Inter-subunit interactions between glutamate-like receptors in Arabidopsis

The plant Glutamate-Like Receptors (GLRs) are homologs of animal ionotropic glutamate receptors (iGluRs), and are hypothesized to be potential amino acid sensors in plants. Genetic studies of proteins from this family implicate individual GLRs in a diversity of physiological roles in plants. Recently, amino-acid gated channel activities have been proven for a few plant GLRs, suggesting that at least some of the functional mechanisms are conserved between plant GLRs and animal iGluRs. Animal iGluRs generally form heterotetramers, and the ligand-binding specificity and channel functionality is determined by interaction between the subunits. In order to investigate whether plant GLRs interact with each other, a modified yeast-2-hybrid system (mbSUS) approach was taken on 15 of the 20 Arabidopsis GLRs to identify potential interaction partners. Using this approach, we have successfully identified GLR subunits that are capable of interacting with multiple other GLRs. Unlike iGluRs, sequence similarity between the subunit was not correlated with the likelihood of interaction among 2 given subunits. Interactions between selected GLRs (GLR1.1, 2.9, 3.2, and 3.4) were further tested in another heterologous expression system, mammalian HEK293 cells, using Förster resonance energy transfer (FRET). Two separate approaches (sensitized FRET and acceptor photobleaching) indicated that GLRs 1.1 and 3.4 are capable of forming homomers, whereas other combinations did not result in detectable FRET between the subunits.