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Frontiers in Reproductive EndocrinologySerono Symposia International
Mechanisms of Hormone Action:Steroid Hormones
Kelly MayoNorthwestern University
General Mechanisms of Actionof Steroid and Peptide Hormones
SteroidHormone S Diffuses across
plasma membrane
SS
Cytoplasmic ornuclear receptor
S
Hormone-activatedreceptor
Binds to DNAin target genes
Regulatesgene transcription
New mRNAs
Synthesis of new proteins
Non-genomic effects via
protein -protein interaction
Biologicaloutput
Biologicaloutput
Changes inenzyme activity
ProteinHormone
PP Binds to cell
surface receptor
Activation of effector enzymes
Receptor-associated changes in enzyme activity
Generation ofsecond messengers
Changes inenzyme activity
Biologicaloutput
BiologicaloutputTFP P
Activation oftranscription
factors
New mRNAs
Synthesis of new proteins
Biologicaloutput
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Select Families of Nuclear Hormone Receptors
Steroid ReceptorsEstrogen Receptor (ER)Androgen Receptor (AR)
Progesterone Receptor (PR)Glucocorticoid Receptor (GR)
Mineralocorticoid Receptor (MR)
Mevalonate PathwayLiver X Receptor (LXR)
Pregnane X Receptor (PXR)Farnesoid X Receptor (FXR)
Peroxisome ProliferatorsPPARa (fibrates)
PPARg (thiazolidinediones)PPAR b/d
Non-Steroid HormonesThyroid Hormone Receptor (TR)
Vitamin D Receptor (VDR)Retinoic Acid Receptor (RAR)
9-Cis Retinoic Acid Receptor (RXR)Ecdysone Receptor (EcR)
Orphan ReceptorsERR family
NGF1-B familyCOUP TF familyNGF1-B family
RVR familySF-1
LRH-1Dax-1
HNF-4GCNF
Ex-Orphan ReceptorsBenzoate X Receptor (BXR)
Steroid and Xenobiotic Receptor (SXR)Constitutive Androstane Receptor (CAR)
Also RXR, LXR, FXR, PPARs
Metabolic Pathways of Nuclear Receptor Ligands
Chawla et al Science 294:1866, 2001
3
Domain Structure of Nuclear Receptors andDimerization and DNA Binding Properties
EE FFDDCCA/BA/B
LBD AF-2 LBD AF-2hingehingeDBDDBDAF-1AF-111 180180 263263 304304 554554 595595
ER
LBD LBD
DBDDBD
Homodimeric Bindingto Inverted RepeatsSteroid Receptors
ER, GR, MR, AR, PRSome Orphan Receptors
LBD RXRRXR
DBDDBD
Heterodimeric Bindingto Direct RepeatsWith RXR Partner
Non-Steroid ReceptorsTR, RAR, VDR, PPAR
LBD
DBDDBD
Monomeric Bindingto Half Site
Orphan ReceptorsLigand Binding Unknown
SF-1, NGF1-B, ERR
?
DNA Binding by Nuclear Hormone Receptors
Sanchez et al, BioEssays 24:244, 2002
4
DNA Binding by Nuclear Hormone Receptors
ER LBD Homodimer NGF1-B LBD MonomerRAR-RXR LBD Heterodimer
HATs
Coactivators
Local ChromatinRemodeling
BasalTxn
FactorsRNAP
Activation of TargetGene Transcription
Nuclear Receptor Signaling Pathways
Nucleus
Cytoplasm
Hhsp90 hsp70
RAPs
Inactive Receptor Complex
HHormone Enters Nucleus
And Interacts with Receptor
H H
H H
Receptor ActivationAnd Dimerization
5
Nuclear Receptor Coactivators and CorepressorsCoactivators
SRC-1, NCoA-1GRIP-1, TIF-2, SRC-3pCIP, ACTR, SRC-3
Many others
SRC-1SRC-1632632 75975911 907907 955955 14401440
NHR NHR InteractionInteraction
LXXLL motifsLXXLL motifs
RIDRID ADAD RIDRIDQ-richQ-richbHLHbHLH PAS-APAS-A PAS-BPAS-B
CBPCBPInteractionInteraction
