Lecture 2. Mechanisms of Hormone action (1).pdf

Mechanisms of hormone action Hormone action hormones combine with hormone receptors initiation of a cascade of reactions in the cell Signal transduction: the process by which information coming from the extracellular enviroment is conveyed to the interior of the cell. information contained in signal...

Mechanisms of hormone action Hormone action hormones combine with hormone receptors initiation of a cascade of reactions in the cell Signal transduction: the process by which information coming from the extracellular enviroment is conveyed to the interior of the cell. information contained in signaling molecules (hormones, growth factors, etc) is passed to specific effector molecules within the cell. Extracellular enviroment stimuli Cell Cell response Signal transduction systems: Over the past 10 years our knowledge of the molecular mechanisms by which extracellular signals are received and transmitted across the cell surface has increased. The molecules involved in these signalling systems known as “signal transduction systems” are major targets for therapeutic agents and therefore of great interest to pharmacologists. Receptors MEMBRANE-BOUND RECEPTORS 7 Transmembrane (TM) “serpentine”/ interact with G proteins Single Transmembrane (TM)/Intrinsic kinase activity (tyrosine kinase) Single Transmembrane (TM)/ interact with tyrosine kinases (Hormone cannot permeate into cell) CYTOSOLIC/NUCLEAR RECEPTORS interact with DNA (Hormone is lipid soluble) 7 TM Receptors Glucagon Vasopressin, ADH ECF plasma membrane ICF Receptors containing intrinsic kinase activity (tyrosine kinase receptors) TM Receptors Insulin, growth factors transmembrane domain ECF plasma membrane ICF Tyrosine kinase Receptors associated with an intracellular tyrosine kinase TM receptors Growth Hormone ECF transmembrane domain plasma membrane TK ICF Intracellular Receptors Glucocorticoids Mineralocorticoids Steroids (sex hormones) Thyroid hormone Hormone binding DNA binding Signaling through receptors coupled to G-proteins 7 TM Receptors Properties - Membrane receptors that span the plasma membrane 7 times. - These receptors are associated with heterotrimeric (they have 3subunits: alpha, beta, gamma) G-Proteins Mechanism of activation of 7TM receptors Ligand/hormone/signaling molecule Receptor G protein Effector Second messenger Response Table 6-2 Criteria for the participation of an intracellular messenger in the action of a hormone (see question and answer at the end of this presentation) The enzyme that produces the messenger should respond to the hormone A change in the concentration of the messenger should precede or occur simultaneously with the intended action of the hormone Inhibitor of the removal or degradation of the messenger should function synergistically with the hormone that promotes its synthesis The biological effect of the hormone should be mimicked directly by the addition to the cell of the messenger itself or a suitable analog. Signaling through 7 TM receptors coupled to Heterotrimeric guanine nucleotide-binding (G) proteins plasma membrane  GDP a subunit GTP/GDP binding site GTPase activity   Signaling through 7 TM receptors Heterotrimeric guanine nucleotide-binding (G) proteins plasma membrane substrate  GTP E2 E1 product E1, E2: Effectors   7 TM Receptors Coupled to G-Proteins 1. The ligand binds to the 7 TM Receptor and causes conformational changes on the receptor 2. This causes GTP to replace GDP on the binding site of the alphasubunit of the G protein and allowing it to dissociate from the beta and gamma subunits 3. The alpha subunit of the G-protein will bind and activate the effector molecule (Adenyl Cyclase or Phospholipase C). Activation / Deactivation G proteins   GTP E1 Pi intrinsic GTPase activity GDP plasma membrane  E1 GDP   Families of G proteins Family Function Gs Adenylyl cyclase (AC) stimulation Gi AC inhibition Gq Phospholipase C (PLC) stimulation G12 sodium/hydrogen exchanger (NHE) regulation cholera toxin pertussis toxin plasma membrane   GTP GDP cholera toxin ADP-ribosylation of Gs (arg) GTPase activity abolished Gs “locked” in active monomeric state Cholera toxin produced by the bacterium Vibrio cholerae causes diarrhea.   