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Introduction to Hormone Signalling Receptors and signals theme ‘Hormone action’ Signalling in Health and Disease 2011 GPCR Tues Wk1 Enzyme - linked receptors G protein - coupled receptors Drug discovery Thurs Wk2 Receptors and drugs Tues Wk 2 nuclear receptors Insulin and Growth factor signalling Hilary term Peptide hormone G protein linked receptors Light amines Ions Amino acids Peptides Lipids Enzymes Odorants Hypothalamic hormones: GnRH Somatostatin CRH TRH GHRH AVP Oxytocin Pituitary hormones: ACTH LH FSH TSH MSH Gut hormones: Secretin CCK Glucagon Parathyroid hormone Substance P Opiates NPY Bombesin Neurotensin Angiotensin II PACAP/VIP Glucagon-like peptide 1 Calcitonin hCG Receptor molecular biology/bioinformatics Thurs/Fri Wk1 Second messengers Mon Wk 2 Genomic and nongenomic steroid signalling Hilary term GnRH and LH receptor structure GnRH receptor no Cter tail 3D LH receptor long Nter tail Antisense knockout of Gα αo prevents TRH stimulation of Ca2+ currents in a pituitary cell line G protein cycle Which G protein? Drugs – cholera toxin Find mRNA of G protein TRH Prepare complementary oligonucleotide probe TSH Inject probe into cell Antisense knockout of specific subunits in cell lines Probe binds to mRNA preventing protein translation Protein ‘knocked out’ Examine functional activity of cell (electrical activity, hormone release) Laugwitz et al, 1996 PNAS tissue expression pattern bioinformatics Evidence for GPCR dimerisation Fluorescence Resonance Energy Transfer Salaphour 2000 full length cloning Reverse pharmacology approach to characterizing orphan GPCR expression putative ligands; biological extracts functional screens, e.g. cAMP natural or surrogate agonist antagonist pharmacology of receptor Transcription via cAMP Clinical evidence of a role for GPCRs in normal and aberrant growth Activating mutations TSH thyroid adenoma, hyperthyroidism LH pseudoprecocious puberty GHRH acromegaly Inactivating mutations CREB = cAMP response element binding protein Greenspan & Strewler GHRH signalling is essential for post-natal expansion of the GH cell population VP V2 Diabetes insipidus TSH Congenital hypothyroidism LH Leydig cell hypoplasia GHRH Pituitary dwarfism Inactivating Mutation in the Growth Hormone-Releasing Hormone Receptor in man: Dwarfism of Sindh MAHESHWARI et al., (J Clin Endocrinol Metab 1998 83: 4065–4074) GH cells No response to GHRH normal GHRH receptor mutation GH cells lit/lit mouse Lin et al, Nature 364, 208 (1993) Grave’s disease Enzyme-linked receptors Predominantly signal growth and differentiation tyrosine kinase linked e.g. insulin, EGF cytokines, growth factors e.g. GH, PRL Growth factor receptors e.g. insulin tyrosine kinase activation Autophosphorylation of the receptor SH2 GRB2 SOS GRB2 binds via SH2 domain and provides ‘scaffolding’ for signal apparatus MAPK MAPK SRF AP1 RAS RAF1 MEK1 Transcription Insulin Signalling activates different pathways in parallel Convergence of GPCR and tyrosine kinase linked receptor pathways Cytokine receptors e.g. GH, PRL GH GH JAK2 JAK2 tyr tyr JAK2 JAK2 tyr P P tyr Ligand activated receptor recruits cytosolic tyr kinase phosphorylation of downstream effectors e.g. STAT, MAPK ‘Classical’ mechanism of steroid action Steroid receptor structure Estrogen receptor Onset >30min Steroid receptor plasma membrane N ter AF-1 NLS C ter A/B C DNA control of gene transcription HRE X gene X steroid nucleus mRNA HRE = hormone response element protein FUNCTION AF-2 D E F ligand agonists A/B protein-protein interactions, transcriptional activation ‘transactivation’ C zinc fingers important for DNA binding D Nuclear localisation signal E Ligand binding, heat shock protein binding, transcription repression, receptor dimerisation F Present in few receptors, minimal function Steroid receptor stabilises the preinitiation complex for transcription Steroid mediated induction of protein synthesis Chick oviduct IIJ IIF SRE SRE IIA IIB IIE IIH progesterone DES mRNA oviduct weight total protein TATA ovalbumin pol II general transcription factors IIJ IIF SRE SRE IIA IIB IIE IIH lysozyme avidin mRNA TATA pol II O’Malley, 1995 Steroid receptors interact with co-regulator proteins ubiquitin ligase, kinase, acetylase coactivation help to recruit pol II O’Malley, 1969 Partial hormone resistance in SRC1 KO males WT Compensation for lack of SRC-1 by increasing expression of other coactivators enzyme remodel chromatin Science (1995) Xu et al, 1998 KO Positive and negative factors on steroid receptor pathway Role of orphan steroid receptors ERR = estrogen receptor related receptor Level Enhancers Opposers receptor ligand ligand independent activators HSPs ligand binding protiens phosphorylation kinases phosphatases heterodimerization RXRs COUP-TF etc nuclear translocation ATP chaperones ATP depletion transactivation coactivators corepression chromatin structure histone acetylase histone deacetylase KO demonstrates a role in early placentation Luo et al, 1997 Rapid action of estradiol in the anterior pituitary 24 ** ** * 8 ** ** ** 16 12 ** 10 min E E-BSA 4 [K+ ] Basal 0 -12 -11 -10 -9 -8 log [Steroid] (M) -7 Christian & Morris, J Physiol 2002 -6 Mean FL1 intensity (units) Ir Ir--PRL (ng mg-1 tissue) 20 120 100 80 60 control + 1 µM E 40 20 0 1 10 100 BSABSA- 0.1 FITC [E[E-BSABSA-FITC] (nM) 100 nM What are the steroid-binding sites in the plasma membrane? Palmitoylation of steroid receptors important for membrane localisation Receptors derived from the nuclear R? Evidence for: effects not evident in receptor knockout tissue immunocytochemistry using antibodies effects blocked by nuclear R antagonists Novel ‘orphan’ receptors? GPCR30- estrogen Modulation of known existing receptors? GABA – neurosteroids, integrin –thyroid hormones 2007 Mutation of residues that are palmitoylated reduces membrane localisation ER associated with cytosolic face of membrane not transmembrane Novel G protein coupled receptor GPR30 binds estrogen Early membrane estrogenic effects required for full expression of slower genomic effects in a nerve cell line Neuroblastoma cell line See Weatherman et al Nature Chem Biol 2:175 (on Weblearn) Transfected with luciferase reporter gene with promotor containing estrogen response elements Measure reporter gene activity Pulse 1 20 min with estrogen Rest 4h Pulse 2h with estrogen Vasudevan et al 2001 Evidence that membrane effects of estrogen permit optimal nuclear effects ‘Although non-classical and genomic mechanisms have been widely viewed as discrete, alternate models of steroid hormone action these data show that they can synergize to potentiate transcription’ Concn response curve Vasudevan et al 2001 Conclusion • Hormones have multiple effects with actions in the membrane complementing those in the nucleus • Actions at the cell membrane are not exclusively mediated by peptide hormones Vasudevan et al 2001