Molecular Genetics of Steroid and Thyroid 
Hormone Receptors 
Ronald M. Evans, Ph.D. — Investigator 
Dr. Evans is also Professor at the Gene Expression Laboratory of the Salk Institute for Biological Studies 
and Adjunct Professor in the Departments of Biology and of Biomedical Sciences at the University of 
California, San Diego. He received his Ph.D. degree in microbiology and immunology from the University 
of California, Los Angeles, School of Medicine. After postdoctoral training with James Darnell at the 
Rockefeller University, he joined the faculty of the Salk Institute. Dr. Evans is a member of the 
National Academy of Sciences. His research interests are in developmental and inducible regulation 
of gene expression. 
AN understanding of the mechanisms by 
which apparently distinct regulatory sys- 
tems integrate to modulate body function and be- 
havior poses one of the most important chal- 
lenges of modern biology. Hence we have 
focused our attention on the action of steroid, 
retinoid, and thyroid hormones in regulatory cel- 
lular and organ physiology. This field has under- 
gone an extraordinary development in the last 
several years as a consequence of the cloning and 
sequencing of the genes encoding the receptors 
for these hormones in target cells. 
It has been demonstrated that these receptors 
are all structurally related and constitute a super- 
family of nuclear regulatory proteins that are ca- 
pable of modulating gene expression in a ligand- 
dependent fashion. One challenge is to define 
each receptor's molecular properties that deter- 
mine its interactions with the transcription ma- 
chinery regulating gene expression. Another 
challenge is to elucidate the contributions of indi- 
vidual regulatory systems to the integrated and 
complex processes associated with cell growth, 
differentiation, and organ function. 
A Novel Retinoic Acid Response Pathway 
The retinoids, a group of compounds that in- 
clude retinoic acid, retinol (vitamin A), and a se- 
ries of natural and synthetic derivatives, exert 
profound effects on development and differentia- 
tion in a wide variety of systems. (The retinoic 
acid receptors are collectively designated RARs.) 
Retinoic acid has also been shown to induce the 
transcription of several genes, suggesting a role 
analogous to those of steroid and thyroid hor- 
mones. In previous studies we described the 
cloning and characterization of a retinoic acid- 
dependent transcription factor referred to as 
RARa. Additional RAR-related genes have been 
isolated, and at least three different RAR subtypes 
(a, /3, and 7) are now known in mice and humans. 
Retinoic acid receptors share homology with 
the superfamily of steroid and thyroid hormone 
receptors and have been shown to regulate 
specific gene expression by a similar ligand- 
dependent mechanism. Complicating these ob- 
servations is our recent identification of a group 
of receptors termed RXRs (retinoid X receptors) , 
which are only distantly related to the RARs. The 
discovery of this second retinoid transduction 
pathway led us to investigate its functional prop- 
erties and determine its relationship to the RARs. 
We now know that there are at least three RXR- 
related genes (termed a, j3, and 7) located at ge- 
netically distinct loci. Northern blot analyses of 
the RXRs indicate that each isoform has a unique 
pattern of expression in adult tissue and is tempo- 
rally and spatially expressed in the embryo. 
These studies suggest a role for RXRs in adult 
physiology and embryonic development. 
Binding experiments demonstrate that the RXR 
protein has low affinity for retinoic acid (RA) 
and, taken together with the transactivation stud- 
ies, indicate that the RXR ligand may be a metabo- 
lite of RA. Based on these assumptions, we de- 
vised a strategy to identify the putative 
metabolite referred to as retinoid X. The implicit 
concept of the strategy was that all-trans RA may 
be converted to retinoid X by a natural cellular 
process. Accordingly, high doses of all-trans RA 
were fed to recipient tissue culture cells. After 
allowing for metabolic conversion, material ex- 
tracted from these cells was fractionated to re- 
solve the various retinoid peaks, and each peak 
was assayed for its ability to activate the RXRs in a 
transfection assay. A specific peak, termed reti- 
noid X, was identified and characterized by mass 
spectrometry. 
Based on this process, we have now demon- 
strated that 9-cis RA is the high-affinity ligand for 
the RXR. While not previously seen in living or- 
ganisms, 9-cts RA is apparently a new and widely 
used vertebrate hormone. It transactivates RXRa 
up to 40 times more efficiently than all-trans RA 
and binds to RXR with high affinity. We also con- 
firmed that all-trans RA shows no detectable 
binding affinity for the RXR. Furthermore, each 
of the RXR subtypes (a, /3, and 7) is activated by 
9-c« RA with increased potency and efficacy rela- 
tive to all-trans RA. 
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