pertussis toxin ADP-ribosylation of Gi(cys) exchange GDP/GTPabolished Gi “locked” in inactive heterotrimeric state Pertussis toxin produced by the bacterium Bordetella pertussis causes whooping cough. Cholera toxin: Gs “locked” in active monomeric state Cholera is an infectious disease caused by eating food or drinking water contaminated with a bacterium called Vibrio cholerae. It causes severe watery diarrhea, which can lead to dehydration and even death if untreated. Signaling through 7 TM receptors G-protein- Adenylyl cyclase plasma membrane adenyl cyclase  ATP PKA (inactive) PKA (active) GTP 3’,5’-AMP (cAMP) Protein phosphorylation Response Gene transcription:role of cAMP plasma membrane  GTP PKA (inactive) ATP adenyl cyclase 3’,5’-AMP (cAMP) PKA (active) CREB- PO32+ CREB nucleus CREB-P CRE Gene transcription New protein Response DNA Signaling through 7 TM receptors G-protein-Phospholipase C  PLC phosphatidyl inositol 4,5 P2 (PIP2) plasma membrane PKC (inactive) PKC (active) GTP IP3 + DAG ER Protein phosphorylation Ca++ CaM kinase (inactive) CaM kinase (active) response Figure 6-12 - Overview Regulation of cAMP levels Role of Phosphodiesterase plasma membrane  adenyl cyclase ATP phosphodiesterase GTP 3’,5’-AMP (cAMP) AMP Signaling molecules regulating ion channels Ion channel Ion channel ligand plasma membrane Response ions Signaling molecules ( Hormones) regulating ion channels Ca++ :an intracellular messenger Intracellular [Ca++ ] (resting) (10-7 M) Extracellular [Ca++ ] 0.01-0.1 uM 2-5mM (10-3 M) Release into cytosol Ca++ channels in PM voltage gated Ca++ channels in PM IP3 gated Ca++ channels in ER ryanodine receptors in SR Sequestration from cytosol Ca++ ATPase in PM and in ER / SR Ca++ :an intracellular messenger Ca++ : an intracellular messenger increased intracellular Ca++ protein bound Ca++ calmodulin (CaM) (4 mol Ca++ / mol protein) activation of CaM -dependent protein kinases troponin C (4 mol Ca++ / mol protein) initiation of muscle contraction free Ca++ activation of PKC exocytosis (insulin release) Calmodulin Signaling through receptors with intrinsic tyrosine kinase (TK) activity General Structure of Tyrosine Kinase Receptors ligand binding domain extracellular domain -S-S-S-S- -S-S- transmembrane domain catalytic domain epidermal growth factor receptor insulin receptor platelet-derived growth factor receptor Tyrosine kinase Receptors hormone hormone kinase domains 1. Ligand Binding 2. Receptor Autophosphorylation 3. Increase TK activity 4. Phosphorylation of Intracellular substrates intrinsic autophosphorylation phosphorylation of effector protein receptor Response Tyrosine kinase Receptors Tyrosine kinase receptors are membrane receptors that have an intrinsic tyrosine kinase activity in their intracellular loop. These receptors activate the following signaling pathways PI3K-Akt signaling pathway Ras-MAPK signaling pathway PI3K-Akt signaling pathway PI3K-Akt signaling cascade Please keep in mind that the PI3K-Akt signaling cascade operates in all the cells and is important for normal cell function but its overactivation is linked to cell proliferation (carcinogenesis) and survival/inhibition of apoptosis. This cascade is overactivated in many cancers The PI3K-Akt cascade is involved in growth factor signaling. PI3K-Akt cascade: (in response to growth factor binding binding) Some growth factors: epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), Insulin-like growth factor (IGF), etc - The ligand (growth factor) binds on the extracellular loop of the receptor - This binding causes a conformational change in the receptor (growth factor receptors work as dimers) - The receptor is auto-phosphorylated (The intracellular domains of the receptor dimer cross phosphorylate each other) - This autophosphorylation increases the tyrosine kinase activity of the receptor and the receptor is thereby fully activated - Phosphorylated growth factor receptor attracts phosphatidylinositol 3-kinase (PI3K) and activates it. (PI3K has a specific domain called Src homology 2 (SH2) domain that recognizes tyrosine phosphorylated residues) - PI3K (a lipid kinase) converts membrane phospholipids to 3-phosphoinositides (PIP₃) Phosphatidylinositol (3,4,5)-trisphosphate, abbreviated PIP₃) - 3-Phosphoinositides bind and activate phosphoinositide-dependent kinase (PDK) - PDK then phosphorylates and activates Akt - Akt is a serine threonine kinase and phosphorylates intracellular proteins which lead to cellular response (the response is increased cell proliferation and survival ). Insulin stimulates glucose uptake by muscle and fat cells by activating the PI3K- Akt signaling pathway PI3K-Akt cascade: (in response to insulin binding) - The ligand (insulin) binds on the extracellular loop of the receptor - This binding causes a conformational change in the receptor - The receptor is auto-phosphorylated (The intracellular domains of the  subunits of the receptor cross phosphorylate each other) - This autophosphorylation increases the tyrosine kinase activity of the receptor and the receptor is thereby fully activated - The fully activated receptor phosphorylates intracellular substrates - In the case of insulin being the ligand, that intracellular substrate is insulin receptor substrate1 (IRS-1) - IRS-1 binds to the receptor and is phosphorylated by its kinase activity - Phosphorylated IRS-1 attracts phosphatidylinositol 3-kinase (PI3K) and activates it (PI3K has a specific domain called Src homology 2 (SH2) domain that recognizes tyrosine phosphorylated residues) - PI3K (a lipid kinase) converts membrane phospholipids to 3-phosphoinositides (PIP₃) Phosphatidylinositol (3,4,5)-trisphosphate, abbreviated PIP₃) - 3-Phosphoinositides bind and activate phosphoinositide-dependent kinase (PDK) - PDK then phosphorylates and activates Akt - Akt is a serine threonine kinase and phosphorylates intracellular proteins which lead to cellular response. Akt causes metabolic responses. - In fat and skeletal muscle cells insulin-activated Akt leads to GLUT4 glucose transporter translocation from an intracellular pool into the plasma membrane and increase in glucose uptake. Ras-MAPK signaling pathway Ras plays a role in proliferation and mutated Ras is involved in carcinogenesis Ras-MAPK cascade: (in response to growth factor binding to its receptor) The ligand (growth factor ) binds on the extracellular loop of the receptor This binding causes a conformational change in the receptor The receptor is auto-phosphorylated (The intracellular domains of the receptor cross phosphorylate each other) This autophosphorylation increases the tyrosine kinase activity of the receptor and the receptor is thereby fully activated The fully activated receptor phosphorylates intracellular substrates Grb2-SOS exists as a complex (Grb2 has a specific domain called Src homology 2 (SH2) domain that recognizes tyrosine phosphorylated residues). Grb2 via its SH2 domains binds to phosphorylated/activated receptor. SOS is a GTP/GDP exchanger. When the Grb2 binds to the phosphorylated growth factor receptor, brings SOS to the proximity of the plasma membrane and SOS places Ras into a GTP-bound form and activates it. Remember: GTP bound Ras is active Ras Activated Ras binds to Raf kinase and activates it Raf kinase phosphorylates MAP kinase kinase (MEK) and activates it Activated MAP kinase kinase (MEK) phosphorylates MAP kinase (MAPK) and activates it MAP kinase (MAPK) phosphorylates proteins that lead to cellular responses insulin insulin -Ras- MAPK pathway Increased receptor tyrosine kinase activity P IRS Ras P IRS Grb2 Sos Raf kinase MAP kinase kinase =MEK MAP kinase = MAPK (ERK) Growth Responses Ras-MAPK cascade: (in response to insulin binding) The ligand (insulin) binds on the extracellular loop of the receptor This binding causes a conformational change in the receptor The receptor is auto-phosphorylated (The intracellular domains of the  subunits of the receptor cross phosphorylate each other) This autophosphorylation increases the tyrosine kinase activity of the receptor and the receptor is thereby fully activated The fully activated receptor phosphorylates intracellular substrates In the case of insulin being the ligand, that intracellular substrate is insulin receptor substrate-1 (IRS-1) IRS-1 binds to the receptor and is phosphorylated by its kinase activity Phosphorylated IRS-1 attracts the Grb2-SOS complex Grb2-SOS exists as a