CBS Intracellular Signalling KEATS 22_23 PDF

Dr Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Department Biochemistry Intracellular Signalling  Teaching Objectives Describe different ways for cells to...

Dr Stuart Knight Foundations of Medical Science Cell Biology and Signalling block Department Biochemistry Intracellular Signalling  Teaching Objectives Describe different ways for cells to signal to each other Explain what is meant by the term ‘second messenger’ and give examples Describe the importance of transmembrane receptors and their associated signalling pathways in controlling gene activity Give examples of the roles of G proteins, cAMP, phospholipase C, diacylglycerol , inositol triphosphate (IP3) and Receptor Tyrosine Kinase (RTK) in signal transduction pathways Outline a metabolic regulated via intracellularly signalling Recognise that overlap of signalling pathways can occur Beware that intracellular signalling is important in the action of drugs  How cells change Cell Division Cell Growth Differentiation Cell Movement  Cellular responses Change metabolic activities – Glucagon switches liver from synthesising glycogen to breaking down glycogen Secrete and release – Binding of antigen to mast cell stimulates the secretion of histamine Changes in gene expression – Epidermal Growth Factor (EGF) activating genes involved in cell growth Sensory perception – Light activation of rhodopsin 4  What factors act as extracellular signals? Amino acids (and derivatives) – glutamate; adrenaline; dopamine, etc. Steroids – oestradiol; testosterone; cortisol; aldosterone, etc. Prostaglandins (eicosanoids) – derived from arachidonic acid Proteins and Peptides – insulin; glucagon; growth factor; EGF, etc. Gases – NO; CO 5  Different ways for cells to signal to each other Endocrine – Signal produced by cells in one part of body and travels in blood to target cells somewhere else Autocrine – Signal acts on the same cell that produces it Paracrine – Signal produced by cell and acts on other cells that are very close Contact dependent – Signal is integral part of one cell and interacts directly with another cell Neuronal – Electrical signal transmitted down cell and message passed to another via synapse 6  Receptor / Signal Cell must express receptor in order to respond to signal Receptors have high selectivity Receptors have high affinity Signal can bind to different types of receptor – β adrenergic receptor (adrenaline) – α adrenergic receptor (adrenaline) Signal is eventually turned off  Location of receptor 1. Cell surface receptor – Hormone is hydrophilic e.g. adrenaline – Binding of hormone triggers response inside cell – Hormone does not “enter” the cell 2. Intracellular receptor – Hormone is hydrophobic e.g. steroid hormones – Hormone crosses the plasma membrane – Hormone binds to receptor in the cytosol and triggers a response inside cell 8  Types of signalling Binding of signal to receptor – Depolarisation of membrane due to flow of ions – [see physiology lectures] acetylcholine – Direct activation of transcription factor steroid – Generation of secondary message inside cell glucagon – cAMP – Direct activation of enzymatic kinase cascade EGF – MAP kinase pathway 9 Depolarisation of membrane due to flow of ions Ion channels e.g. acetylcholine binding to nicotinic acetylcholine receptor Na+, K+, Ca2+ 10  Direct activation of transcription factors Steroid hormones contain a hormone binding domain, a DNA binding domain and a domain for interacting with other transcription factors Binding of steroid induces conformational change that allows DNA binding and activation of transcription of target genes Sequence specific DNA binding domain – hormone response elements in sequence of target genes They are ligand–dependent transcription factors 11  Secondary message inside cell Signal = first messenger plasma membrane R cyclic AMP cyclic GMP IP3/DAG nitric Ca2+ oxide 12  2nd messengers generated by enzymes G-protein-coupled Receptor (GPCR) – Activation of adenylyl cyclase (adenylate cyclase) adenosine 3’:5’- cyclic monophosphate (cAMP) – Activation of phospholipase C inositol 1,4,5-trisphosphate (IP3) 1,2-diacylglycerol (DAG) 