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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

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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

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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

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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

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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

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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

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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?

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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

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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

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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

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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.