Chambon and Evans set to work deconstructing the glucocorticoid and estrogen receptors. By creating mutations at different spots and probing which activities the resulting proteins lost, they dissected the receptor into three domains: one bound hormone, one bound DNA, and one activated target genes. The structure of each domain strongly resembled the analogous one in the other receptor.
Chambon and Evans wanted to match other members of the growing receptor gene family with their chemical triggers. Because the DNA- and ligand-binding regions functioned independently, it was possible to hook the DNA-binding domain of, say, the glucocorticoid receptor to the ligand-binding domain of another receptor whose ligand was unknown. The ligand for that receptor would then activate a glucocorticoid-responsive test gene.
Evans would use this method to identify ligands for several novel members of the nuclear receptor family, and both he and Chambon exploited it to discover a physiologically crucial receptor. In the late 1970s, scientists had suggested that the physiologically active derivative of vitamin A, retinoic acid, could exert its effects by binding to a nuclear receptor. This nutrient is essential from fertilization through adulthood, and researchers were eager to understand its activities on a molecular level. During embryonic development, deficiency of retinoic acid impairs formation of most organs, and the compound can hinder cancer cell proliferation. So Chambon set out to find a receptor that responded to retinoic acid. He isolated a member of the nuclear receptor gene family whose production increased in breast cancer cells that slowed their growth upon exposure to the chemical. Simultaneously, Evans identified the same protein. He tested whether more than a dozen compounds activated an unknown receptor and one passed: retinoic acid.
Remarkably, in 1986, the two scientists had independently — and unbeknownst to each other — identified the same retinoic acid receptor, a molecule of tremendous significance. The discovery of this molecule provided an entry point for detailing vitamin A biology.
Rx for lonely receptors: RXR
The list of presumptive nuclear receptors was growing quickly as scientists realized that the common DNA sequences provided a handle with which to grab these molecules from the genome. Because their chemical activators weren’t known, they were called ‘orphan’ receptors, and researchers were keen on ‘adopting’ them to ligands. Some of these ligands, they reasoned, would represent previously unknown classes of gene activators. The test system that Chambon and Evans used to match up retinoic acid with its receptor, in which they stitched an unknown ligand-binding domain to a DNA-binding domain for a receptor with known target sequences, could be harnessed to accomplish this task.
Evans had identified some potential nuclear receptors from fruit flies. He decided to pursue a human orphan receptor that closely resembled one of these receptor genes, reasoning that a protein that functioned in both flies and mammals was likely to perform an important job.
This receptor responded to retinoic acid in intact cells but did not bind it in the test tube, so Evans called it the retinoid X receptor (RXR), thinking that its ligand was some retinoic acid derivative. In cells, enzymes convert retinoic acid to metabolites and it seemed possible that one of these compounds was RXR’s ligand. In 1992, Evans’s group and one at Hoffmann-La Roche discovered that 9-cis-retinoic acid, a stereoisomer of retinoic acid, could activate RXR, identifying the first new receptor ligand in 25 years. This finding launched the orphan receptor field because it provided strong evidence that the strategy could unearth previously unknown ligands.
In the meantime, Chambon had found that the purified retinoic acid receptor, in contrast to the estrogen receptor, did not bind efficiently to its target DNA. Other nuclear receptors, too, needed help grasping genes. In the test tube, the retinoic acid, thyroid hormone, and vitamin D3 receptors could attach well to their target DNA only when supplemented with cellular material, which presumably contained some crucial substance. Chambon and Michael Rosenfeld independently purified a single protein that performed this feat, and it turned out to be none other than RXR. This ability of RXR to pair with other receptors — forming so-called heterodimers — would turn out to be key for switching on many orphan receptors. These heterodimeric couplings yield large numbers of distinct gene-controlling entities.
Chambon revealed the power of mixing and matching in these molecular duos through his thorough and extensive genetic manipulations in mice. He has shown that vitamin A exerts its wide-ranging effects on organ development in the embryo through the action of eight different forms of the retinoic acid receptor and six different forms of RXR, interacting with each other in a multitude of combinations.
Clinical applications of the superfamily work
The concept of RXR as a promiscuous heterodimeric partner for certain nuclear receptors led to the unexpected identification of a number of clinically relevant receptors. These proteins include the peroxisome proliferator-activated receptor (PPAR), which stimulates fat-cell maturation and sits at the center of Type 2 diabetes and a number of lipid-related disorders; the liver X receptors (LXRs) and bile acid receptor (FXR), which help manage cholesterol homeostasis; and the steroid and xenobiotic receptor (PXR), which turns on enzymes that dispose of chemicals that need to be detoxified, such as drugs.
