Cellular Signaling PDF - School of Osteopathic Medicine

School of Osteopathic Medicine Cellular Signaling Christopher Beevers, Ph.D. School of Osteopathic Medicine May not be used or reproduced without written permission from the SOM 1 Topic #1 General Principles of Cellular Signaling School of Osteopathic Medicine 2 Cellular Signaling I. Produ...

School of Osteopathic Medicine Cellular Signaling Christopher Beevers, Ph.D. School of Osteopathic Medicine May not be used or reproduced without written permission from the SOM 1 Topic #1 General Principles of Cellular Signaling School of Osteopathic Medicine 2 Cellular Signaling I. Production, release, detection, & response to extracellular signaling molecules is a key regulatory & control mechanism in human body A. Carry information that controls numerous biochemical & physiological processes (e.g., gene expression, metabolism, & growth & differentiation) B. Large variety of molecules are utilized (e.g., small organic molecules, lipids, peptides, proteins, etc.) II. Signaling cells (produce & secrete extracellular signaling molecule, i.e., the ligand) → Target cells (respond to ligand via a receptor) School of Osteopathic Medicine 3 Types of Cellular Signaling (d) Juxtacrine signaling School of Osteopathic Medicine 4 Two Main Receptor Types Cell Surface Receptors Induce Signal Transduction Intracellular Receptors Directly Regulate Gene Expression School of Osteopathic Medicine 5 Topic #2 Signaling Molecules & Cell Surface Receptors School of Osteopathic Medicine 6 Cell Surface Receptor Signaling 2. Reception 3. Transduction 4. Response 5. Termination (removal of signal) 1. Synthesis (in signaling cell), Release (by signaling cell), & Transport (in body fluids) School of Osteopathic Medicine 7 Naming Signal Transduction Pathways I. Based on receptor class (e.g., GPCR pathways utilize G protein-coupled receptors) II. Based on ligand (e.g., Hedgehog pathway utilizes extracellular signaling molecule called Sonic Hedgehog) III. Based on key intracellular component (e.g., NF-B pathway utilizes intracellular protein called nuclear factor kappa B) School of Osteopathic Medicine 8 Common Features of Signal Transduction Pathways I. 2 major types of cellular responses are induced by activation of signal transduction pathways A. Regulation of pre-existing proteins B. Changes in gene expression II. 1 receptor can activate multiple signal transduction pathways, each of which can elicit different cellular responses School of Osteopathic Medicine 9 Major Types of Cell Surface Receptors I. G protein-coupled receptor (GPCR) A. Possesses 7 transmembrane domains & is associated with trimeric G proteins B. Utilizes cAMP/PKA & IP3/DAG/Ca2+ pathways II. Receptor tyrosine kinase (RTK) A. Multi-subunit complex possessing intrinsic protein tyrosine kinase enzyme activity (i.e., phosphorylate Tyr residues of proteins) B. Utilizes Ras/MAPK, PI3K, JAK/STAT, & IP3/DAG/Ca2+ pathways III. Tyrosine kinase-associated receptor A. Multi-subunit complex associated with protein tyrosine kinase enzymes B. Utilizes the same pathways as RTKs IV. Receptor protein tyrosine phosphatase (RPTP) A. Possesses 1 transmembrane domain & intrinsic protein tyrosine phosphatase enzyme activity (i.e., dephosphorylate Tyr residues of proteins) 10 School of Osteopathic Medicine Major Types of Cell Surface Receptors (2) I. Receptor serine/threonine kinase A. Possesses 1 transmembrane domain, but can be multi-subunit complex B. Possesses intrinsic protein serine/threonine kinase enzyme activity (i.e., phosphorylate Ser & Thr residues of proteins) C. Utilizes Ras/MAPK & Smad pathways II. Receptor guanylyl cyclase A. Possesses 1 transmembrane domain & intrinsic guanylyl cyclase enzyme activity (i.e., produces cyclic guanosine monophosphate, cGMP) B. Utilizes cGMP/PKG pathways III. Death receptor A. Possesses 1 transmembrane domain