FOR, a Novel Orphan Nuclear Receptor Related to Farnesoid X Receptor

Han-Jong Kim,David D. Moore,Yun-Bo Shi,Young-Woo Seo,Heonjoong Kang,Won-Sun Kim,Hyuk-Bang Kwon,Tosikazu Amano,Dong Hwan Won,Hueng-Sik Choi,Jee-Young An,Ann Marie Zavacki,Sabyasachi Sanyal

doi:10.1074/jbc.m111795200

Abstract

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that the FOR gene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situ hybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer protein gene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.

Highlights

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR
We describe the isolation and characterization of a novel Xenopus orphan nuclear receptor, FOR, that associates with retinoid X receptor (RXR) and shares extensive sequence similarity to the orphan nuclear receptor FXR
A single nucleotide insertion was found at the position of amino acid number 501 in the C-terminal region of FOR2, relative to FOR1, which caused an addition of 15 amino acids and eliminated the classical AF-2 consensus motif found in FOR1 (Fig. 1A)

Summary

Introduction

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. The nuclear receptors modulate target gene transcription by direct binding to specific DNA sequences, called hormone response elements (HRE), which are generally located in the promoter of the specific target genes Both classic nuclear hormone receptors and orphan nuclear hormone receptors consist of four or five different modules or domains; A/B, C, D, E, and F [1]. A large number of orphan nuclear receptor genes have been discovered by several different approaches These include 1) screening cDNA libraries with conventional receptor cDNA probes at relaxed stringency [5] or with degenerate oligonucleotides based on the conserved regions [6], 2) performing PCR with degenerate oligonucleotide PCR primers from the DBD [7, 8], 3) screening cDNA libraries using nuclear receptor ligand binding domains (LBD) or receptor interaction domains of coactivators as bait in a yeast two-hybrid system [9, 10]

Methods

Results

Conclusion