates the autoinhibition for the subsequence Rac1 activation. Numbers denote the amino acid residue number in ELMO1. RBD: Ras-binding domain; EID: ELMO inhibitory domain; ELMO: ELMO domain; PH: Pleckstrin homology domain; EAD: ELMO autoregulatory domain; PXXP: PXXP motif.