Breast CancerResearch and Treatment18: 67-71, 1991. © 1991KluwerAcademic Publishers. Printedin the Netherlands. 13th San Antonio Breast Cancer Symposium - Plenary lecture
Steroid hormone receptors as transactivators of gene expression Bert W. O'Malley Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
Key words: DNA binding, enhancers, gene regulation, genetic diseases, heat shock proteins, response elements, steroid receptors Abstract
In the two decades since the discovery of the steroid hormone receptors, a great deal has been learned about their structure, their relationships with each other, and the target sequences (response elements) at which they regulate expression of specific genes. Analysis of receptor sequences has confirmed the existence of several domains with distinct functions in each receptor molecule, and has also indicated that the steroid receptors are members of a 'superfamily' which also includes receptors for vitamin D, retinoic acid, thyroid hormone and its related oncogene v-erbA, and a substantial number of 'receptors' whose ligands are still unidentified. The response element sequences are also closely related, and we are beginning to understand the interaction of the receptors with these response elements and with proteins bound nearby such as transcription factors. Certain naturally occurring genetic diseases have been identified with specific receptor mutations. The future promises greater understanding not only of the detailed action of the receptors of this superfamily, but of their precise role in fertility, development, and disease.
Over the past two decades, a great deal of evidence has accumulated in favor of the hypothesis that steroid hormones act at the level of nuclear D N A to regulate gene expression [1-4]. The earliest studies were qualitative and involved experiments which showed that steroid hormones: (1) caused accumulation of new species of hybridizable RNAs which did not exist prior to stimulation; (2) caused stimulation of synthesis of new specific proteins; (3) caused a corresponding increase in the cellular levels of specific mRNAs; and (4) stimulated the rate of transcription of certain nuclear genes [5]. At this point, the early 1970's, the primary pathway for steroid hormone action was defined as follows: steroid--~ (steroid-receptor)--~ (steroid-receptorDNA)---> mRNA--> protein ~ functional response (Fig. 1) [4]. Steroid enters cells by passive diffusion
and allosterically activates receptors in either the cytoplasm or nucleus. The activated receptor binds usually at the 5'-flanking region of target genes and stimulates transcription and protein synthesis. The first receptors were subsequently purified to near homogeneity and characterized as to size, charge, etc. Antibodies were developed, structural domains were postulated by proteolytic analyses, and assays of sex steroid receptors became commonplace in the diagnosis and assignment of therapy for breast cancer. Investigators isolated specific target genes for steroid hormones, defined their structure, and proved that cis-acting regulatory sequences were located nearby such genes, usually in the 5'-flanking sequences. When such sequences, termed steroid response elements (SREs), are occupied by receptors, these genes come under hormone control.
Address for offprints: B.W. O'MaIley,Department of Cell Biology,BaylorCollegeof Medicine, One BaylorPlaza, Houston,Texas 77030, USA
68
B W O'MaIly
S-{'- R l n a c t i v e
Activ 2
Protein
,
mRNA
,
mRNA PrecursorJ
Fig. 1. Molecularpathway for steroid hormone action. S = steroid hormone.
