Identification, cloning, and expression of human estrogen receptor-α36, a novel variant of human estrogen receptor-α66,☆☆

https://doi.org/10.1016/j.bbrc.2005.08.226Get rights and content

Abstract

The identification and subsequent cloning of the 66-kDa human estrogen receptor (here termed hER-α66), its 46-kDa splice variant hER-α46, and the closely related hER-β have had a profound impact on the generation of new understanding of estrogen-mediated functions and led to progress in diagnosis and treatment of human breast cancer. However, a persistent problem has been that not all findings previously reported in estrogen-stimulated cell proliferation can be explained through the known properties of the different estrogen receptors described. As the consequence of a search for alternative mechanisms to account for these different findings, we have now identified, cloned, and expressed in HEK 293 cells a previously unrecognized 36-kDa variant of hER-α66, termed hER-α36. hER-α36 differs from hER-α66 since it lacks both transcriptional activation domains (AF-1 and AF-2) but it retains the DNA-binding domain, and partial dimerization and ligand-binding domains of hER-α66. It also contains three myristoylation sites postulated to direct ER-α36 to the plasma membrane. It is concluded that ER-α36 is a unique variant of ER-α66; ER-α36 is predicted to function as a dominant-negative effector of hER-α66-mediated estrogen-responsive gene pathways and has the potential to trigger membrane-initiated mitogenic estrogen signaling.

Section snippets

Materials and methods

Homology search and cloning of ER-α36. The sequence homology searches were performed with Blastn and Blastp programs of National Center for Biotechnology Information (NCBI) and used to look for short, nearly exact matches using DNA sequences (20–50 bp in length) encoding the ligand-binding domain of ER-α66. We found one full-length cDNA clone (GenBank Accession No. BX640939) from a human uterus cDNA library that was matched exactly to a DNA sequence encoding the ligand-binding domain of hER-α66

Identification of a novel isoform of hER-α66

Previous reports indicated that three predominant bands of 35–39, 46, and 66 kDa were present in Western blots probed with the anti-ER-α antibodies raised against the ligand-binding domain of hER-α66 [16], [18]. Although these and other reports consistently noted a predominant band at 35–39 kDa, there were no reports that described what this protein band represented. In confirmation, we observed three protein bands (36, 46, and 66 kDa) in Western blot analysis of ER-positive MCF7 breast cancer

Discussion

In this study, a novel variant of hER-α66 termed hER-α36 has been identified and cloned; the cDNA that was cloned expresses the predicted 36-kDa protein in HEK 293 cells; Western blot analysis of lysates prepared from HEK 293 cells expressing the cloned cDNA revealed that the hER-α36 protein was recognized by an antibody raised against an epitope common to hER-α36, hER-α46, and hERα-66. The novel hER-α isoform is the product of a transcript initiated from a previously unidentified promoter in

Acknowledgments

We thank Dr. Zafar Nawaz for the expression vectors. The technical assistance of Megan Coleman is also acknowledged. This work was supported by National Institutes of Health Grant RO1 CA84328 (to Z.-Y.W.) and RO1s CA84400 and CA66029 (to T.F.D.).

References (18)

There are more references available in the full text version of this article.

Cited by (340)

  • An overview on Estrogen receptors signaling and its ligands in breast cancer

    2022, European Journal of Medicinal Chemistry
    Citation Excerpt :

    The closing pocket site of the ligand is considered essential for its direct interaction in the transactivation function AF-2 [32]. ER-α36, a novel variant of ER-α and an isoform of ER-α66, was first of all cloned and identified by Wang et al., in 2005 [33]. Human ER-α36 has a molecular weight of approximately 35.7 kDa and lacks both transcriptional activation domains (AF-1 and AF-2), but contains partial ligand-binding domains, DNA binding domains and dimerization domains.

  • Cancer stem cells in TNBC

    2022, Seminars in Cancer Biology
    Citation Excerpt :

    TNBC cells, being negative for ER, PR, and HER2 expression, are generally believed to lack intracellular estrogen signal transduction and are therefore insensitive to endocrine therapy. Interestingly, the novel estrogen variant ER-α36, which lacks the transcriptional activator domains AF-1 and AF-2, has recently been discovered [16]. The well-known TNBC cell lines MDA‑MB‑231 and MDA‑MB‑436 all expressed high levels of ER‑α36, which can activate the MAPK/ERK and PI3K/AKT signaling pathway downstream of the wild-type estrogen receptor ER-a66 [17,18].

View all citing articles on Scopus

This is manuscript number 17432-MEM from The Scripps Research Institute.

☆☆

Abbreviations: ER, estrogen receptor; AF, activation function; E2β, 17β-estradiol; E2α, 17α-estradiol; HEK 293 cells, human embryonic kidney 293 cells; NO, nitric oxide; eNOS, endothelial NO synthase.

View full text