Retinoic acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new class of nuclear receptor isoforms

Abstract

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

Figure 1

Isolation of novel RARα isoforms. (A) Schematic representation of the genomic structure of the human RARα gene (adapted from refs 24–26 and our unpublished observations). Exons, numbered 1–10, are represented as black boxes. Exons encoding the different domains, A–F, are indicated. Two promoters, P1 and P2, located in front of exons 1 and 3, give rise to the alternative domains A1 and A2, present in the isoforms RARα1 and RARα2 [detailed in (C)]. (B) Trace from the sequencing of the junction between exons 2 and 6 of the RARα1ΔBC isoform. The predicted amino acid sequence of the junction following the open reading frame is indicated. (C) Schematic representation of the human RARα isoforms. The predicted sequence of RARα1ΔB has an altered reading frame from R60 until the stop codon at position 100, designated the C′ domain. Oligonucleotides used as RT–PCR primers, R6 and RARD/E, or as Southern blotting probes, RAR21 and RAR22, are drawn at their approximated positions. (D) Predicted amino acid sequence of novel isoforms RARα1ΔBC and RARα1ΔB. Amino acids of the junction [detailed in (B)] are underlined.

Figure 2

RARα1ΔBC mRNA is expressed at the highest levels in fresh hematopoietic cells. (A) Southern blot of semi-quantitative competitive RT–PCR products using different quantities of total RNA (1–0.1 µg, indicated at the top of the blots) from one B-CLL patient (no. 1) and one normal purified CD19⁺ B cell sample (no. 1) using primers R6 and RARD/E. Specific bands corresponding to RARα1, RARα1ΔB and RARα1ΔBC were detected using the RAR21 probe (left). The same blot was reprobed with RAR22 to the A1–D junction (right), which is specific for the RARα1ΔBC isoform. The size of the PCR products is indicated on the right. (B) As (A) with 1 µg RNA from different human tissues using the RAR21 probe.

Figure 3

The RARα1ΔBC protein is expressed in hematopoietic myeloid cells and is nuclear. (A) Western blot analysis using 10 µg whole cell extracts from COS cells transfected with pSG5-RARα1 (lane 1) or pSG5-RARα1ΔBC (lane 2), BM purified CD34⁺ cells (lane 7), BMMC (lane 8) and cytoplasmic and nuclear extracts from HL60 (lanes 3 and 4, respectively) and NB4 (lanes 5 and 6, respectively) cells, using the RARα antibody RPα(F). (B) Western blot analysis of purified recombinant proteins (RARα1, RXRα and RARα1ΔBC) with RARα(F) (lanes 1–3) or RPα1Δ made against the A1–D junction (lanes 4–6), showing that this antibody is specific for the RARα1ΔBC isoform. (C) Immunofluorescence analysis of COS cells transfected with RARα1, labeled with a mAb against RARα (Ab9α) and visualized with cyanin 5 (cy5)-conjugated anti-mouse antibody. (D) As (C) except that COS cells were transfected with RARα1ΔBC, labeled with RPα1Δ and visualized with a FITC-conjugated anti-rabbit antibody, showing that RARα1ΔBC, like RARα1, is located in the nucleus.

Figure 4

RARα1ΔBC alone does not bind a DR5 response element, but can bind RXRα and be found in the complex that binds DR5. (A) EMSA using purified recombinant proteins (RARα1, RXRα and RARα1ΔBC) shows that RARα1ΔBC does not bind the RARE DR5 (lane 2), unlike RARα1 and RXRα (lanes 3 and 4, respectively). When RARα1ΔBC and RXRα are incubated together (lane 5), a retarded complex is observed; this complex was shifted with a RARα mAb (Ab9α, lane 6) and a RXRα mAb (4RX3A2, lane 7). A complex is also observed when RARα1 and RXRα are co-incubated (lane 8), which shifted with the same antibodies (lanes 9 and 10). (B) GST ‘pull down’ experiments using immobilized GST–RXRα or GST proteins and whole cell extracts from COS cells transfected with RARα1 (lanes 1–3) or RARα1ΔBC (lanes 4–6). Like RARα1, RARα1ΔBC directly interacts with RXRα in vitro.

Figure 5

Transactivation of a RARE through combinations of the RXRα, RARα1 and RARα1ΔBC receptors upon binding to the retinoids ATRA (white bars), RAR agonist CD336 (red bars) or RXR agonist CD2809 (green bars). RARα1ΔBC alone does not produce significant activity of the RARE₃-tk-luc reporter in COS cells (lane 4); the RXRα/RARα1ΔBC heterodimers transactivate a RARE upon binding to ATRA, CD336 or CD2809 (lane 7); RXRα/RARα1 transduces the transactivating signal mediated by ATRA and CD336 (lane 5); RARα1 fixation inhibits the transactivation mediated by CD2809 binding to RXRα (lanes 5 and 3, green bars, respectively). Luciferase activities were normalized to β-galactosidase activities. A representative experiment, done in triplicate, is shown. All the retinoids were used at 10^–6 M. The results are expressed as fold induction produced by the transfected receptors related to pSG5 empty vector in the presence of ATRA.