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Signal Transduction and the Related Disorders Department of Pathophysiology Shanghai Jiao-Tong University School of Medicine CHAPTER 1 General Introduction of Cell Signal Transduction Concept of Cell Signaling The process in which cells sense the extracellular stimuli through membranous or intracellular receptors, transduce the signals via intracellular molecules , and thus regulate the biological function of the cells Signal molecules Physical signals Light, electronic, mechanic, UV, heat, volume or osmotic, etc Chemical signals Hormones, neurotransmitters, Growthe factors, cytokines, odor molecules, ATP, active oxygen, drugs, toxins, etc Modes for the function of endogenous signals Endocrine: Act on a far away organ via blood circulation Paracrine: Act on a nearby target Autocrine: Act on itself after secreted Synaptic: Presynaptic to postsynaptic, Endocrine Autocrine Paracrine Synaptic The primary pathways of cell signalling G-protein-mediated pathway Adenylate cyclase mediated pathway Phospholipase mediated pathway Small G-protein-mediated pathway Non-G-protein-mediated pathway Receptor tyrosine kinase mediated pathway Receptor serine/threonine kinase mediated pathway Receptor guanilate cyclase mediated pathway Intracellular (unclear) receptor mediated pathway G-protein-mediated pathway G-proteins, coupled with members of the seven transmembrane domain of the receptor superfamily, are regulatory proteins that act as molecular switches. They control a wide range of biological processes Classification of G-protein High moleular weight G-protein (trimeric GTP-binding regulatory protein) Low moleular weight G-protein Ras Regulation of G-Protein Activity G protein-coupled receptors exhibit a common structural motif consisting of seven membrane spanning regions. Receptor occupation promotes interaction between the receptor and the G protein on the interior surface of the membrane. This induces an exchange of GDP for GTP on the G protein subunit and dissociation of the subunit from the heterodimer. Depending on its isoform, the GTP- subunit complex mediates intracellular signaling either indirectly by acting on effector molecules such as adenylyl cyclase (AC) or phospholipase C (PLC), or directly by regulating ion channel or kinase function. Regulation of G-Protein Activity Regulation of G-Protein Activity Regulation of G-Protein Activity Regulation of G-Protein Activity G-protein-mediated pathway Adenylate cyclase mediated pathway Phospholipase mediated pathway Small G-protein-mediated pathway G-protein-mediated pathway Adenylate cyclase mediated pathway cAMP can activate protein kinase A(PKA), which can phosphorylate CREB( binding protein of cAMP-respones element)and initiate gene transcription. CRE is cAMP response element in DNA. G-protein-mediated pathway Phospholipase C mediated pathway Non-G-protein-mediated pathway Receptor tyrosine kinase mediated pathway Receptor serine/threonine kinase mediated pathway Receptor guanilate cyclase mediated pathway Intracellular (unclear) receptor mediated pathway Receptor tyrosine kinase mediated pathway Receptor tyrosine kinases transmit signals across the plasma membrane, from the cell exterior to the cytoplasm. The interaction of the external domain of a receptor tyrosine kinase with the ligand, often a growth factor, up-regulates the enzymatic activity of the intracellular catalytic domain, which causes tyrosine phosphorylation of cytoplasmic signaling molecules. Receptor tyrosine kinase mediated pathway Receptor tyrosine kinases transmit signals across the plasma membrane, from the cell exterior to the cytoplasm. The interaction of the external domain of a receptor tyrosine kinase with the ligand, often a growth factor, up-regulates the enzymatic activity of the intracellular catalytic domain, which causes tyrosine phosphorylation of cytoplasmic signaling molecules. Mechanism of Tyrosine Kinase Receptors When hormone binds to the extracellular domain the receptors aggregate Mechanism of Tyrosine Kinase Receptors When the receptors aggregate, the