A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors

Abstract

The orphan receptors Rev-erbα and Rev-erbβ are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbα is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbβ. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbα and Rev-erbβ proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbα is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbα deletion mutants showed that the NLS is located whithin a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbβ fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located whithin the DBD. GFP/DBD.RORα or GFP/DBD.RORβ remained cytoplasmic, in contrast to GFP/DBD.Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD.Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD.RORα towards the Rev-erbα residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

“…The mechanism for nonnuclear localization of HNF4␣ induced by PKC may involve interference with the positively charged residues (R76, R77, R80, and K81) that surround S78, which comprise an NLS. This region has also been shown to be an NLS for other NRs (60)(61)(62)(63)(64), although until now there has not been a detailed examination of the effect of PKC phosphorylation of 'S78' on nuclear localization. Here, we show that 'S78' (T162) in the RXR␣ DBD is phosphorylated by PKC and that introduction of a single negative charge at that position results in a remarkable cytoplasmic localization.…”

“…The sequence of the other NR DBDs with a Ser or Thr at that position also match the PKC consensus but to varying degrees. This region of the NR DBD has been shown to be important for nuclear localization of RXR, VDR, SXR, and the Rev-erb receptors (60)(61)(62)(63)(64). In addition, the conserved serine has been shown to be a target of PKC phosphorylation in VDR (S51) and PPAR␣ (T129) (35,38).…”

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

“…Further, although Rev-erb␣/␤ have been shown to be predominantly nuclear proteins (87), the effect of heme on Rev-erb␤ subcellular localization has not been tested. We transiently transfected HEK293 cells with expression vectors for the wildtype, H568F, and H568F/C384A forms of Rev-erb␤, fractionated the cells into cytosolic and nuclear compartments, and determined the relative amounts of Rev-erb␤ in each fraction (Fig.…”

Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1

“…However, we did not detect significant NLS activity in the corresponding regions of LRH-1. The absence of a NLS in the LBD has also been reported for another orphan receptor Rev-erb [41]. Structural and biochemical analysis showed that the activation of mouse LRH-1 is ligand-independent, whereas the binding of phosphatidyl inositol can regulate the activity of human LRH-1 and mouse and human SF-1 [13,42].…”