complex (Grb2 has a specific domain called Src homology 2 (SH2) domain that recognizes tyrosine phosphorylated residues) SOS is a GTP/GDP exchanger. When the Grb2 binds to IRS-1, brings SOS to the proximity of the plasma membrane and SOS put Ras into a GTP-bound form. Increases GTP binding to the small G-protein Ras and activates it. Remember: GTP bound Ras is active Ras Activated Ras binds to Raf kinase and activates it Raf kinase phosphorylates MAP kinase kinase (MEK) and activates it Activated MAP kinase kinase (MEK) phosphorylates MAP kinase (MAPK) and activates it MAP kinase (MAPK) phosphorylates proteins that lead to cellular responses Signaling through receptors associated with intracellular tyrosine kinases (TKs) General Structure of Receptors associated with TK extrecellular domain ligand-binding transmembrane domain growth hormone (somatotropin) receptor prolactin receptor erythropoietin receptor interleukin receptors Receptors associated with tyrosine kinase TM receptors Growth Hormone ECF transmembrane domain plasma membrane TK ICF Signal Transduction by Receptors associated with a TK growth hormone growth hormone receptor ATP Tyr-PO32 2O P-Tyr3 Tyr- -Tyr -Tyr JAK -Tyr-PO32 STAT JAK Tyr- JAK non-receptor protein tryrosine kinase Tyr ATP interactions with IRS, GRB, and G proteins ATP ADP regulation of gene expression STAT Tyr-PO32 transcription factor JAK/STAT pathway : activated by growth hormone (GH) binding to its receptor. GH GH GH GH transmembrane domain Nucleus JAK P P STAT STAT P P Gene transcription JAK-STAT Pathway JAK: Janus-Activated Kinase STAT: Signal Transducer and Activator of Transcription 1. Growth Hormone binds to its receptor 2. This causes a conformational change in the receptor bringing together the two JAKS associated with the receptor **JAK is a tyrosine kinase 3. JAK autophosphorylation 4. JAK activation 5. JAK phosphorylates tyrosine residues of the receptor 6. STAT (signal transducer and activator of transcription) binds to phosphorylated tyrosine residues of the receptor and is phosphorylated 7. Phosphorylated STAT dissociates from the receptor and STAT dimerizes and translocates to the nucleus 8. STATs regulate gene transcription ANP- cGMP signaling pathway ANP: Atrial Natriuretic Peptide ANP- Cyclic GMP (cGMP) signaling cascade Cyclic nucleotides have been extensively studied as second messengers of intracellular events initiated by activation of many types of hormone and neurotransmitter receptors. Cyclic guanosine monophosphate (cGMP) serves as a second messenger in a manner similar to that observed with cAMP. Peptide hormones, such as the natriuretic factors, activate receptors that are associated with membrane-bound guanylate cyclase (GC). Receptor activation of GC leads to the conversion of GTP to cGMP. Nitric oxide (NO) - Cyclic GMP (cGMP) signaling cascade Several families of phosphodiesterases (PDE-I-VI) act as regulatory switches by catalyzing the degradation of cGMP to guanosine-5’-monophosphate (5’-GMP). Three prescription drugs sildenafil (Viagra®) vardenafil (Levitra®) tadalafil (Cialis®) enhance the effects of NO by inhibiting the enzyme that normally breaks down cGMP. Nitric oxide (NO) - Cyclic GMP (cGMP) signaling cascade in vasodilation. When needed, certain drugs with vasoactive properties are released into the blood. These substances interact with specific receptors located at the endothelial layer of the vessel producing an elevation of calcium concentration inside the cells. Calcium activates an enzyme (endothelial nitric oxide synthase or eNOS) that makes nitric oxide (usually abbreviated as NO). Nitric oxide reach neighbour cells (other endothelial cells, blood cells and smooth muscle cells of the vessel). In smooth muscle cells, nitric oxide stimulate other enzyme: soluble guanylyl cyclase. Guanylyl cyclase synthesizes cyclic GMP which is able to activate protein kinase G. This enzyme mediates vasodilation, that is the increase in the diameter of the blood vessel and in consequence the decrease in blood pressure. Cycl ic GM P Site Crea ted by Dr. Luis Agul ló (last upda te on 28122007 ) Ani mati on of the Nitr ic Oxid e/Cy clic GM P Path way Fig. 1. The cycli c GM P path way in vaso dilati on. Whe