B. Utilizes death-domain accessory protein & caspase pathways IV. Ligand-gated ion channel A. Multi-subunit complex that forms gated pore through plasma membrane B. Controls ion flow into & out of cells School of Osteopathic Medicine 11 Receptor-Ligand Interactions I. Receptors display binding specificity (1 ligand/receptor; ligands are more promiscuous) II. Reversible binding is mediated by noncovalent interactions (e.g., H-bonding, ionic interactions, hydrophobic interactions, van der Waals forces) KD  Affinity School of Osteopathic Medicine KD = [L] w/ 50% R occupancy 12 Saturation Kinetics of Cell Surface Receptors Cellular response to ligand binding remains stable at this point despite continued ↑ [Ligand] [Ligand] School of Osteopathic Medicine 13 Cellular Response to RL Interaction I. Effector specificity: specific RL interactions cause specific cellular responses A. E.g., acetylcholine (ACh) receptors 1. Nicotinic muscular (NM) ACh receptor (ligand-gated ion channel) → induces striated muscle cell contraction 2. M2 muscarinic ACh receptor (mAChR) (GPCR) → decreases heart contractility 3. M1 & M3 mAChRs (GPCRs) → induce pancreas release of digestive enzymes II. Multiple RL interactions can induce the same cellular responses A. E.g., epinephrine, glucagon, & ACTH (in liver) all activate cAMP pathway School of Osteopathic Medicine 14 Percent RL Interaction & Percent Cellular Response KD School of Osteopathic Medicine 15 Sensitivity I. This is dependence of cellular response on cell’s receptor # for a particular ligand A. ↑ receptor #, ↑ sensitivity to ligand (& vice versa) II. Regulation of receptor # & expression are key for controlling physiological & developmental events School of Osteopathic Medicine 16 Topic #3 Intracellular Signal Transduction School of Osteopathic Medicine 17 1st Messengers to 2nd Messengers to Signal Transduction Proteins I. 1st messengers = extracellular signaling molecules II. 2nd messengers = intracellular signaling molecules III. 3 main groups of signal transduction proteins involved in intracellular signaling School of Osteopathic Medicine Ca2+ Phosphoinositides 18 GTPase Switch Proteins (G Proteins) I. Trimeric (large) G proteins (direct receptor interactions) II. Monomeric (small) G proteins (indirect receptor interactions) School of Osteopathic Medicine 19 Protein Kinases & Protein Phosphatases Ser/Thr or Tyr School of Osteopathic Medicine May be intrinsic to the receptor, receptorassociated, or intracellularly located 20 Interaction & Regulation of Signal Transduction Pathways I. Pathway interaction is critical A. Activation of single R type can produce multiple 2nd messengers B. Multiple signaling pathways can induce same cellular response 1. Allows for fine-tune control of complex processes (not all-or-nothing control) II. Pathway regulation is important A. 2nd messenger degradation B. Signal transduction protein deactivation C. Receptor desensitization (chemical structure modification, & endocytosis leading to sequestration or degradation) School of Osteopathic Medicine 21 Topic #4 E: extracellular segments H: transmembrane segments C: cytosolic segments Basics of G Protein-Coupled Receptors School of Osteopathic Medicine 22 GPCR & Trimeric G Protein Structure E: extracellular segments H: transmembrane segments C: cytosolic segments → • Remain associated together • Occasional mediator of effector protein School of Osteopathic Medicine • GTPase switch protein • Usual mediator of effector protein 23 Some Major Classes of Trimeric G Proteins & Their Major Effector Proteins G Class Gs Gi Gq Associated Effector Protein Effect on 2nd Messenger Receptor Example Adenylyl Cyclase ↑ cAMP 1-adrenoceptor 2-adrenoceptor Glucagon receptor Adenylyl cyclase ↓ cAMP K+ channel (activated by Change in membrane G subunit) potential Phospholipase C cGMP Gt (Transducin) phosphodiesterase School