The nuclear receptor superfamily: a personal retrospect on the first two decades

Abstract

Although lipophilic hormones (steroids, thyroid hormones, and vitamin D3) were isolated in the first part of the 20th century, their mediators remained elusive until the synthesis of radiolabeled estradiol by Jensen and Jacobsen in 1960, which allowed the identification of one or more estradiol-binding proteins. The finding that this protein was translocated from cytoplasm to nucleus upon hormone administration suggested that there was a link between control of transcription and the physiological action of the hormone (1). This concept was supported by the work of the Clever and Ashburner laboratories (2) showing that chromosomal puffing in insect polytene chromosomes was triggered by ecdysteroids. The subsequent identification of transcriptionally responsive genes in vertebrate steroid hormone target tissues, together with the finding that the glucocorticoid receptor (GR), could exhibit a modular structure with separable hormone- and DNA-binding sites (3), and also that a MMTV (mouse mammary tumor virus) GR response element (GRE) had the characteristic properties of a transcriptional enhancer element, led to a model of steroid hormone action by postulating that steroid hormone receptors are ligand-regulated enhancer-binding transcriptional factors (Refs. 4 and 5 and references therein). At this stage, the cloning of steroid receptors became an imperious necessity, because reverse genetic studies were obviously required to elucidate the molecular basis of this model. Twenty years have passed since the human GR cDNA was cloned by Ron Evans and his collaborators (6, 7); concomitantly, using antibodies prepared by Geoffrey Greene and Elwood Jensen against purified human estrogen receptor (ER), together with oligonucleotide probes derived from its amino acid sequence, we isolated and sequenced ER cDNA clones (8) and showed that one of them contained the entire open reading frame of the ER now known as ERα (9, 10). The GR and the ER, which incidentally were the first RNA polymerase II transcription factors to be cloned, exhibited a surprising homology with the v-erbA viral oncogene whose cellular counterpart, the c-erbA protein, was shown shortly afterward to be the thyroid hormone receptor (TR) (11, 12). This provided the first indication that steroid receptors could belong to a superfamily of structurally related receptors interacting with chemically unrelated ligands (13). A comparison of the above receptors with the chicken ER revealed six regions of homology (A to F, from the N-terminal to the C-terminal end), two of which (C and E) being highly conserved (14). In vitro studies showed that regions E and C were the ligand binding domain (LBD) and the DNA binding domain (DBD), respectively (15). This was unequivocally confirmed by the end of 1986, when Stephen Green demonstrated in our laboratory that a chimeric receptor, in which he had swapped region C of ER for that of GR, could bind estradiol but not activate a chimeric estradiol-responsive gene (vit-tk-CAT), whereas it activated a glucocorticoid-responsive gene (MMTV-CAT) in the presence of estradiol, but not in the presence of a glucocorticoid (16). Using a reciprocal construct, Vijay Kumar et al. (17) demonstrated that, in the presence of a glucocorticoid, the chimeric receptor could activate the expression of the vit-tk-CAT reporter gene, but not of MMTV-CAT. These experiments not only unequivocally demonstrated the modular structure of NRs, in which the DBD and the LBD can function independently (see also Ref. 18), but even more importantly they indicated how such chimeric receptors could be used to characterize the ligand requirement of a novel putative member of the NR superfamily: hooking the LBD of a novel receptor candidate to the DBD of either the ER or the GR would result, upon addition of a cognate agonistic ligand, in activation of an estrogen- or a glucocorticoid-responsive reporter gene, respectively, unless this ligand is endogenously produced by the transfected cultured cells, or the LBD of the novel receptor is constitutively transcriptionally active. The value of this approach was illustrated shortly afterward by the independent discovery of the first RAR [retinoic acid (RA) receptor] α by Martin Petkovitch in our laboratory (19) and Vincent Giguère in Ron Evans’ laboratory (20), which was followed by the subsequent finding of two additional RAR isotypes (subtypes), RARβ (21), and RARγ (22, 23). Clearly, in 1987, the stage was set to clone all members of the NR superfamily, based on sequence homology of their DBDs and LBDs. This was achieved in 1999: 48 human and 49 mouse NRs have been cloned (examination of the human and mouse genome sequences did not reveal any additional member of the superfamily), among which 24 are orphan receptors for which no ligand have yet been discovered (24, 25). Which ones will remain true orphans needs to be established (25, 26). The functional dissection of the ER demonstrated that it is a ligand-regulat