n need ed, cert ain drug s with vaso activ e prop ertie s are rele ased into the bloo d. Thes e subst ance s inter act with spec ific rece ptors locat ed at the endo theli al laye r of the vess el prod ucin g an elev ation of calci um conc entr ation insid e the cells. Calc ium activ ates an enzy me (end othel ial nitri c oxid e synt hase or eNO S) that mak es nitri c oxid e (usu ally abbr eviat ed as NO). Nitri c oxid e reac h neig hbou r cells (oth er endo theli al cells , bloo d cells and mus cle cells of the vess el). In myo cyte s (mus cle cells ), nitri c oxid e stim ulate othe r enzy me: solu ble guan ylyl cycl ase. Gua nylyl cycl ase synt hesi zes cycli c GM P whic h is able to activ ate prot ein kina se G. This enzy me medi ates vaso dilati on, that is the incr ease in the diam eter of the bloo d vess el and in cons eque nce the decr ease in bloo d pres sure. Steroid hormones: mechanism of action Signaling through intracellular receptors hormone enters the cell and binds with the specific receptor ◆ ◆ hormone/receptor diffuses into the nucleus activates gene transcription to form messenger RNA (mRNA) ◆ the mRNA diffuses into the cytoplasm and promotes translation to form new proteins ◆ Steroid hormone control of gene transcription hormone receptor inactive gene HRE nucleus promoter active gene hormone mRNA synthesis cytosol blood Hormone response elements GRE (glucocorticoid) ERE (estrogen) TRE (thyroid hormone) Intracellular receptor structure DNA binding hormone binding hormone receptor Zinc finger motifs in DNA binding domain C Zn ++ C C Zn ++ C C C C C Hormone response elements DNA 5’-AGAACA nnn TGTTCT ------3’ Specific sequences HRE DNA binding domain of steroid H receptor Receptor/hormone complex DNA Steroid hormone action HSP: heat shock protein Steroid hormone Steroid hormone receptor Transcription of early regulatory genes Early regulatory or early response genes c-myc,c-fos, c-jun growth regulating or proto-oncogenes direct synthesis of mRNA and protein that are transcription factors these transcription factors regulate transcription of other genes ( “late” genes) early gene mRNA seen within minutes “late” gene mRNA 1 h or longer required to be seen Early regulatory or early response genes Signaling molecule (Hormone) action could affect: cell membrane permeability levels of a metabolite enzyme function gene transcription protein -protein interaction cellular localization of substrate cytoskeletal arrangement Protein phosphorylation Important modification of many cellular proteins Important in many signal transduction pathways Net levels of phosphorylation determined by: The activity of both Kinases Phosphatases Kinases Add phosphate groups to ser/thr or tyr residues Serine/ Threonine kinases Tyrosine kinases Phosphatases Remove phosphate groups from ser/thr or tyr residues Serine/ Threonine Phosphatases (PSPs) Tyrosine Phosphatases (PTPs) Phosphorylation /dephosphorylation of proteins protein kinase ATP ADP protein-PO3 2- protein Pi phosphoprotein phosphatase Amplification during signal transduction hormone Amplification Amplification receptor Aden Cyclase cAMP PKA Amplification Amplification modified enzyme metabolic product Crosstalk in signal transduction R1 G1 E1 M1 R2 G2 E2 M2 R3 G3 E3 M3 R: receptor G: G protein E: effector M: messenger Cellular response Multiple receptors for the same ligand Figure 6-18 Receptor agonists and antagonists Receptor Antagonists Reversible antagonists compete for agonist binding at the same molecular binding site but their binding is not permanent and can be displaced when agonist concentration increase Irreversible antagonists covalently bind to the receptor binding site and prevent agonist binding Receptors: Binding Parameters Radiolabelled Ligands (Hormones) are used to measure binding properties of hormones on intact cells, isolated membranes or cell fractions, or with purified receptor proteins. Receptor-Ligand complex (Bound) separated from Free Ligand by sedimentation, precipitation, or microfiltration Plots yield Bmax and Kd Receptor binding characteristics Bmax Bmax Fig 9-1 HLSC 3P09 Second messenger question 1. What is second messenger? Give 4 criteria for a substance to act as a second