13  G-protein-coupled receptor 7 transmembrane-spanning G-protein e.g. β-adrenergic receptor coupled receptors (adrenaline) eg -adrenergic receptor NH3+ transmembrane -helix Exterior 1 2 3 4 5 6 7 Cytosol G-protein COO- interaction domain 14  Guanine nucleotide binding proteins (G-proteins)   g   g GDP INACTIVE GTP ACTIVE Heterotrimeric complex Dissociates when GTP binds Free active G subunit activates effector enzymes Complex re-associates when GTP hydrolysed to GDP by a 15 GTPase activity GPCR Signalling to Effector Enzymes 1. Signal (e.g. adrenaline) binds to receptor 2. G-protein (GDP bound) associates with receptor 3. GTP/GDP exchange on G-protein (GTP bound) 4. G-proteins dissociates into  (GTP bound) and g subunits 5.  subunit (with GTP bound) activates effector enzyme 6. Effector enzyme produces 2nd messenger 7. GTP hydrolysed to GDP, G-protein complex re- associates, signalling ends 16  Stimulation of adenylyl cyclase Signal 1-3 R s s  g cyclase  g GDP GDP/GTP Signal 4-6 R s cyclase s  g GTP GTP ATP cAMP Signal Off 7 R cyclase s  g GDP 17 ATP cAMP dependent protein kinase A (PKA) Tetrameric enzyme, 2 regulatory (R) and 2 catalytic (C) subunits (R2 C2) cAMP binds to the regulatory subunit and tetramer dissociates Catalytic monomers (C) are now active enzymes cAMP dependent protein kinase (PKA) cAMP C C R cAMP R ACTIVE C R R C cAMP INACTIVE cAMP mediated effects on glycogen breakdown phosphorylase protein kinase A (PKA) phosphorylase kinase b kinase a P ATP ADP ACTIVE phosphorylase kinase a phosphorylase b phosphorylase a ACTIVE P ATP ADP phosphorylase a GLUCOSE -1 - GLYCOGEN PHOSPHATE glycogen protein kinase A (PKA) glycogen synthase a synthase b P INACTIVE ATP ADP  Signal amplification via kinase cascade 1 hormone glucagon glucagon receptor ATP cAMP 40 molecules inactive PKA active PKA 10 enzymes 250 inactive phosphorylase kinase active phosphorylase kinase enzymes inactive phosphorylase active phosphorylase 40,000 enzymes The numbers are not accurate but are designed to illustrate amplification  cAMP ATP AMP adenylyl cyclase phosphodiesterase 21  cAMP and gene transcription PKA phosphorylates CREB (cAMP response element binding protein) CREB binds to specific sequences in target genes and stimulates transcription Long term adaptation to starvation: changes in gene expression 22  GPCR and IP3/DAG Some GPCR contain Gαq (Gq) subunit Dissociated Gq activates phospholipase C Phospholipase C cleaves inositol phospholipids in membrane – Diacylglycerol (DAG) – Inositol 1, 4, 5 trisphosphate (IP3) IP3 activates Ca2+ channel in endoplasmic reticulum – Ca2+ concentration increases in cytosol DAG together with Ca2+ activates protein kinase C e.g. α1-adrenergic (adrenaline) receptor 23  Receptor Activation of Phospholipase C Figure 16-25 Essential Cell Biology (© Garland Science 2010)  Direct activation of enzymatic kinase cascade Binding of EGF triggers the autophosphorylation of tyrosine residues in cytoplasmic domain of receptor : Receptor Tyrosine Kinase (RTK) Adaptor proteins contain phosphotyrosine binding domains – SH2 (src – homology 2) – PTB (phosphotyrosine binding) Adaptor proteins Grb2 and Sos bind to receptor This complex activates the exchange GDP-Ras GTP- Ras 25  Ras is a G-protein Ras is monomeric G-protein GTP-Ras triggers a kinase cascade MAPKKK (mitogen activated protein kinase kinase kinase) activates MAPKK that activates MAPK that activates transcription factor Ras-MAP kinase pathway This is not a secondary messenger 26  Overlap One hormone different receptors Receptors trigger different pathways Convergence – Different signals trigger different pathways but cause the same effect in the cell Cross talk – Different signals trigger different pathways that block each other – EGF signalling via PTK and Ras-MAP kinase but adrenaline inhibiting one of the steps via the action of PKA 27  Summary Binding of extracellular signal to receptor triggers response in target cell G-protein coupled receptors (GPCR) – Generate cAMP – leading to activation of PKA – Generate IP3 , DAG and Ca2+ – leading to activation of PKC Receptor tyrosine kinase (RTK) – Direct activation of kinase cascade without secondary messenger 28