of Osteopathic Medicine 2-adrenoceptor M2 mAChR ↑ IP3 & DAG 1-adrenoceptor M1 & M3 mAChRs ↓ cGMP Rhodopsin 24 Mechanism of Action of GPCR School of Osteopathic Medicine 25 Topic #5 ATP → cAMP + PPi GPCRs That Regulate Adenylyl Cyclase School of Osteopathic Medicine 26 Adenylyl Cyclase (AC) AC ATP → cAMP + PPi School of Osteopathic Medicine 27 Epinephrine & Gs I. Hormone produced by adrenal gland that is body’s fight-or-flight response mediator II. Activates 1-adrenoceptor (expressed in heart, kidneys, neurons, & adipocytes) & 2-adrenoceptor (expressed in smooth muscle, liver, heart, skeletal muscle, neurons) A. Epi → 1/2 → Gs → AC → ↑[cAMP]Intracellular School of Osteopathic Medicine 28 Clinical Application: -Adrenoceptors are Pharmacological Targets I. Isoproterenol: agonist (i.e., activator) of 1- & 2adrenoceptors A. Utilized in therapy of emergency cardiovascular situations to ↑ heart contractility & rate II. Propranolol: antagonist (i.e., inhibitor) of -adrenoceptors A. Utilized in therapy of hypertension, cardiac arrhythmias, & angina School of Osteopathic Medicine 29 Clinical Application: Cholera I. Severe form of diarrhea that can lead to death by rapid dehydration A. Caused by bacterium Vibrio cholerae 1. Produces & secretes cholera toxin (CT; protein complex) in small intestine a. Internalized into intestinal mucosal cells where it uses nicotinamide adenine dinucleotide (NAD+) to add ADP-ribose (reaction is called ADPribosylation) to Gs i. Allows activation of AC, but inhibits GTPase activity a) Causes continuous cAMP production which causes excessive water & electrolyte secretion into intestinal lumen School of Osteopathic Medicine 30 School of Osteopathic Medicine 31 Clinical Application: Toxic Thyroid Nodules I. Benign thyroid adenomas (i.e., tumors) A. Sometimes caused by Gs mutations that inactivate GTPase activity 1. Causes overproduction of cAMP in absence of extracellular signaling molecule (i.e., thyroid-stimulating hormone, TSH) activation a. Results in excessive thyroid hormone production, abnormal cell proliferation, & adenoma formation School of Osteopathic Medicine 32 Clinical Application: Pseudohypoparathyroidism I. Condition caused by Gs mutations that prevent its association with parathyroid hormone (PTH) receptor A. Makes cells resistant to the action of PTH 1. Extracellular signaling molecule produced by parathyroid gland that regulates maintenance of [Ca2+]Blood B. Symptoms include mild skeletal deformities, short stature, muscle spasms, cramping, developmental delay, & seizures School of Osteopathic Medicine 33 Epinephrine & Gi I. Activates 2-adrenoceptor (expressed in neurons, pancreas, platelets, ciliary epithelium, & smooth muscle) A. Epi → 2 → Gi AC (↓ [cAMP]Intracellular) School of Osteopathic Medicine 34 Clinical Application: 2-Adrenoceptor is Pharmacological Target I. Clonidine: agonist of 2adrenoceptor A. Utilized in therapy of hypertension, open-angle glaucoma, & other important indications II. Yohimbine: antagonist of 2adrenoceptor A. Utilized in the therapy of impotence & as aphrodisiac School of Osteopathic Medicine 35 Clinical Application: Whooping Cough I. Acute inflammation of upper respiratory tract causing severe coughing attacks, inspiratory whoop, & fainting & vomiting after coughing A. Caused by bacterium Bordetella pertussis 1. Produces & secretes pertussis toxin (PT; protein complex) in upper respiratory system a. Internalized into upper respiratory cells where it uses NAD+ to do ADP-ribosylation of Gi i. Keeps it in GDP-bound state, preventing it from inhibiting AC a) Leads to continuous cAMP production School of Osteopathic Medicine 36 Fine-Tune Control of Adenylyl Cyclase School of Osteopathic Medicine 37 Protein Kinase A (PKA) School of Osteopathic Medicine 38 Protein Kinase A (2) AC Ser/Thr Ser/Thr School of Osteopathic Medicine 39 PKA Regulation of Glycogen