Molecular biologists became interested in these receptors for steroid hormones as they came to realize that they were the most intensively studied and highly purified transactivation factors for control of eucaryotic transcription, and that they were the specific activators of an emerging and fascinating genetic cis-element, the enhancer. In the past 5 years, a great deal more has been learned of the structure-function relationships of steroid receptors and the mechanisms by which they interact with DNA. Biochemical studies in the late t970's suggested that steroid receptors, thyroid hormone receptors, and receptors for vitamins such as vitamin D belonged to a single family of gene regulatory proteins. Furthermore, it was thought that these proteins were organized into domains which contained the functions of (1) specific and high affinity ligand binding, (2) specific DNA binding, and (3) 'transcriptional modulation'. Molecular cloning of the receptors confirmed the 'superfamily' concept. Molecular cloning and sequence analyses not only substantiated the existence of domains but showed that they could be rearranged as independent cassettes within their own molecules or as hybrid molecules with other regulatory peptides [6]. Perhaps the most intensively studied domain has been that responsible for DNA binding. This domain has been shown to be a cysteine-rich region which is capable of binding zinc in a manner which creates two peptide projections referred to as 'zinc fingers'. These zinc fingers promote the interactions of receptors with target enhancers and clearly mark each as a member of this evolutionarily conserved family. Each zinc finger is important for high affinity binding to target DNA sequences, although certain experiments indicate that the first (N-terminal) finger plays a greater role in specific sequence recognition. A surprising observation has
been that certain oncogenes such as v-erbA are also members of this receptor gene family. The avian erythroblastosis virus appears to have captured the cellular gene coding for thyroid hormone receptor, and this retrovirus uses this mutated molecule for its own oncogenic purposes. One of the most revealing observations to emanate from the molecular analyses of the receptor superfamily of genes using recombinant DNA methods is the existence of many new members of this receptor family whose functions are as yet undetermined [6]. As evidenced by cloning of their cellular cDNAs, more receptors with unknown function appear to exist than the total known number of ligand-activated members of the family. This means that many new hormones, many perhaps non-steroidal or nutritional in nature, wait to be discovered. The sequences of the target enhanced sequences referred to as steroid response elements (SREs) regulated by steroid hormones have been described for most members of this family to date [6-9]. A summary of consensus regulatory sequences for genes responsive to glucocorticoid and progesterone (GRE/PRE), estrogen (ERE), and thyroid hormone (TRE) is listed in Fig. 2. In general, there are 15 base pair consensus, composed of two half-sites of six base pairs arranged in a dyad axis of symmetry (inverted repeats) around a few central base pairs of random composition. The SREs for various receptors share similarities in sequence and, in fact, the identical sequence allows activation by glucocorticoid, progesterone, and androgen receptors. One copy of such an SRE is usually sufficient to bring a promoter under moderate hormonal control, and two copies often provide a synergistic response to the cognate hormone. The precise mechanism of interaction of receptors with their target SREs has come under close scrutiny
69
Steroid hormone receptors
GRE/PRE
G/T G/T
T
A
C
A/C
N3
T
G
T
TIC
C
T
ERE
-
G/T
G
T
C
A
N3
T
G
A
C
C
--
TRE
A
G
A
T
C
A
N1
G
G
A
C
G
T
Fig. 2. C o n s e n s u s sequences for steroid response elements: G R E / P R E = glucocorticoid/progesterone response element; E R E = estrogen response element; T R E = thyroid h o r m o n e response element.
of late. After cytoplasmic synthesis, many steroid receptors form transient complexes with heat shock proteins, such as hsp90. Certain extracts of receptors for glucocorticoid, progesterone, and estrogen are prepared from cells in vitro as such aggregates. In cells, this interaction may promote proper folding and stability of the molecule; in complex with hsp90, receptors cannot bind to DNA. When a receptor is complexed with a heat shock protein, hormone binding drives in vitro dissociation of the complex. The exact meaning of this putative association of receptors with heat shock proteins is unclear at present, but is the subject of intensive study. Recent evidence shows that glucocorticoid, progesterone, and estrogen receptors bind to their SREs as dimers, one molecule to each half-site. This interaction appears to be cooperative, at least for glucocorticoid and progesterone receptors, and is shown schematically in Fig. 3. In this manner, receptor dimers bind with greater affinity and stability to their SREs than do monomers [10]. Interactions between receptor dimers at separate SREs allow also a higher order cooperative interaction which stabilizes the two dimers into a tetrameric structure which has a 100-fold greater affinity for its SRE sequences than does a single dimer [11]. Such protein-protein interactions may occur among homologous receptor complexes, heterologous receptor complexes, or receptor-promoter/TATA complexes. These interactions are thought to stabilize transcription factors at their DNA sites and promote a high degree of initiation of transcription at nearby genes (Fig. 3). Receptors may enhance transcription by stabilizing general transcription factors (e.g. TFIID, IIA, IIB, IIE/F, RNA polymerases, etc.) at the TATA box directly. Alternatively, stabilization may be 'referred' through interaction of receptors first with proteins bound to up-
stream promotors (e.g. COUP, NF-1, etc.) [12]. Incompatible complexes or proteins which disrupt such interaction should promote negative control by decreasing the chance that RNA polymerase will initiate transcription at a gene. Protein-protein interaction is the currently popular hypothesis for formation of stable transcription complexes at regulated genes. A great deal more, however, needs to be known about chromatin structure in regions of genetic control. Recent experiments suggest that specific phasing of nucleosomes around select SREs may allow recognition by receptors. Certain studies indicate that hormone stimulation may lead to structural arrangements of nucleosomes which promote transcriptional changes at target genes. The definitive role of ligand in receptor activaRegulatory Elements
Transcriptional Enhancement
~ Fold
~
COUP
TATA
~
mIRNA
22,l?,",'~;'o
SRE
m
OV SRE
~ 1 ~ ,
5G-Fold rnRN A
Fig. 3. Cooperative e n h a n c e m e n t of transcription proceeds from stable occupation of S R E by steroid receptors. C O U P = chicken ovalbumin upstream p r o m o t e r response element; S R E = steroid response element; TATA = downstream promoter element; O V = ovalbumin gene.
70
B W O'Mally Nucleus
Cytoplasm
]
I
1
I DNA I
(Inactive)
+H
,.
I
,A.P,D.At'nac'ive' +.
(Salt;&)
(8-10S)
(4S)
(4S)
Conclusion S t e p 1: H e a t Shock P r o t e i n D i s s o c i a t i o n S t e p 2: A I I o s t e r i c A c t i v a t i o n
for T r a n s a c t i v a t i o n
of G e n e s
Fig. 4. Hypothetical kinetic intermediates in steroid hormone activation of receptor, hsp = heat shock protein; R = receptor; SRE = steroid response element.
tion remains still a bit of a mystery. Certainly receptors are inactive in cells in the absence of hormone. Hormone administration in vivo leads to formation of a bound protein complex at SREs of hormone-regulated genes, as evidenced by in vivo footprint analyses. Crude receptor extracts of cells bind DNA poorly until they undergo a temperature and salt aided 'activation' which is driven by hormone [13]. As a first step, activation is promoted by disaggregation of receptors from heat shock and/or other proteins (Fig. 4). Antihormones promote such disaggregation but do not activate target genes, indicating an additional level of ligand-induced allosteric control. Recent evidence implicates a second step in which some allosterically altered form of receptor becomes a suitable substrate for a protein kinase and is activated by phosphorylation. Finally, the role of steroid receptors has been difined now for a genetic disease of hormone resistance. Receptor defects had been suspected to be the culprit for testicular feminization, for vitamin D resistant rickets, and in the case of hypercortisolism without Cushing's syndrome. Our recent familial studies have proven that vitamin D resistance can result from a single amino acid mutation at the tip of either 'zinc finger' of the DNA binding domain of its receptor [14]. These observations were important in proving that mutations in human steroid receptors, or in any transcription factor, can cause a human genetic disease. Many recent re-
ports also show that genetic mutations in the androgen receptor are a frequent cause of the androgen insensitivity syndrome, commonly called testicular feminization. Studies of the molecular mechanisms of steroid hormone action have had an immense impact on the field of endocrinology, advancing it as a legitimate discipline wherein technologies inherent to biochemistry, molecular biology, biophysics, pharmacology, and immunology can be unified toward the study of cellular physiology and regulatory biology. Much excitement has already occurred, but the near future holds promise for a greater understanding of fertility, early embryonic development, genetic disease, stress, eating and nutritional disorders, emotional and depressive disorders, cancers, and aging via research in this field. Our fruits to date should not only allow the discovery of a series of new hormones but also the rational design and synthesis of new agonists and antagonists for therapy of the above-mentioned disorders.
References 1. Jensen EV, Suzuki T, Kawashima T, Stumpf WE, Jungblut PW, DeSombre ER: A two-step mechanism for the interaction of estradiol with rat uterus. Proc Natl Acad Sci USA 59: 632-638, 1968 2. Gorski J, Tort D, Shyamala G, Smith D, Notides A: Hormone receptors: Studies on the interaction of estrogen with the uterus. Rec Prog Horm Res 24: 45-80, 1968
Steroid hormone receptors 3. O'Malley BW, Means AR: Female steroid hormones and target cell nuclei. Science 183: 610-620, 1974 4. O'MaUey BW, Roop DR, Lai EC, Nordstrom JL, Catterall JF, Swaneck GE, Colbert DA, Tsai M-J, Dugaiczyk A, Woo SLC: The ovalbumin gene: Organization, structure, transcription, and regulation. Rec Prog Horm Res 35: 1-42,
10.
1979 5. O'Malley BW, McGuire WL, Kohler PO, Korenman SG: Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. Rec Prog Horm Res 25: 105160, 1969 6. Evans RM: The steroid and thyroid hormone receptor superfamily. Science 240: 889-895, 1988 7. Payvar F, DeFranco D, Firestone GL, Edgar B, Wrange O, Okret S, Gustafsson J-A, Yamamoto K: Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35: 381-392, 1983 8. Renkawitz R, Schiitz G, yon der Ahe D, Beato M: Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding. Cell 37: 503-510, 1984 9. Jantzen H-M, Strfihle U, Gloss B, Stewart F, Schmid W, Boshart M, Miksicek R, Schiitz G: Cooperativity of the
11.
12.
13.
14.
71
glucocorticoid response elements located far upstream of the tyrosine aminotransfcrase gene. Cell 49: 29-38, 1987 Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson J-A, Tsai M-J, O'Malley BW: Molecular interactions of steroid hormone receptor within its enhancer element: Evidence for receptor dimer formation. Cell 55: 361-369, 1988 Tsai SY, Tsai M-J, O'Malley BW: Cooperative binding of hormone receptors contributes to transcriptional synergism at target enhancer elements. Cell 57: 443-448, 1989 Klein-Hitpass L, Tsai SY, Weigel NL, Allan GF, Riley D, Rodriguez R, Schrader WT, Tsai M-J, O'Malley BW: The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60: 247-257, 1990 Bagchi MK, Tsai SY, Tsai M-J, O'Malley BW: Identification of a functional intermediate in receptor activation in progesterone-dependent cell-free transcription. Nature 345: 547-550, 1990 Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, O'Malley BW: Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 242: 1702-1705, 1988
Steroid hormone receptors as transactivators of gene expression Bert W. O'Malley Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
Key words: DNA binding, enhancers, gene regulation, genetic diseases, heat shock proteins, response elements, steroid receptors Abstract
In the two decades since the discovery of the steroid hormone receptors, a great deal has been learned about their structure, their relationships with each other, and the target sequences (response elements) at which they regulate expression of specific genes. Analysis of receptor sequences has confirmed the existence of several domains with distinct functions in each receptor molecule, and has also indicated that the steroid receptors are members of a 'superfamily' which also includes receptors for vitamin D, retinoic acid, thyroid hormone and its related oncogene v-erbA, and a substantial number of 'receptors' whose ligands are still unidentified. The response element sequences are also closely related, and we are beginning to understand the interaction of the receptors with these response elements and with proteins bound nearby such as transcription factors. Certain naturally occurring genetic diseases have been identified with specific receptor mutations. The future promises greater understanding not only of the detailed action of the receptors of this superfamily, but of their precise role in fertility, development, and disease.
Over the past two decades, a great deal of evidence has accumulated in favor of the hypothesis that steroid hormones act at the level of nuclear D N A to regulate gene expression [1-4]. The earliest studies were qualitative and involved experiments which showed that steroid hormones: (1) caused accumulation of new species of hybridizable RNAs which did not exist prior to stimulation; (2) caused stimulation of synthesis of new specific proteins; (3) caused a corresponding increase in the cellular levels of specific mRNAs; and (4) stimulated the rate of transcription of certain nuclear genes [5]. At this point, the early 1970's, the primary pathway for steroid hormone action was defined as follows: steroid--~ (steroid-receptor)--~ (steroid-receptorDNA)---> mRNA--> protein ~ functional response (Fig. 1) [4]. Steroid enters cells by passive diffusion
and allosterically activates receptors in either the cytoplasm or nucleus. The activated receptor binds usually at the 5'-flanking region of target genes and stimulates transcription and protein synthesis. The first receptors were subsequently purified to near homogeneity and characterized as to size, charge, etc. Antibodies were developed, structural domains were postulated by proteolytic analyses, and assays of sex steroid receptors became commonplace in the diagnosis and assignment of therapy for breast cancer. Investigators isolated specific target genes for steroid hormones, defined their structure, and proved that cis-acting regulatory sequences were located nearby such genes, usually in the 5'-flanking sequences. When such sequences, termed steroid response elements (SREs), are occupied by receptors, these genes come under hormone control.