tyrosine kinase domains phosphorylate the C terminal tyrosine residues Mechanism of Tyrosine Kinase Receptors This phosphorylation produces binding sites for proteins with SH2 domains. GRB2 is one of these proteins. GRB2, with SOS bound to it, then binds to the receptor complex. This causes the activation of SOS. Mechanism of Tyrosine Kinase Receptors SOS is a guanyl nucleotide-release protein (GNRP). When this is activated, it causes certain G proteins to release GDP and exchange it for GTP. Ras is one of these proteins. When ras has GTP bound to it, it becomes active. Mechanism of Tyrosine Kinase Receptors Activated ras then causes the activation of a cellular kinase called raf-1 Mechanism of Tyrosine Kinase Receptors Raf-1 kinase then phosphorylates another cellular kinase called MEK. This cause the activation of MEK Mechanism of Tyrosine Kinase Receptors Activated MEK then phosphorylates another protein kinase called MAPK causing its activation. This series of phosphylating activations is called a kinase cascade. It results in amplification of the signal Mechanism of Tyrosine Kinase Receptors Among the final targets of the kinase cascade are transcriptions factors (fos and jun showed here). Phosphorylation of these proteins causes them to become active and bind to the DNA, causing changes in gene transcription Signal Transduction Through Receptor Tyrosine Kinases Receptor serine/threonine kinase mediated pathway (1) Type I and type II receptors for TGF(beta) in a cell prior to binding of the growth factor. (2) Binding of growth factor results in clustering of type I and type II receptors, and phosphorylation of type I receptors by type II receptors. (3) The activated type I receptors then phosphorylate particular receptor-mediated Smads. (4) These Smads then bind to other Smads (co-Smads), and together they enter the nucleus. Intracellular (nuclear) receptor mediated pathway Basic Structure of nuclear receptor Hormone-bindind domain DNA-binding domain Transcriptionactivating domain nuclear receptor mediated pathway The signal pathway by steroid hormones The signal pathway by steroid hormones Networks of Signal Transduction 600 G protein-coupled receptors + Multiple gene families and combinations of G protein subunits 20G isoforms 6 G isoforms 12 G isoforms + Multiple gene families for selected effector proteins Adenylyl cyclases Phospholipases Ion channels Cascade structure of cellular signal pathways The magnitude of amplification within this cellular cascade structure often exceeds 10+4. That is, the binding of one molecule of ligand to a cell-surface receptor leads a change of 10,000-fold in the intracellular concentration of a metabolic product. CHAPTER 2 Dysfunction of cellular signal transduction in diseases Aberrant Signal in cell signaling Aberrant Receptor in cell signaling Aberrant G-protein in cell signaling Aberrant Intracellular Signaling Multiple Abnormalities in cell signaling Aberrant Signal in cell signaling ischemia, epilepsy, neurodegenerative diseases extracellular glutamate/aspartic acid NMDAR activation Ca2+ influx [Ca2+]i , activation of enzymes excitatory intoxication Aberrant Receptor in cell signaling Receptor-based diseases Alterations in number, structure or function of receptors will lead to disorder in cellular signal transdution Up-regulation/hypersensitivity Down-regulation/desensitization Receptor Gene Mutation Myasthenia Gravis Myasthenia Gravis is an autoimmune receptor disorder in which antibodies form against acetylcholine(Ach) nicotinic postsynaptic receptors at the neuromuscular junction mechanism anti n-AchR AchR Ach The Neuromuscular Junction Contraction of muscle fiber influx of Na manifestations Drooping of the eyelids Double vision Difficulty smiling, speaking, swallowing Difficulty raising the arms Difficulty walking Difficulty breathing if chest muscle are affected Autoimmune Thyroid Diseases hyperthyroidism (Grave's disease) hypothyroidism (Hashimoto's thyroiditis) mechanism Stimulatory Ab Blocking Ab TSH-R TSH-R Gs Gq 295~302 385~395 AA residues PLC AC IP3 DG Ca2+ PKC Binding of TSH to R↓ cAMP Thyroid proliferation & secretion hyperthyroidism hypothyroidism manifestations Grave's disease Stimulatory antibodies mimic the function of TSH Stimulating thyroid hormone synthesis, secretion, and thyroid growth female:male incidence -- 5:1 to 10:1 diffusely enlarged goiter manifestations Hashimoto's thyroiditis Inhibitory antibodies antaonize the function of TSH Inhibiting thyroid hormone synthesis, secretion, and thyroid growth Thyroid gland is gradually destroyed Myxedema Receptor Gene Mutation-Genetic insulin-resistant diabetes Diabetes Mellitus Type 1 Diabetes Mellitus Type 2 Non-Insulin dependent diabetes mellitus, NIDDM NIDDM is a chronic metabolic syndrome defined by resistance to the hormone insulin. This leads to inappropriate hyperglycaemia (increased blood sugar levels) and deranged metabolism of carbohydrate, fats and proteins. mechanism The cause of Diabetes Mellitus Type 2 is not known, but it may involve a defect or change in the insulin receptor (IR). Genetic insulin-resistant diabetes IR gene mutations synthesis transfer to the membrane Disturbances in affinity to insulin RPTK activation proteolysis Diabetes Mellitus Type 2 G protein-based disease GHRH pituitary tumor Pituitary GHRH Receptor Gsα (+ ) cAMP (-) GH secretion Gi GHRH--Growth-hormone-releasing hormone somatostatin GH--Growth-hormone mechanism Gs gene mutation GTPase activity Persistent activation of Gs Persistent activation of AC cAMP Pituitary proliferation and secretion Acromegaly or Gigantism manifestations G-protein modification—— cholera lumen of intestine Na+ H2O Cl- cAMP ↑ ↑ ↑ CT CT--Cholera toxin Gs AC Gs ribosylation at Arg201 manifestations Diarrhea Dehydration Circulation failure Aberrant Intracellular Signaling The intracellular signaling involves various messengers, transducers and transcription factors. Disorders can occur in any of these settings. WNT sinal pathway Cancer Wnt-1 was found as an oncogene activated by the Mouse Mammary Tumor Virus in murine breast cancer. APC was first isolated as a tumor suppressor gene in human colon cancer. After establishing that APC and beta-catenin bind to each other activating mutations in the human beta-catenin gene were found in human colon cancer and melanomas .These mutations alter specific beta-catenin residues important for GSK3 phosphorylation and stability .The role for Frat/GBP in cancer is illustrated by its activation by proviral insertion in mouse lymphomas. Interestingly, mutations in the human AXIN1 gene were reported in human hepatocellular carcinomas. TCF1 can also act as a tumor suppressor gene , as Tcf1 mutant mice develop adenomas in the gut and mammary glands Multiple Abnormalities in Signaling Pathway In the development of diseases, the aberrant cellular signal transduction usually involves multiple molecules or pathways. Such diseases include type-2 diabetes mellitus, cancers, hypertension, and so on Multifactor Aberrancies and Cancer (Enhancement of proliferating signals) Ligands (GFs) Receptors (overexpression, activation of TPK) Intracellular transducers: Ras mutation Ras-GTPase MEK ERK Proliferation Ras activation Cancer Raf Multifactor Aberrancies and Cancer (Deficits in proliferation-inhibiting signal) (TGF-β)2 Cell memberane GS Ⅱ Ⅰ Ⅱ Ⅰ Smad2 SARA (—) Smad6,7 Smad2 -P Smad4 Smad4 Cytosal Smad2 -P Nuclear membrane P300 P300 Smad4 Smad2 -P Fast2 Fast2 P15、P21 Principles for Treatment To regulate the level of extracellular molecules To regulate the structure and the function of receptors To regulate the level and modifications of intracellular messenger molecules and transducers To regulate the level of nuclear transcription factors Target Therapy The Philadelphia Chromosome: t(9;22) Translocation Chr.9+ Chr.9 Chr.22 Ph bcr abl bcr-abl FUSION PROTEIN WITH TYROSINE KINASE ACTIVITY Structure of BCR-ABL Fusion Proteins p210Bcr-Abl Fusion Protein Tyrosine Kinase Extracellular space Cytoplasm Y177 BAP-1 SH3 SH2 SH1 GRB2 CBL SHC CRKL Gleevec®-Tyrosine Kinase Inhibitor Bcr-Abl Bcr-Abl Substrate Substrate P P P ATP P STI571 Y = Tyrosine P = Phosphate Goldman JM. Lancet. 2000;355:1031-1032.