Metabolism cAMP ← AC ← Gs ← GPCR ← Epi/Glucagon School of Osteopathic Medicine 40 PKA Regulation of Gene Expression CRE-Binding Protein (Transcription Factor) CREB-Binding Protein (Coactivator) BTM School of Osteopathic Medicine cAMP-Response Element Expression of Target Genes → Altered Cellular Function → Changes in Neuronal Plasticity & Long-Term Memory Formation Changes in Circadian Clock 41 Roles of cAMP in Different Tissues Tissue/Cell Type Agents ↑ [cAMP] Liver Glucagon & Epinephrine Skeletal Muscle Epinephrine Adipose Tissue Epinephrine Lipolysis Renal Tubular Epithelium Antidiuretic Hormone (ADH; Vasopressin) Water Reabsorption Intestinal Mucosa Vasoactive Intestinal Polypeptide (VIP), Adenosine, & Epinephrine Water & Electrolyte Secretion Vascular Smooth Muscle Epinephrine (‐ adrenoceptors) Relaxation (Vasodilation) Growth Inhibition Epinephrine (‐ adrenoceptors) Prostacyclin & Prostaglandin E Relaxation (Bronchodilation) Maintenance of Inactive State Adrenal Cortex ACTH Hormone Secretion Melanocytes Melanocyte‐Stimulating Hormone (MSH) Melanin Synthesis Bronchial Smooth Muscle Platelets Thyroid‐Stimulating Hormone (TSH) School of Osteopathic Medicine Thyroid Gland Effects of ↑ [cAMP] Glycogenolysis Gluconeogenesis Glycogenolysis Glycolysis Hormone Secretion Biological Importance of cAMP 42 Signal Amplification School of Osteopathic Medicine • Cascades • More steps = more amplification + regulation 43 Regulation of Adenylyl Cyclase-Stimulating GPCRs I. ↓ GPCR affinity for L (occurs upon Gs GTP binding) II. Gs GTP hydrolysis III. cAMP phosphodiesterase (cAMP → 5′-AMP) IV. Heterologous desensitization A. Activated PKA phosphorylates GPCR (feedback suppression) 1. Phosphorylated GPCR can still bind L, but cannot activate Gs V. Homologous desensitization A. Epi-bound -adrenoceptors phosphorylated by -adrenergic receptor kinase (BARK) 1. Phosphorylated residues are bound by -arrestin a. Blocks Gs interaction & targets receptor for endocytosis & lysosomal degradation School of Osteopathic Medicine 44 School of Osteopathic Medicine 45 Topic #6 GPCRs That Regulate Ion Channels School of Osteopathic Medicine 46 M2 mAChR I. Muscarinic (responsive to muscarine) vs. nicotinic (responsive to nicotine) AChRs II. M2 mAChR (GPCR expressed in heart, neurons, & smooth muscle) bound & activated by ACh III. Gi inhibits AC; activation of K+ channel by G causes long hyperpolarization of cardiac muscle cells, slowing rate of heart contraction School of Osteopathic Medicine 47 Clinical Application: mAChRs are Pharmacological Targets I. Atropine: antagonist of mAChRs A. Utilized to treat cardiorespiratory disorders, excessive salivation, organophosphate poisoning, & in ophthalmological procedures II. Scopolamine: antagonist of mAChRs A. Utilized to prevent & treat nausea/vomiting (particularly caused by motion sickness) III. Ipratropium: antagonist of mAChRs A. Utilized to treat respiratory conditions such as COPD & asthma School of Osteopathic Medicine 48 Rhodopsin I. GPCR expressed in flattened-membrane disks in outer segment of rod cells of retina (innermost, light-sensitive tissue layer of eye) A. It is a photoreceptor (no molecular ligand) that is stimulated by dim light (e.g., moonlight) 1. Rod cells mediate dim light vision (as opposed to cone cells which mediate color & bright light vision) B. Composed of opsin (GPCR) covalently bound to 11-cis-retinal (1 form of vitamin A) School of Osteopathic Medicine 49 School of Osteopathic Medicine 50 Rhodopsin Activation School of Osteopathic Medicine 51 PDE = cGMP Phosphodiesterase Gt = Transducin Rhodopsin Mechanism of Action School of Osteopathic Medicine 52 Rhodopsin Regulation GAP GAP School of Osteopathic Medicine 53 Rhodopsin Regulation & Light/Dark Adaptation School of Osteopathic Medicine 54 Clinical Application: Nyctalopia I. Also known as night blindness A. Inability to see