Address for offprints: B.W. O'MaIley,Department of Cell Biology,BaylorCollegeof Medicine, One BaylorPlaza, Houston,Texas 77030, USA
68
B W O'MaIly
S-{'- R l n a c t i v e
Activ 2
Protein
,
mRNA
,
mRNA PrecursorJ
Fig. 1. Molecularpathway for steroid hormone action. S = steroid hormone.
Molecular biologists became interested in these receptors for steroid hormones as they came to realize that they were the most intensively studied and highly purified transactivation factors for control of eucaryotic transcription, and that they were the specific activators of an emerging and fascinating genetic cis-element, the enhancer. In the past 5 years, a great deal more has been learned of the structure-function relationships of steroid receptors and the mechanisms by which they interact with DNA. Biochemical studies in the late t970's suggested that steroid receptors, thyroid hormone receptors, and receptors for vitamins such as vitamin D belonged to a single family of gene regulatory proteins. Furthermore, it was thought that these proteins were organized into domains which contained the functions of (1) specific and high affinity ligand binding, (2) specific DNA binding, and (3) 'transcriptional modulation'. Molecular cloning of the receptors confirmed the 'superfamily' concept. Molecular cloning and sequence analyses not only substantiated the existence of domains but showed that they could be rearranged as independent cassettes within their own molecules or as hybrid molecules with other regulatory peptides [6]. Perhaps the most intensively studied domain has been that responsible for DNA binding. This domain has been shown to be a cysteine-rich region which is capable of binding zinc in a manner which creates two peptide projections referred to as 'zinc fingers'. These zinc fingers promote the interactions of receptors with target enhancers and clearly mark each as a member of this evolutionarily conserved family. Each zinc finger is important for high affinity binding to target DNA sequences, although certain experiments indicate that the first (N-terminal) finger plays a greater role in specific sequence recognition. A surprising observation has
been that certain oncogenes such as v-erbA are also members of this receptor gene family. The avian erythroblastosis virus appears to have captured the cellular gene coding for thyroid hormone receptor, and this retrovirus uses this mutated molecule for its own oncogenic purposes. One of the most revealing observations to emanate from the molecular analyses of the receptor superfamily of genes using recombinant DNA methods is the existence of many new members of this receptor family whose functions are as yet undetermined [6]. As evidenced by cloning of their cellular cDNAs, more receptors with unknown function appear to exist than the total known number of ligand-activated members of the family. This means that many new hormones, many perhaps non-steroidal or nutritional in nature, wait to be discovered. The sequences of the target enhanced sequences referred to as steroid response elements (SREs) regulated by steroid hormones have been described for most members of this family to date [6-9]. A summary of consensus regulatory sequences for genes responsive to glucocorticoid and progesterone (GRE/PRE), estrogen (ERE), and thyroid hormone (TRE) is listed in Fig. 2. In general, there are 15 base pair consensus, composed of two half-sites of six base pairs arranged in a dyad axis of symmetry (inverted repeats) around a few central base pairs of random composition. The SREs for various receptors share similarities in sequence and, in fact, the identical sequence allows activation by glucocorticoid, progesterone, and androgen receptors. One copy of such an SRE is usually sufficient to bring a promoter under moderate hormonal control, and two copies often provide a synergistic response to the cognate hormone. The precise mechanism of interaction of receptors with their target SREs has come under close scrutiny
69
Steroid hormone receptors
GRE/PRE
G/T G/T
T
A
C
A/C
N3
T
G
T
TIC
C
T
ERE
-
G/T
G
T
C
A
N3
T
G
A
C
C
--
TRE
A
G
A
T
C
A
N1
G
G
A
C
G
T
Fig. 2. C o n s e n s u s sequences for steroid response elements: G R E / P R E = glucocorticoid/progesterone response element; E R E = estrogen response element; T R E = thyroid h o r m o n e response element.
