A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors.
The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha 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-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta 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 within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta 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. RORalpha towards the Rev-erbalpha 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.
Chopin-Delannoy
S
S
Institut de Biologie de Lille, CNRS UMR 8526, Institut Pasteur de Lille, 1 rue Calmette, 59021 Lille cedex, France.
Thénot
S
S
Delaunay
F
F
Buisine
E
E
Begue
A
A
Duterque-Coquillaud
M
M
Laudet
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D017434:Protein Structure, Tertiary; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000906:Antibodies; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C064353:NR1D1 protein, human; D019913:Nuclear Localization Signals; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins