En int r7 Intracellular Hormone Receptors

Intracellular Hormone Receptors Steroid versus Peptide Hormones Mechanism of Action of Steroid Receptors Cellular Localization and Structure of Steroid Receptors How Steroid Receptors Initiate Transcription Role of Hormone Response Elements (HREs) Interactions of Steroid Receptors with Other Pathways Regulation of Steroid Receptors Action of Steroid Hormones versus Gonadotropin Hormones Peptide Hormones Half-life in circulation Speed of action Duration of effect Location of receptor Post-receptor regulation Signal amplification short fast short membrane high high Steroids long slow long inside low low Mechanism of Action of Steroid Receptors Gene Transcription RNA mRNA R Protein Steroid Binding protein Cellular Localization of Steroid Receptors Early Studies (1968): Tissue homogenize centrifuge Cytoplasmic fraction Nuclear fraction unoccupied occupied Localization of Protein by Immunocytochemistry Incubate cell specimen with antibody which specifically detects the protein Wash away unbound antibody Use a second labeled antibody to detect the first antibody protein Cellular Localization of Steroid Receptors Immunocytochemical studies indicated that all steroid receptors are nuclear Receptor antibody Color reaction Cellular Localization of Steroid Receptors Cell enucleation experiments also indicated that all steroid receptors were nuclear centrifugation Cellular Localization of Steroid Receptors It was thought that occupied receptor had higher affinity for the nucleus than unoccupied receptor, and was thus less likely to leak out during homogenization step in earlier studies homogenize Cellular Localization of Steroid Receptors More recent immunocytochemistry experiments reveal cytoplasmic estrogen receptors in certain regions of the hypothalamus under certain conditions Low ER Expression Increased ER Expression Cellular Localization of Steroid Receptors It is generally thought that unoccupied steroid receptors can exist in the cytoplasm, while occupied receptors act in the nucleus on target DNA When bound to hormone, cytoplasmic receptors move into the nucleus How does binding of Activated Steroid Receptor to HREs Enhance Transcription? HREs are enhancer sequences: they are orientation- and distance-independent Binding of activated receptor to HRE may stabilize the interactions between TATA box, Transcription Factor IID, and RNA polymerase II 5’-flanking region intron exon ERE TATA BOX Do steroids always act through classical steroid receptor mechanisms? Some effects of steroids are observed in minutes, too quickly to be explained by regulation of transcription Rapid effects of steroids may involve changes in ion channels and membrane permeability, such as influencing membrane potentials in CNS neurons In human sperm, a membrane-bound progesterone “receptor” has been described, which may mediate the effects of P on sperm maturation progesterone Ca++ Interactions of Steroid Receptors with Regulatory Elements of other Signaling Pathways • I previously mentioned that the transcriptional activity of the chick PR is increased by cAMP in the absence of progesterone Protein kinase A inhibitors block this effect We also know that: • Glucocorticoid receptors interact with the transcription factor AP1 • Progesterone receptors can be activated by dopamine (a neurotransmitter) • Thyroid hormone receptor activity is inhibited by AP1 Molecular Mechanism through which Glucocorticoids inhibit Inflammatory Responses Macrophages Interleukin-1 Protein Kinase C Pathway Increased Expression of jun, fos (-) Glucocorticoid Receptor dimerization AP1 collagenase Inhibition of AP-1 by GR Glucocorticoid receptor may bind jun, decreasing the formation of AP-1 (jun/fos dimer) This results in less AP-1 to bind to the the AP-1 enhancer site on the 5’ flanking region of the collagenase gene However, DNA footprinting studies show that AP-1 still DOES bind the AP-1 site during GR-induced inhibition of AP-1 stimulated transcription GR may inhibit transcriptional activation by AP-1 once bound to the site Activation of Progesterone Receptor by Dopamine in absence of Progesterone Dopamine is a common neurotransmitter in the brain Dopamine D1 receptor agonists mimic the effects of progesterone on female mating behavior The effects of dopamine can be blocked by a progesterone receptor antagonist and by antisense oligonucleotides to the progesterone receptor Activation of Progesterone Receptor by Dopamine in Absence of Progesterone -O’Malley Female Lordotic Response C P D1 D1 + RU486 Use of Antisense Oligonucleotides to Block Gene Expression complementary DNA sequence AAAAA , PR mRNA Translation Start site mRNA degradation block translation Block Gene Expression Blockade of Dopamine Activation of PR by Antisense Oligonucleotides Female Lordotic Response C P D1 D1 + antisense oligo to PR Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003 Treatment of brain cells with estrogen results in rapid phosphorylation (15 min) of CREB This effect is blocked by an estrogen receptor antagonist (ER dependent) This effect is dependent upon the activity of the mitogen-activated protein kinase (MAPK) pathway This effect is NOT dependent upon activity of protein kinase A Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003 E2 E2:ER MAPK pathway phosphorylation of CREB gene transcription (CRE) Regulation of Steroid Hormone Receptor Expression In general, tissue-specific and hormone-mediated regulation of steroid hormone receptors is not as dramatic as that for peptide hormone receptors Autoregulation: Ligand influences expression of own receptor Autoregulation can occur at several levels: - transcriptional: control of gene expression - post-transcriptional: modulation of mRNA stability - post-translational: rate of receptor degradation Examples: -Estradiol decreases uterine ER expression -Estradiol increases ER in pituitary, liver Regulation of Steroid Hormone Receptor Expression Heterologous regulation - Regulation by other steroids: -Estrogen up-regulates progesterone receptor in breast, uterus, hypothalamus -Progesterone down-regulates estrogen receptor -Androgen down-regulates estrogen receptor Influences of Other Signaling Pathways Steroid receptor expression may also be influenced by the protein kinase pathways Example: Activation of PKC decreases estrogen receptor mRNA stability, resulting in decreased synthesis Next Time… First Midterm Exam [...]... by binding to the same enhancers - Act as repressor or enhancer, depending on the physiological signals - thus, the response element serves as either enhancer or silencer Lipophillic Hormone Responses to hydrophobic hormones are mediated by intracellular receptors Plasma membrane Target cell Cytoplasm Intracellular receptor Nuclear envelope Transcription Promoter Hormone binds intracellular receptor... receptors appears to be an important step in receptor activation Steroid Receptors bind to Hormone Response Elements (HREs) on DNA Following hormone binding, intracellular receptors act as transcription factors, binding to hormone response elements (HREs) on the 5’ flanking region of target genes HRE 5’ flanking region target gene Steroid Receptors bind to Hormone Response Elements (HREs) on DNA Hormone. .. P? Gene Transcription H H HRE 5’ flanking region target gene H Not All Intracellular Receptors are Associated with HSPs HSPs bind to glucocorticoid, mineralocorticoid, androgen, progesterone, and estrogen receptors in absence of hormone However, receptors for thyroid hormone, retinoic acid, and vitamin D are not bound to HSPs This second group of receptors is bound to their hormone response element... membrane 2 Form hormone- receptor complexes that function as activators by binding to enhancers  hormone response elements 3 Sex hormones: estrogens and androgens; glucocorticoids, cortisol, vitamin D  Ca2+ metabolism; thyroid hormone, retinoic acid  developmental factors 1 1 The majority of nuclear receptors bind to respective enhancer elements and repress transcription - In the presence of hormone, they... target genes, but are inactive until hormone binds to them Is Removal of HSPs Sufficient to Induce Activation of Steroid Hormone Receptors? Removal of HSPs is not sufficient to induce transcriptional response - requires ligand Steroid receptors with HSP bound can still bind DNA Activation of steroid receptors may require a ligand-induced phosphorylation step Ligand-independent activation of receptors. .. Related: all are intracellular receptors which act as transcription factors, regulating target gene expression The Superfamily includes: glucocorticoid receptor mineralocorticoid receptor progesterone receptor (A and B) androgen receptor estrogen receptor (alpha and beta) thyroid hormone receptor vitamin D receptor retinoic acid receptor Nuclear Receptors Proteins interact with steroids and other hormones... Androgen, Glucocorticoid, Mineralocorticoid Consensus HREs AGAACAnnnTGTTCT Estrogen AGGTCAnnnTGACCT Thyroid hormone Retinoids Vitamin D AGGTCATGACCT Palindromic Sequences Allow Binding of Receptors as Dimers 5’ -AGAACAnnnTGTTCT- 3’ H NNN A T C G A T A T G C A T H Transcription TATA EXON 1… Sharing of HREs by Different Steroid Receptors Note that there are 8 classes of steroid receptors, but only 3 consensus... diffuses across cell membrane and enters cytoplasm 3 G binds to GR  changes conformation  dissociates from hsp90 4  exposes a nuclear localization signal (stretch of aas) on GR 5 G-GR (hormone- receptor complex, HR) enters nucleus, dimerizes with another HR 6 HR dimer binds to enhancer /hormone- response element upstream of hormone activated gene 7 Binding of HR dimer to enhancer  activates transcription... activity of receptor hypervariable DBD HBD How Are Steroid Hormone Receptors Activated? We know that when bound to hormone, the hormone- receptor complex initiates transcription of target genes But how? - Role of Heat Shock Proteins (HSPs) - Role of Hormone Response Elements (HREs) Role of HSPs Unbound receptor is associated with several HSPs Binding of hormone to receptor results in loss of HSPs, followed... estradiol HBD HBD HBD How does binding of Activated Steroid Receptor to HREs Enhance Transcription? HREs are enhancer sequences: they are orientation- and distance-independent Binding of activated receptor to HRE may stabilize the interactions between TATA box, Transcription Factor IID, and RNA polymerase II 5’-flanking region intron exon ERE TATA BOX