well at night or in areas with poor lighting 1. Inability to quickly adapt from a well-illuminated to a poorly illuminated environment (i.e., poor dark adaptation) II. 1st detectable sign of vitamin A deficiency A. Lack of adequate dietary vitamin A intake results in lack of retinal to assist opsin in the retina 1. Treatment with vitamin A supplementation rapidly corrects the condition School of Osteopathic Medicine 55 Topic #7 GPCRs That Regulate Phospholipase C School of Osteopathic Medicine 56 Phosphatidylinositol-Based Signaling School of Osteopathic Medicine 57 IP3/DAG Pathway & 2+ Ca /PKC Beta (PLC) -Adrenoceptor Original [Cytosolic Ca2+] restored by Ca2+-ATPases (Pump Ca2+ out of cell & into ER lumen) Cell growth/metabolism Glycogen metabolism Transcription factors IP3 degraded to 1,4-bisphosphate & Pi School of Osteopathic Medicine 58 Ca2+ as a 2nd Messenger Striated (skeletal) muscle cells Acetylcholine School of Osteopathic Medicine Contraction 59 Integrated Control of Glycogen Metabolism School of Osteopathic Medicine 60 2+ Ca /Calmodulin I. Calmodulin A. Cytosolic protein that binds 4 Ca2+ with positive cooperativity II. Ca2+/calmodulin complex mediates activity of many factors A. E.g., activation of cAMP/cGMP PDEs B. E.g., activation of calcineurin 1. Phosphatase that dephosphorylates & activates a T-cell-specific transcription factor (NFAT) School of Osteopathic Medicine 61 Ca2+/Calmodulin & Myosin Light-Chain (MLC) Kinase Smooth Muscle Cells School of Osteopathic Medicine 62 Ca2+/Calmodulin & Gene Expression Expression of Target Genes → Altered Cellular Function → Changes in Neuronal Plasticity & Long-Term Memory Formation Changes in Circadian Clock (CaMK = Calmodulin Kinase) CRE School of Osteopathic Medicine 63 Ca2+/Calmodulin & Blood Vessel Diameter (EndothelialNitric Nitric Oxide Oxide Synthase) (Endothelial Synthase) (Nitric Oxide) (Soluble Guanylyl Cyclase) (Soluble Guanylyl Cyclase) Vascular Smooth Muscle Cells PDE-5 Inhibition of Actin/Myosin, School of Osteopathic Medicine , Vasodilation 64 Clinical Application: NO Pathway is Pharmacological Target I. Nitroglycerin & nitroprusside sodium: metabolized into NO A. Utilized in the treatment of cardiovascular conditions such as hypertensive crisis & angina II. Sildenafil (Viagra®) & tadalafil (Cialis®): antagonists of PDE-5 A. Utilized in the treatment of arterial pulmonary hypertension & erectile dysfunction School of Osteopathic Medicine 65 Topic #8 Non-GPCR Cell Surface Receptors that Control Gene Expression School of Osteopathic Medicine 66 Non-GPCR Cell Surface Receptor Signaling I. Primary role is to influence gene expression II. There are many ligands, many receptors, several signal transduction pathways, & multiple transcription factors involved III. Same receptor in different cells can activate different pathways resulting in different cellular responses School of Osteopathic Medicine 67 Receptor Tyrosine Kinase School of Osteopathic Medicine 68 RTK Ligands & Receptors I. Epidermal Growth Factor (EGF) & EGFR II. Nerve Growth Factor (NGF) & NGFR III. Platelet-Derived Growth Factor (PDGF) & PDGFR IV. Fibroblast Growth Factor (FGF) & FGFR V. Insulin & IR VI. Insulin-Like Growth Factor (IGF) & IGFR School of Osteopathic Medicine 69 RTK Docking/Adaptor Proteins School of Osteopathic Medicine 70 (Monomeric G Protein) Ras-MitogenActivated Protein Kinase (MAPK) Signaling (MAPKKK, MAP3K) (Also ASK, MEKK, MLK) (MAPKK, MAP2K) (Erk, JNK, p38) Expression of Target Genes → Altered Cellular Function → Changes in Cell Cycle Progression Changes in Cell Growth, Division, & Proliferation Changes in Cell Survival Changes in Inflammatory Response School of Osteopathic Medicine 71 Clinical Application: RTKs & Cancer I. Mutated RTKs can induce pro-cellular proliferation signals in absence of their ligands A. E.g., human epidermal growth factor receptor 2 (HER2) mutations