of late. After cytoplasmic synthesis, many steroid receptors form transient complexes with heat shock proteins, such as hsp90. Certain extracts of receptors for glucocorticoid, progesterone, and estrogen are prepared from cells in vitro as such aggregates. In cells, this interaction may promote proper folding and stability of the molecule; in complex with hsp90, receptors cannot bind to DNA. When a receptor is complexed with a heat shock protein, hormone binding drives in vitro dissociation of the complex. The exact meaning of this putative association of receptors with heat shock proteins is unclear at present, but is the subject of intensive study. Recent evidence shows that glucocorticoid, progesterone, and estrogen receptors bind to their SREs as dimers, one molecule to each half-site. This interaction appears to be cooperative, at least for glucocorticoid and progesterone receptors, and is shown schematically in Fig. 3. In this manner, receptor dimers bind with greater affinity and stability to their SREs than do monomers [10]. Interactions between receptor dimers at separate SREs allow also a higher order cooperative interaction which stabilizes the two dimers into a tetrameric structure which has a 100-fold greater affinity for its SRE sequences than does a single dimer [11]. Such protein-protein interactions may occur among homologous receptor complexes, heterologous receptor complexes, or receptor-promoter/TATA complexes. These interactions are thought to stabilize transcription factors at their DNA sites and promote a high degree of initiation of transcription at nearby genes (Fig. 3). Receptors may enhance transcription by stabilizing general transcription factors (e.g. TFIID, IIA, IIB, IIE/F, RNA polymerases, etc.) at the TATA box directly. Alternatively, stabilization may be 'referred' through interaction of receptors first with proteins bound to up-
stream promotors (e.g. COUP, NF-1, etc.) [12]. Incompatible complexes or proteins which disrupt such interaction should promote negative control by decreasing the chance that RNA polymerase will initiate transcription at a gene. Protein-protein interaction is the currently popular hypothesis for formation of stable transcription complexes at regulated genes. A great deal more, however, needs to be known about chromatin structure in regions of genetic control. Recent experiments suggest that specific phasing of nucleosomes around select SREs may allow recognition by receptors. Certain studies indicate that hormone stimulation may lead to structural arrangements of nucleosomes which promote transcriptional changes at target genes. The definitive role of ligand in receptor activaRegulatory Elements
Transcriptional Enhancement
~ Fold
~
COUP
TATA
~
mIRNA
22,l?,",'~;'o
SRE
m
OV SRE
~ 1 ~ ,
5G-Fold rnRN A
Fig. 3. Cooperative e n h a n c e m e n t of transcription proceeds from stable occupation of S R E by steroid receptors. C O U P = chicken ovalbumin upstream p r o m o t e r response element; S R E = steroid response element; TATA = downstream promoter element; O V = ovalbumin gene.
70
B W O'Mally Nucleus
Cytoplasm
]
I
1
I DNA I
(Inactive)
+H
,.
I
,A.P,D.At'nac'ive' +.
(Salt;&)
(8-10S)
(4S)
(4S)
Conclusion S t e p 1: H e a t Shock P r o t e i n D i s s o c i a t i o n S t e p 2: A I I o s t e r i c A c t i v a t i o n
for T r a n s a c t i v a t i o n
of G e n e s
Fig. 4. Hypothetical kinetic intermediates in steroid hormone activation of receptor, hsp = heat shock protein; R = receptor; SRE = steroid response element.
