A genome threading algorithm was employed by Inpharmatica to identify a
number of proteins with a predicted structure similar to that of the ligand binding
domain (LBD) of nuclear receptors (NRs), an approach that has been successfully
used to annotate the yeast transcription factor Oaf1 (Phelps et al, 2006). This work
focuses on one such protein termed NR3, which is identical to TRPC4AP or TRUSS,
a protein proposed to function as a scaffold protein in cell signalling processes and as
a cell cycle regulator. It is conceivable that NR3 does not function as a transcription
factor in contrast to bona fide NRs and that the putative LBD may function as an
allosteric switch to control functional activity.
To investigate the idea that NR3 may possess a fold similar to the LBD of NRs
preliminary structural work has been undertaken, which has suggested the putative
LBD folds into an autonomous domain as it is region resistant to proteolysis. In
addition, the potential role of the putative LBD fold as a molecular switch was
examined by using constitutively active fusion proteins in reporter gene assays. It was
determined that the putative NR3 LBD acts in a repressive manner, potentially due to
the alteration in subcellular localisation exerted by the putative NR3 LBD on the
fusion protein. To further assess the role of the putative LBD a ligand screen was
undertaken to identify compounds that may reduce its repressive activity, however no
ligand was identified and it is conceivable NR3 may act in a ligand independent
manner similar to some orphan receptors.
Initial analysis of NR3 function indicates that its expression may have a positive
effect on cell proliferation. To further assess the role of NR3 protein interaction
assays were established to screen for binding partners. This identified the E3 ubiquitin
ligase component DNA damage-binding protein 1 (DDB1) as an interacting protein
involved in the regulation of cell cycle progression and DNA repair. Mapping studies
suggest NR3 binds to the substrate docking site of DDB1 and further analysis showed
NR3 to be ubiquitinated, affecting the stability of the protein. It is reported that the
arylhydrocarbon receptor binds to a DDB1 complex, which then acts as a ligand
regulated E3 ubiquitin ligase complex (Ohtake et al, 2007). This raises the possibility
that NR3 may act in functionally analogous manner. To address NR3 function within
the whole organism a targeting vector designed to inactivate the NR3 gene has been
generated and currently a conditional knockout mouse line are being bred.