HATHAT
CorepressorsNCoRSMRTREA
TRUP, SURF-3
NCoRNCoR11 19441944 24532453
NHR NHR InteractionInteraction
LXX IXX I/L helicesLXX IXX I/L helices
Su(H)DSu(H)D RIDRIDRIDRIDRepression DomainsRepression Domains
9292 10351035751751SID2SID2SID1SID1
Sin 3Sin 3InteractionInteraction
Integrators/ComplexesCBP, p300
TRAPs, DRIPsSWI/SNF
pCAF/CARM
Coregulators and Chromatin Remodeling
HRNCoR
HR
Sin3
HDAC
SWI/SNGBRG/BGM
HRSRC-1
CBP
CARM-1 BTFsRNAP
TRAPDRIP
Med/ARC
Inactive
Remodeling
ActiveHormone
CorepressorsHDACs
ChromatinModifyingATPases
CoactivatorsHATS/MTs
Txn Complexes
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ER LBDTR LBD
Some Nuclear Receptor Ligand-Binding Domains
H12
H1
RXR LBD
Weatherman et al, Ann Rev Biochem 68: 559, 1999
RAR LBD
H3H3
H5H5
H4H4
H12H12(AF-2)(AF-2)
ER + 4-HydroxytamoxifenER + 4-Hydroxytamoxifen
Structures of Agonist and Antagonist BoundEstrogen Receptor Ligand Binding Domain
H3H3
H5H5
H4H4
H12H12(AF-2)(AF-2)
NRII BoxNRII Box
ER + DiethylstilbestrolER + Diethylstilbestrol+ NR Box II Peptide+ NR Box II Peptide
Shiau et al Cell 95:927-937, 1998
7
Multiple MechanismsGenomic actions
Complex response elementsNon-genomic actions
CoregulatorsTissue specific coregulators
Combinatorial coregulator codeAgonist versus antagonist actions
Complexities in Steroid Hormone Action: Estrogen
Receptor GenesER-a and ER-b
Receptor VariantsDiverse promoters
Alternative RNA splicingPost-translational modifcation
InteractorsERR Orphan Receptors
Tissue-Specific Responses
SERMS
Generation of Diversity in Nuclear Receptors:Multiple Estrogen Receptor Genes
ER-a
ER-b
DBD LBD
23% 86% 24% 58% 12%
AF-1 AF-2
Homology:
ER-a predominates: Kidney, Adrenal, Pituitary, Testis, Epididymis
Equivalent expression: Mammary gland, Uterus, Bone, Heart, Gut, Brain
ER-b predominates: Prostate, Ovary, Lungs, Bladder
8
Generation of Diversity in Nuclear Receptors:Multiple Estrogen Receptor Gene Promoters
Human ER-a Promoters
Kos et al, Mol Endocrinol 15:2057, 2001
Potential Implications:Tissue- or cell-specific ER expression
Developmental-specific ER expressionAlternative splicing of ER transcripts
Generation of Diversity in Nuclear Receptors:Multiple Estrogen Receptor Splice Variants
1 2 3 4 5 6 7 8
DBD LBD
ER-a
ER-a
d4(P,H)
d3(P)
d3,4(P,H)
d5,6(P,H)
TERP(P)
1 2 3 4 5 6 7 8
DBD LBD
ER-b
18aa
18aa
ER-b
1-d3(P,H)
1-d4(B)
2(P>H)
2-d3(P>H)
Shupnik, J Neuroendocrinol 14:85, 2002
9
Generation of Diversity in Nuclear Receptors:Phosphorylation of the Estrogen Receptor
Lannigan, Steroids 68:1, 2003
AF-1 DBD AF-2
AF-1 DBD AF-2
In response to estrogen binding:
In response to second messenger pathways:
Ser104/106
Ser118
Ser118
Ser167
Ser236
MAPK?CDK2
PKAMAPK RSK
Giguere, Trends Endocrinol Metab 13:220, 2002
Generation of Diversity in Nuclear Receptors:Interaction with Other Nuclear Receptor Pathways
ERa
ERb
ERRa
ERRb
ERRg
AF-1 DBD LDB/AF-2
68% 37%
ERa
RERa
AGGTGAnnnTGACCTERE
TnAAGGTCAERRE
ERa
ERRa
ERa,b
ERRa,b,g
Estradol DES/OHT
CoA
CoR
EREERREOther
Cell-Specific Responses
NaturalLigands?
10
Falkenstein et al, Physiol Rev 52:523, 2000
Non-Genomic Actions
ER
E2
Membrane-Bound ER
E2
Novel ER?