are common in breast cancer 1. Trastuzumab: monoclonal antibody (mab) that binds & neutralizes HER2 a. Utilized to treat HER2-dependent cancers School of Osteopathic Medicine 72 Clinical Application: RTKs & Cancer (2) I. Overexpression of RTKs &/or ligands can lead to aberrant autocrine signaling via RasMAPK pathways A. Present in many types of cancer II. Ras mutations (block GTPase function) & Raf mutations keep these proteins in constitutively active signal transduction state A. Present in many types of cancer School of Osteopathic Medicine 73 RTK & PLC School of Osteopathic Medicine 74 Action of PI3K PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) School of Osteopathic Medicine 75 PI3K & Akt/PKB 5 P 5 P 5 P 5 P PIP3 School of Osteopathic Medicine 76 Akt/PKB Signaling GLUT4 mTORC1 School of Osteopathic Medicine 77 PI3K Signaling Regulation © Cayman Chemical Company School of Osteopathic Medicine 78 Clinical Application: Leprechaunism I. Also known as Donohue syndrome A. Caused by mutations in IR 1. Affected infants are born with severe metabolic derangement 2. Patients suffer with growth retardation, large malformed ears, absence of subcutaneous fat, & severe insulinresistant diabetes 3. Typically die in 1st years of life School of Osteopathic Medicine 79 Clinical Application: PI3K Pathway-Related Diseases I. Deletion of PTEN gene is feature of many cancers A. Results in unregulated PI3K-signaling II. Mutations in TSC2 (protein inhibitor of mTORC1) causes tuberous sclerosis A. Condition characterized by growth of benign tumors in brain, spinal cord, nerves, eyes, lung, heart, kidneys, & skin III. Sirolimus (rapamycin): antagonist of mTORC1 A. Utilized to prevent kidney transplant rejection School of Osteopathic Medicine 80 Tyrosine Kinase-Associated Receptor I. Cell surface receptor whose intracellular domains are associated with protein tyrosine kinase enzymes A. E.g., cytokine receptors (CR) 1. Bound & activated by cytokines (extracellular signaling molecules utilized by the immune system) a. E.g., erythropoietin (EPO), granulocytecolony stimulating factor (G-CSF), interferons (IFNs), & interleukins (ILs) 2. Associated with Janus kinase (JAK) enzymes School of Osteopathic Medicine 81 Cytokine Receptor Signaling 4. Ras-MAPK; PLC/Ca2+; PI3K; JAK/STAT School of Osteopathic Medicine 82 EpoR I. EPO is produced & secreted by renal cortex in response to ↓[O2]Blood II. Binds to EPO receptor (EpoR) on erythroid progenitor cells A. Activates the JAK2/STAT5 pathway 1. STAT5 ↑ expression of Bcl-XL a. Anti-apoptotic protein; its action stimulates proliferation & differentiation of erythroid progenitor cells into mature RBCs School of Osteopathic Medicine 83 School of Osteopathic Medicine 84 Western Blot Analysis of EpoR Signaling JAK/STAT Signaling PI3K Signaling (Erk) Ras/MAPK Signaling (Erk) School of Osteopathic Medicine 85 EpoR Signaling Regulation Short-Term Regulation Long-Term Regulation Ubiquitination & Proteasomal Degradation of JAK2 School of Osteopathic Medicine 86 Clinical Application: Polycythemia Vera I. Neoplastic condition characterized by abnormal increase in circulating RBC # for no obvious external cause A. Often caused by mutated JAK2 that is constitutively active in absence of EPO stimulation 1. Causes excessive proliferation & differentiation of erythroid progenitor cells School of Osteopathic Medicine 87 Clinical Application: Cytokines in Pharmacology I. Epoetin alfa: recombinant form of EPO utilized in the therapy of amenia arising from kidney disease, cancer chemotherapy, & zidovudine therapy II. Filgrastim: recombinant G-CSF utilized in the therapy of neutropenia caused by cancer chemotherapy, radiation exposure, & bone marrow transplantation preparation School of Osteopathic Medicine 88 RTK & CR Pathways Review School of Osteopathic Medicine 89 NF-B I. Heterodimer (p50 & p65 subunits) that