messenger. You have just discovered a new hormone and you hypothesize that it acts through cAMP. How would you determine whether cAMP acts as a second messenger for the hormone? Describe/design different studies you could perform to study your hypothesis. Answer: Second messenger is a chemical substance produced within the cell in response to a ligand (signaling molecule, hormone) binding to PM receptor. This substance then mediates the hormones action (induces a cellular response in the target cell). The ligand is the first messenger and cannot cross the plasma membrane (it is not lipid soluble; it is water soluble) to get into the cell. By binding to the receptor and initiating a cascade of events the ligand/first messenger leads to the generation of the second messenger, a water-soluble chemical generated inside the cell that can diffuse and influence the activity of intracellular effector molecules/proteins. Examples: cyclic AMP (cAMP), IP-3, DAG, Ca2+ Criteria: The enzyme that produces the messenger should respond to the hormone. A change in the concentration of the messenger should precede or occur simultaneously with the intended action of the hormone. Inhibitor of the removal or degradation of the messenger should function synergistically with the hormone that promotes its synthesis. The biological effect of the hormone should be mimicked directly by the addition to the cell of the messenger itself or a suitable analog. Since the hormone acts by employing c AMP as a second messenger the Signaling Cascade is as follows: Hormone binds to 7 transmembrane Adenylate cyclase generates c AMPK which acts as second messenger and binds and activates protein kinase A (PKA) PKA then phosphorylates proteins leading to the cell response. for the experimental design: Answer (continue): As an example, we can think of the action of glucagon on liver cells/hepatocytes. You can design experiments utilizing hepatocytes in cell culture and your signaling molecule/hormone is glucagon. Give explanation of comparing control, untreated cells to stimulated cells. Specify which protein/molecule in the pathway is important to identify based on each criterion (ex, AC is enzyme, cAMP is second messenger, phosphodiesterase is inhibitor) For criteria 1: The enzyme that produces the messenger should respond to the hormone. The enzyme is Adenylate cyclase: therefore, you will design an experiment that will measure Adenylate cyclase activity. Have control and hormone (glucagon) treated cells (hepatocytes), use an established assay/protocol that measures Adenylate cyclase activity. For criteria 2: A change in the concentration of the messenger should precede or occur simultaneously with the intended action of the hormone. The messenger is cAMP; therefore, the experiment will measure cAMP levels. Have control and hormone (glucagon) treated cells (hepatocytes), use an established assay/protocol that measures cAMP levels. Answer (continue): For criteria 3: Inhibitor of the removal or degradation of the messenger should function synergistically with the hormone that promotes its synthesis. The molecule that degrades the messenger / cAMP, is phosphodiesterase. Therefore, in the experiment you should use a phosphodiesterase inhibitor. Have control, phosphodiesterase inhibitor and hormone (glucagon)-treated cells (hepatocytes). Use an established assay/protocol that measures a hormone response (glucose production). For criteria 4: The biological effect of the hormone should be mimicked directly by the addition to the cell of the messenger itself or a suitable analog. The biological effect of glucagon on hepatocytes is to increase endogenous glucose output, increase gluconeogenesis, and Increase glycogenolysis. The net effect of glucagon on hepatocytes is to increase glucose output. You could therefore utilize an assay the measures glucose release by the cells into the media. Adding a plasma membrane permeable cAMP analogue to the cells should mimic the effect of the hormone. cAMP is not water soluble and cannot cross the plasma membrane, so companies have created an analog which is able to enter the PM without a receptor/carrier protein, and it can therefore activate the signaling cascade. the end