tion remains still a bit of a mystery. Certainly receptors are inactive in cells in the absence of hormone. Hormone administration in vivo leads to formation of a bound protein complex at SREs of hormone-regulated genes, as evidenced by in vivo footprint analyses. Crude receptor extracts of cells bind DNA poorly until they undergo a temperature and salt aided 'activation' which is driven by hormone [13]. As a first step, activation is promoted by disaggregation of receptors from heat shock and/or other proteins (Fig. 4). Antihormones promote such disaggregation but do not activate target genes, indicating an additional level of ligand-induced allosteric control. Recent evidence implicates a second step in which some allosterically altered form of receptor becomes a suitable substrate for a protein kinase and is activated by phosphorylation. Finally, the role of steroid receptors has been difined now for a genetic disease of hormone resistance. Receptor defects had been suspected to be the culprit for testicular feminization, for vitamin D resistant rickets, and in the case of hypercortisolism without Cushing's syndrome. Our recent familial studies have proven that vitamin D resistance can result from a single amino acid mutation at the tip of either 'zinc finger' of the DNA binding domain of its receptor [14]. These observations were important in proving that mutations in human steroid receptors, or in any transcription factor, can cause a human genetic disease. Many recent re-
ports also show that genetic mutations in the androgen receptor are a frequent cause of the androgen insensitivity syndrome, commonly called testicular feminization. Studies of the molecular mechanisms of steroid hormone action have had an immense impact on the field of endocrinology, advancing it as a legitimate discipline wherein technologies inherent to biochemistry, molecular biology, biophysics, pharmacology, and immunology can be unified toward the study of cellular physiology and regulatory biology. Much excitement has already occurred, but the near future holds promise for a greater understanding of fertility, early embryonic development, genetic disease, stress, eating and nutritional disorders, emotional and depressive disorders, cancers, and aging via research in this field. Our fruits to date should not only allow the discovery of a series of new hormones but also the rational design and synthesis of new agonists and antagonists for therapy of the above-mentioned disorders.
References 1. Jensen EV, Suzuki T, Kawashima T, Stumpf WE, Jungblut PW, DeSombre ER: A two-step mechanism for the interaction of estradiol with rat uterus. Proc Natl Acad Sci USA 59: 632-638, 1968 2. Gorski J, Tort D, Shyamala G, Smith D, Notides A: Hormone receptors: Studies on the interaction of estrogen with the uterus. Rec Prog Horm Res 24: 45-80, 1968
Steroid hormone receptors 3. O'Malley BW, Means AR: Female steroid hormones and target cell nuclei. Science 183: 610-620, 1974 4. O'MaUey BW, Roop DR, Lai EC, Nordstrom JL, Catterall JF, Swaneck GE, Colbert DA, Tsai M-J, Dugaiczyk A, Woo SLC: The ovalbumin gene: Organization, structure, transcription, and regulation. Rec Prog Horm Res 35: 1-42,
10.
1979 5. O'Malley BW, McGuire WL, Kohler PO, Korenman SG: Studies on the mechanism of steroid hormone regulation of synthesis of specific proteins. Rec Prog Horm Res 25: 105160, 1969 6. Evans RM: The steroid and thyroid hormone receptor superfamily. Science 240: 889-895, 1988 7. Payvar F, DeFranco D, Firestone GL, Edgar B, Wrange O, Okret S, Gustafsson J-A, Yamamoto K: Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35: 381-392, 1983 8. Renkawitz R, Schiitz G, yon der Ahe D, Beato M: Sequences in the promoter region of the chicken lysozyme gene required for steroid regulation and receptor binding. Cell 37: 503-510, 1984 9. Jantzen H-M, Strfihle U, Gloss B, Stewart F, Schmid W, Boshart M, Miksicek R, Schiitz G: Cooperativity of the
11.
12.
13.
14.
71
glucocorticoid response elements located far upstream of the tyrosine aminotransfcrase gene. Cell 49: 29-38, 1987 Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson J-A, Tsai M-J, O'Malley BW: Molecular interactions of steroid hormone receptor within its enhancer element: Evidence for receptor dimer formation. Cell 55: 361-369, 1988 Tsai SY, Tsai M-J, O'Malley BW: Cooperative binding of hormone receptors contributes to transcriptional synergism at target enhancer elements. Cell 57: 443-448, 1989 Klein-Hitpass L, Tsai SY, Weigel NL, Allan GF, Riley D, Rodriguez R, Schrader WT, Tsai M-J, O'Malley BW: The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60: 247-257, 1990 Bagchi MK, Tsai SY, Tsai M-J, O'Malley BW: Identification of a functional intermediate in receptor activation in progesterone-dependent cell-free transcription. Nature 345: 547-550, 1990 Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, O'Malley BW: Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 242: 1702-1705, 1988