functions as transcription factor & master regulator of immune system A. Held in inactive state in cytosol by I-B B. Activation is triggered by virus infection, ionizing radiation, cytokines (TNF- & IL1), & presence of bacteria & fungi School of Osteopathic Medicine 90    NF-B Pathway      School of Osteopathic Medicine 91 Receptor Protein Tyrosine Phosphatase (RPTP) I. Asprosin A. Protein hormone produced & secreted from white adipose tissue B. Binds & activates receptor-type tyrosine-protein phosphatase delta (PTPRD) in brain AgRP neurons 1. Possesses intrinsic tyrosine phosphatase activity 2. Activated receptor dephosphorylates key proteins (e.g., STAT3; causes its deactivation), leading to ↑ AgRP neuron firing a. This ↑ appetite School of Osteopathic Medicine 92 School of Osteopathic Medicine 93 Topic #9 Other Non-GPCR Cell Surface Receptors School of Osteopathic Medicine 94 Receptor Guanylyl Cyclase I. Atrial natriuretic peptide (ANP) A. Peptide hormone produced & secreted from cardiac atrial cells B. Binds & activates natriuretic peptide receptor A (NPR1) in vascular smooth muscle cells, kidneys, & adrenal glands 1. Possesses intrinsic guanylyl cyclase activity 2. Activated receptor produces cGMP which binds & activates protein kinase G (PKG) a. This ultimately leads to vasodilation, ↑ renal Na+ excretion, & inhibition of renin/aldosterone secretion (all these cause drop in arterial blood pressure) School of Osteopathic Medicine 95 ANP NPR1 School of Osteopathic Medicine 96 Death Receptor I. Fas (CD95, APO-1) A. Ubiquitously expressed cell surface receptor particularly abundant in thymus, liver, & kidneys B. Bound & activated by Fas ligand (FasL) 1. Homotrimeric (3 copies of same molecule) transmembrane protein 2. Interaction with FasL (in membrane of signaling cell) induces homotrimerization of Fas (in membrane of target cell) a. This is example of juxtacrine signaling School of Osteopathic Medicine 97 Death Receptor (2) I. Each Fas molecule possesses an intracellular death domain A. Trimerization brings 3 death domains together, stimulating their activity 1. Recruit, bind, & activate FADD (adaptor protein) a. FADD recruits & binds procaspase-8 (inactive protease), inducing its conversion to caspase-8 (active protease) i. Caspase-8 directly activates caspase-3 (protease that cleaves key cellular proteins) or indirectly activates it through the tBID-Mitochondrion-Cytochrome cCaspase-9 pathway a) The activity of caspase-3 leads to apoptosis (programmed cell death) of target cell School of Osteopathic Medicine 98 Fas/FasL Pathway School of Osteopathic Medicine 99 Clinical Application: Autoimmunity I. Inappropriate immune response (antibodies from B cells & cytotoxic activity from T cells) against normal healthy cells, tissues, & organs of body (autoantigens) A. Can lead to large variety of autoimmune disorders with wide range of severity II. Fas/FasL pathway has roles in both preventing & promoting autoimmunity School of Osteopathic Medicine 100 Clinical Application: Autoimmunity (2) I. Preventing autoimmunity A. Antigen presenting cells (APCs) expressing FasL can induce apoptosis of autoreactive T cells expressing Fas B. Activated T cells expressing FasL can induce apoptosis of autoreactive B cells expressing Fas & dendritic cells (type of APC; can stimulate autoimmune response) expressing Fas II. Promoting autoimmunity A. APCs expressing FasL can induce differentiation of naïve T cells expressing Fas into Th17 cells (produce IL-17, pro-inflammatory cytokine implicated in autoimmunity) School of Osteopathic Medicine 101 Fas/FasL Pathway Regulation School of Osteopathic Medicine 102 Ligand-Gated Ion Channel: nAChR I. Nicotinic acetylcholine receptor A. Cell surface protein complex that forms channel for Na+ (moves into cell) & K+ (moves out of cell) through plasma membrane B. In absence of ACh, channel is