A novel human RARβ protein is identified in breast cancer cell lines. Western blots of nuclear (N) and cytoplasmic (C) fractions of normal HMECs (AG11132) and breast cancer cell lines (Hs578T, MCF-7, MDA-MB-231, MDA-MB-435, and ZR-75–1) used a polyclonal antibody raised against a C-terminal RARβ epitope. The positions of molecular mass markers are indicated to the right. An ≈48.8-kDa protein (RARβ₂) is detected exclusively in the nucleus of normal HMECs. RARβ proteins with molecular masses of ≈58.8 kDa (RARβ*) and 40.6 kDa (RARβ₄) are apparent in the nucleus and cytoplasm of tumor cell lines only (five of five). The total protein stain and topoisomerase II (with a mass of 170 kDa) immunoblot from duplicate samples are included as a loading control and to confirm the specificity of nuclear protein extraction, respectively.

Northern and RT-PCR analysis of RARβ₂ and RARβ₄ expression in mammary cells. (A) Northern blot of RNA from normal HMECs (AG11132) and breast tumor cell lines (Hs578T, MDA-MB-231, MDA-MB-435, MCF-7, and ZR-75–1) hybridized with either probe RARβ₂ or probe RARβ₂/β₄. The sizes of the resulting bands are indicated to the right. There were no RARβ transcripts visible in MCF-7 and MDA-MB-231 cells. Two RARβ transcripts are present at high levels in Hs578T and MDA-MB-435 cells, and at low levels in ZR-75–1 and normal AG11132 cells with probe RARβ₂/β₄. Only the higher molecular mass (3.1-kb) mRNA hybridized to probe RARβ₂. Membranes were subsequently stripped and rehybridized with 36B4, a loading control. (B) RARβ₂ and RARβ₄ expression was analyzed by using a sensitive RT-PCR assay. The region amplified spans the RARβ₄ splice junction. RNAs used in this experiment were separate aliquots of the same RNA used in the Northern blot assay. After transfer onto a membrane, blots were hybridized with an oligonucleotide specific for RARβ₂ or for RARβ₂/β₄, followed by autoradiography. The sizes of the resulting bands are indicated to the right. Passages 19 and 9 (p19, p9) were examined in the normal HMECs.

Comparison of RARβ₂ and RARβ₄ mRNA and protein structures. Protein domains (A–F) of the RARβ isoforms are depicted to scale above (RARβ₂) or below (RARβ₄) diagrams representing their respective mRNA coding sequences. The molecular masses (in kDa) are the theoretical values predicted on the basis of amino acid sequence. RARβ₂ nucleotide sequence from 269 to 625 is deleted from RARβ₄ by alternative splicing in exon 5. The nucleotide positions of the translation start and stop sites are indicated with short arrows along the mRNA diagrams. Note that the RARβ₂ translation start site is included within the sequence deleted from RARβ₄. The RARβ₄ translation begins within the C region, resulting in loss of the AF-1 and half of the DNA-binding domain. Oligonucleotide primers used for cloning or detection of RARβ₂ and RARβ₄ transcripts are denoted by > and <. The positions of sequence complementary to RARβ₂- or RARβ₂/β₄-specific probes are indicated by bars at the exon-5 end.

In vitro translation of RARβ₄ candidate ORFs. (A) Transcripts used for in vitro translation assays are diagrammed. The translation start site for each transcript is indicated by an arrow, and the resulting protein domains (A–F) are denoted by open boxes. (B) Comparison of in vitro translation products RARβ₂, RARβ₄259wt, RARβ₄259mut, and RARβ₄448 with nuclear protein extracts from normal HMECs (AG11132) and breast cancer cells (MDA-MB-435). After SDS/PAGE, proteins were immunoblotted using the anti-RARβ antibody. Migration of molecular mass markers is indicated in kDa to the right of the figure. The 40.6-kDa protein observed in breast tumor cells (MDA-MB-435) corresponds to the product of translation initiated at nucleotide 448 (RARβ₄448). The 58.8-kDa RARβ isoform in tumor cells is the size obtained from in vitro translation of the full RARβ₂ coding sequence. The 40.6-kDa RARβ protein was a product of all of the in vitro translation reactions, probably caused by leaky translation by the reticulocyte lysate.

Protein localization of (His)₆-RARβ₂ and (His)₆-RARβ₄ in normal HMECs and breast tumor cells. Immunocytochemistry was performed on normal HMECs (AG11132) and breast tumor cells (MDA-MB-231) transiently transfected with pTag(RARβ₂) and pTag(RARβ₄). (His)₆-tagged proteins were detected with a FITC-conjugated secondary antibody (green) with a 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). The majority of the (His)₆-RARβ₂ protein is nuclear in the normal (A) and the breast tumor cells (D). (His)₆-RARβ₄ protein in normal (B) and tumor (E) cells was detected both in the nucleus and in filamentous structures in the cell cytoplasm. The image in B was deconvolved to confirm nuclear localization of the FITC signal (C). Nuclear (His)₆-RARβ₄ localized to structures resembling nuclear bodies (an example is indicated with an arrow). (Bars = 5 μm.)

uORFS in the 5′ untranslated region of the RARβ₄ transcript. Nucleotides are numbered relative to the transcription start site. Nucleotides 269–625 are removed from exon 5 in the RARβ₄ transcript, resulting in a novel exon 5 and exon 6 junction (*). Translation of RARβ₄ begins at +448 of the RARβ₄ transcript, indicated above right-angled arrow. uORFs in the 5′ (UTR) are indicated above their coding sequence (filled arrowheads = exon junctions).