Novel Estrogen Receptor
E2
Other Proteins
Interaction with OtherMembrane Proteins
Generation of Diversity in Nuclear Receptors:Multiple Mechanisms of Action
Genomic Actions
ERE
E2
SP1/AP1
E2
NF-kB
E2
X
Classical Non-ClassicalX ERE
Ligand-Independent
Kinase
Generation of Diversity in Nuclear Receptors:Combinatorial use of Coregulatory Proteins
CoActivatorsp160/SRC family
CBP/p300TRAP/DRIP
“SRA”Differential Regulation of Receptors
Tissue-Specific Expression“Combinatorial Code”
Factor-SpecificModification
(PhosphorylationMethylation)
CoRepressors
NCoR/SMRTSin3/HDACs
“REA”
Receptor and Tissue-Selective Effects
11
Selective Estrogen Receptor Modulators (SERMs)
Examples of SERMs:Tamoxifin (breast cancer)
Antagonist in breast, but agonist in bone/endometrium
Roloxifene (osteoporosis)Agonist in bone, antagonist in breast/endometrium
Determinants of SERM Action:Each ligand (SERM) will induce a unique conformation of the estrogen
receptor that impacts its interaction with coregulatory proteins
Each tissue or cell type will contain a unique complement of coregulatoryProteins and a distinct pattern of activation state of these proteins
Cell-Selective Actions of SERMs
Jordan Cancer Cell 1:215, 2002
12
Additional Pathways of Intracellular Hormone Action
Arylhydrocarbon Receptor Intracellular dioxin receptor
Ligand activated transcription factorBinds to xenobiotic response element
Nitric Oxide Receptor Cytoplasmic form of guanylyl cyclasea/b heterodimer with heme cofactor
Increases cGMP and PKG activity
Extrinsic Signals
Sterol SensingP roteolysis of membrane-bound SREBP
bHLH domain regulates transcriptionProteolysis regulated by sterols
Oxygen SensingProlyl and Asn hydroxylases regulated by O2
Hydroxylation regulates HIF-aHeterodimer with ARNT regulates transcription
Intrinsic Signals
Mutations of Hormones, Receptorsand Signaling Proteins in Reproductive DiseaseHormonesFSH Delayed puberty, primary amenorrhea in females; male hypogonadismLH Luteal insufficiency, infertility in female; delayed puberty, azoospermia in maleMIS Persistence of Mullerian duct derivatives in males
ReceptorsGnRH-R Partial to complete hypogonadotropic hypogonadism, males and femalesFSH-R Primary or secondary amenorrhea in females, variable/mild oligospermia in malesLH-R (Loss) Amenorrhea or oligomenorrhea in females, range of defects to complete feminization in malesLH-R (Gain) Male-limited precocious puberty, no phenotype in femalesEstrogen R Normal puberty, tall stature and unfused epiphyses in maleAndrogen R Many mutations, broad range of phenotypes to complete feminization in malesMIS R-II Persistence of Mullerian duct derivatives in malesRET Multiple endocrine neoplasia type 2
Signaling ProteinsGs protein a McCune-Albright Syndrome (gain), male precocious puberty (loss/gain)Gi protein a Ovarian and adrenal tumors?Smads Mutations in many cancers, including Smad4 mutation in seminoma testicular germ cell tumor
Transcription FactorsDax-1 Hypogonadotropic hypogonadism/adrenal failure in male SF-1 XY sex reversal/adrenal failure Prop-1 Variable hypogonadotropic hypogonadism in males and females
13
Emerging and Future Issues in Hormone Action
•Cross-talk between different signaling pathways•Integration of multiple signals in target cell
•Generation of diverse responses from common stimuli•Combinatorial codes for signaling diversity
•Spatial regulation of signaling complexes•Temporal dynamics of cell signaling
•Discovering new signaling pathways•Discovering ligands for orphan receptors
•Structural solutions to membrane receptors•Mechanistic structural studies on signaling molecules
•Genetic approaches to hormone action•Hormone action and human disease
•Rationale drug design
Additional Readings on Steroid Hormone Action•Evans (1988) The steroid and thyroid hormone receptor superfamily. Science 240:889.•Tsai and O’Malley (1994) Molecular mechanisms of action of steroid/thyroid receptor superfamilymembers. Ann Rev Biochem 63:451.•Rosenfeld and Glass (2001) Coregulator codes of transcriptional regulation by nuclear receptors. JBiol Chem 276:36875.•McKenna and O’Malley (2002) Combin atorial control of gene expression by nuclear receptors andcoregulators. Cell 108:465.•Katzenellenbogen et al (2000) Estrogen receptors: selective ligands, partners and distinctivepharmacology. Rec Prog Horm Res 55:163.•Sanchez et al (2002) Diversity in the mechanisms of gene regulation by estrogen receptors.Bioessays 24:244.•Nilsson et al (2001) Mechanisms of estrogen action. Physiol Rev 81:1535.•McDonnell (1999) The molecular pharmacology of SERMs. Trends Endocrinol Metab 10:301.•Jordan (2002) The secrets of selective estrogen receptor modulation: cell-specific coregulation.Cancer Cell 1:215.•Weatherman et al (1999) Nuclear receptor ligands and ligand-binding domains. Ann Rev Biochem68:559.•Kumar and Thompson (1999) The structure of nuclear hormone receptors. Steroids 64:310.•Chawla et al (2001) Nuclear receptors and lipid physiology: opening the X-files. Science 294:1867.•Willson and Moore (2002) Genomics versus orphan nuclear receptors: a half-time report, MolEndocrinol 16:1135.•Yudt and Cidlowski (2002) The glucocorticoid receptor: coding a diversity of proteins and responsesthrough a single gene. Mol Endocrinol 16:1719.•Gellman (2002) Molecular biology of the androgen receptor. J Clin Oncol 20:3005.•Conneely et al (2002) Reproductive functions of progesterone receptors. Rec Prog Horm Res 57:339.