Because the nuclear receptors wield such physiological power, they have provided excellent targets for disease treatment. The anti-diabetes compounds glitazones, for example, work by stimulating PPAR, and the clinically used lipid-lowering medications called fibrates work by binding a closely related receptor, PPAR. Retinoic acid therapy has dramatically altered the prognosis of people with acute promyelocytic leukemia by triggering specialization of the immature white blood cells that accumulate in these individuals. The three-dimensional structure of nuclear receptors with and without their ligands, which Chambon and his colleagues first solved, promises to accelerate drug discovery in the whole field.
Nuclear hormone receptors have touched on human health in other ways as well. Genetic perturbations in the genes for these proteins cause a variety of illnesses. For example, certain forms of rickets arise from mutations in the vitamin D receptor and several disorders of male sexual differentiation stem from defects in the androgen receptor.
The discoveries of Jensen, Chambon, and Evans revealed an unimagined superfamily of proteins. At the start of this work almost 50 years ago, no one would have anticipated that steroids, thyroid hormone, retinoids, vitamin D, fatty acids, bile acids, and many lipid-based drugs transmit their signal through similar pathways. Four dozen human nuclear receptors are now known, and scientists are working out the roles of these proteins in normal and aberrant physiology. These discoveries have revolutionized the fields of endocrinology and metabolism, and pointed toward new tactics for drug discovery.
by Evelyn Strauss
Key publications of Pierre Chambon
Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J.M., Argos, P., and Chambon, P. (1986). Human oestrogen cDNA: sequence, expression, and homology to v-erb-A. Nature. 320, 134–139.
Petkovich, M., Brand N.J., Krust, A., and Chambon, P. (1987). A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 330, 444–450.
Green, S. and Chambon, P. (1987). Oestradiol induction of a glucocorticoid-responsive gene by a chimaeric receptor. Nature. 325, 75–79.
Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J.Y., Staub, A., Garnier, J.M., Mader, S., and Chambon, P. (1992). Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 68, 377–395.
Bourguet, W., Ruff, M., Chambon P., Gronemeyer H., and Moras, D. (1995). Crystal structure of the ligand binding domain of the human receptor RXRD. Nature. 375, 377–382.
Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. FASEB J. 10, 940–954.
Key publications of Ronald Evans
Hollenberg, S., Weinberger, C., Ong, E.S., Cerelli, G., Thompson, E.B., Rosenfeld, M.G., and Evans, R.M. (1985). Primary structure and expression of a functional human glucocorticoid receptor of cDNA. Nature. 318, 635–641.
Giguere, V., Ong, E.S., Segui, P., and Evans, R.M. (1987). Identification of a receptor for the morphogen retinoic acid. Nature. 330, 624–629.
Mangelsdorf, D.J., Ong, E.S., Dyck, J.A., and Evans, R.M. (1990). Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 345, 224–229.
Kliewer, S.A., Umesono, K., , and Evans, R.M. (1992). Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone, and vitamin D3 signaling. Nature. 355, 446–449.
Heyman, R.A., Mangelsdorf, D.J., Dyck, J.A., Stein, R.B., Eichele, G., Evans, R.M., and Thaller, C. (1992). 9-cis Retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 68, 397–406.
Mangelsdorf, D.J. and Evans, R.M. (1995). The RXR heterodimers and orphan receptors. Cell. 83, 841–850.
Keypublications of Elwood Jensen
Jensen, E.V. and Jacobson, H.I. (1960). Fate of steroid estrogens in target tissues. In Biological Activities of Steroids in Relation to Cancer, G. Pincus and E.P. Vollmer, eds., Academic Press, New York, pp. 61–174.
Jensen, E.V. and Jacobson, H.I. (1962). Basic guides to the mechanism of estrogen action. Rec. Prog. of Hor. Res. 18: 387–414.
Jensen, E.V., Suzuki, T., Kawashima, T., Stumpf, W.E., Jungblut, P.W., and DeSombre, E.R.(1968). A two-step mechanism for the interaction of estradiol with rat uterus. Proc. Nat.l Acad. of Sci. USA. 95, 632–638.
Jensen, E.V. and DeSombre, E.R. (1973). Estrogen-receptor interaction: Estrogenic hormones effect transformation of specific receptor proteins to a biochemically functional form. Science. 182, 126–134.
Jensen, E.V., Block, G.E., Smith, S., and DeSombre, E.R. (1973). Hormonal dependency of breast cancer. Rec. Res. of Canc. Res. 42, 55–62.
Jensen, E.V., Greene, G.L., Closs, L.E., DeSombre, E.R., and Nadji, M. (1982). Receptors reconsidered: a 20-year perspective. Rec. Prog. of Hor. Res. 38, 1–34.