MOLECULAR GENETICS OF STEROID AND THYROID HORMONE RECEPTORS 
Ronald M. Evans, Ph.D., Investigator 
A central question in eukaryotic molecular biol- 
ogy is how specific DNA-binding proteins bind reg- 
ulatory sequences in target genes to influence cell 
function and fate. Dr. Evans and his colleagues have 
demonstrated that steroid, thyroid hormone, and 
retinoid (vitamin A) receptors are composed of a 
common structure and comprise a supergene fam- 
ily of ligand-dependent transcription factors. These 
receptors, in combination with their hormones, 
constitute critical regulators of complex develop- 
mental and physiologic processes. With regard to 
the products of this supergene family, one chal- 
lenge is to understand the molecular properties of 
each receptor that determine its interactions with 
the transcriptional machinery regulating gene ex- 
pression. A second, and perhaps more daunting, 
challenge is to understand the contribution of indi- 
vidual regulatory systems to the integrated and 
complex biologic responses inherent in develop- 
ment and homeostasis. These hormone receptor 
genes have been cloned and molecular techniques 
have been employed to characterize the structure- 
function relationships of individual receptor mole- 
cules, as the essential first step toward understand- 
ing higher levels of control. 
I. Determinants of Target Gene Specificity. 
The molecular specificity of the receptors for ste- 
roid and thyroid hormones is achieved by their se- 
lective interaction with DNA-binding sites, referred 
to as hormone response elements (HREs). HREs 
can differ in primary nucleotide sequence as well as 
in the spacing of their dyadic half-sites. Previously it 
was shown that the modular structure of the 
glucocorticoid and thyroid hormone receptors can 
be exploited in the study of DNA-binding specificity. 
In vitro mutagenesis has been used to interconvert 
the binding specificities of the steroid and thyroid 
hormone receptors. The target gene specificity of 
the glucocorticoid receptor can be converted to 
that of the estrogen receptor by changing three 
amino acids clustered in the first zinc finger region 
of the DNA-binding domain. A single Gly-to-Glu 
transition in this region produces a receptor that 
recognizes both glucocorticoid and estrogen re- 
sponse elements. Further replacement of five 
amino acids in the stem of the second zinc finger 
region transforms the specificity to that of the thy- 
roid hormone receptor. These findings localized 
the structural determinants required for discrimina- 
tion of HRE sequence and half-site spacing and sug- 
gest a simple pathway for the coevolution of recep- 
tor DNA-binding domains and hormone-responsive 
gene networks. 
II. Retinoic Acid Receptor (RAR). 
Retinoic acid, a metabolite of vitamin A, is capa- 
ble of inducing a complex array of developmental 
and physiologic responses in vertebrate cells and 
tissues. The actions of retinoids are believed to be 
mediated through nuclear receptors, which modu- 
late the expression of specific gene and target cells. 
Thus the discovery by Dr. Evans and his colleagues 
of the RAR offers, for the first time in a vertebrate 
system, the hope of analyzing the mechanisms of 
morphogenesis by identifying a set of developmen- 
tally controlled genes. Retinoic acid is known to 
have effects on the vertebrate limb pattern in devel- 
opment and regeneration, supporting a model in 
which a gradient of retinoic acid serves as a 
morphogen to supply positional information differ- 
entially to a developing limb. One prediction of this 
model is that the putative RAR must be expressed 
in the developing and regenerating limb anlage. 
The expression of the RAR has been investigated in 
the adult newt, whose amputated limbs are capable 
of regenerating and upon which retinoic acid can 
act to alter pattern. In situ hybridization studies 
confirm the presence of the RAR in the regenerat- 
ing limb and localize expression specifically in the 
regenerating mesodermal cells that control limb 
pattern. It was also demonstrated that the RAR 
gene is not active in the adult limb, becomes acti- 
vated after amputation (but only at the site of the 
stump) and remains active during the regeneration 
process, and then turns off once the limb is com- 
plete. These results indicate that the morphogenic 
field is established through differential activation of 
preexisting RARs. Despite the large amount of 
knowledge about the action of retinoids, specific 
genes that are activated by the receptor have yet to 
be identified. However, because of structural ho- 
mology of the RAR to the thyroid hormone recep- 
tor, it was possible to demonstrate that both 
retinoic acid and thyroid hormones can induce 
gene expression through a common HRE. This in- 
duction is unexpected, because thyroid hormones 
and retinoids are proposed to elicit different effects 
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