closed (no ion movement through membrane) 1. Upon ACh binding, channel opens, allowing ion movement through membrane School of Osteopathic Medicine 103 nAChR Types I. Nicotinic neuronal (NN) A. Found in neurons & adrenal glands 1. When activated, it stimulates action potential generation in neurons II. Nicotinic muscular (NM) A. Found in skeletal neuromuscular endplates (interface between skeletal muscle cells & innervating motoneuron axon terminals) 1. When activated, it stimulates action potential generation in skeletal muscle cells, leading to skeletal muscle contraction School of Osteopathic Medicine 104 School of Osteopathic Medicine 105 AChR Regulation I. Controlled by degradation of ACh in synaptic cleft (space between ACh-releasing neuron axon terminal & target cell, e.g., skeletal muscle cell or next neuron) A. Occurs through the action of acetylcholinesterase (AChE) School of Osteopathic Medicine 106 NM AChR School of Osteopathic Medicine 107 Clinical Application: Myasthenia Gravis I. Rare autoimmune disorder primarily caused by autoantibodies against NM AChRs A. The hallmark of disorder is muscle weakness that worsens after activity & improves after rest 1. Patients have issues with eye/eyelid movement, facial expressions, chewing/swallowing, talking, arm/hand/finger/leg/neck weakness, & shortness of breath a. In severe cases, respiratory muscle weakness necessitates ventilatory assistance School of Osteopathic Medicine 108 Clinical Application: NN AChR is Pharmacological Target I. Nicotine: agonist at almost all NN AChRs, particularly in the central nervous system (CNS) A. It exerts stimulant or sedative effects based on dosage B. It stimulates release of dopamine & endogenous opioids which activate the reward system (neural structures involved in desire or craving for reward, motivation, pleasure) 1. Accounts for addictive qualities of nicotine School of Osteopathic Medicine 109 Clinical Application: NM AChR is Pharmacological Target I. Curare: plant extract poison used on arrow tips by many Central & South American indigenous peoples A. Main toxin (d-tubocurarine) functions as competitive antagonist of NM AChRs in neuromuscular junction B. Causes paralysis (including of diaphragm) II. Vecuronium: nondepolarizing neuromuscular blocker that functions as competitive antagonist of NM AChRs in neuromuscular junction A. Utilized as muscle relaxer during general anesthesia for endotracheal intubation & mechanical ventilation School of Osteopathic Medicine 110 Clinical Application: AChE is Pharmacological Target I. Carbamates: reversible AChE inhibitors A. Neostigmine: utilized in treatment of myasthenia gravis & urinary retention B. Rivastigmine: utilized in treatment of Alzheimer’s- & Parkinson’s-induced dementia School of Osteopathic Medicine 111 Clinical Application: AChE is Pharmacological Target (2) I. Organophosphate A. Organic molecule containing central phosphate ester bonds B. Used as insecticide (e.g., parathion, malathion), flame retardants, & nerve agents (e.g., Sarin, VX) C. Form a covalent bond with Ser residue of AChE 1. They act as irreversible inhibitors 2. Prevent breakdown of ACh in synaptic clefts, leading to excessive ACh-mediated stimulation of nAChRs & mAChRs throughout body a. Treated with atropine (antimuscarinic actions) & pralidoxime (AChE regenerator; removes organophosphates from AChE) School of Osteopathic Medicine 112 Ligand-Gated Ion Channel: GABAA Receptor I. Cell surface protein complex that forms channel for Cl- (into cell) through plasma membrane of CNS neurons A. In absence of -aminobutyric acid (GABA), channel is closed (no Clmovement through membrane) 1. Upon 2 GABA molecules binding, channel opens, allowing Clmovement through membrane a. Activation of receptor inhibits neuron excitability by preventing action potential generation School of Osteopathic Medicine 113 Clinical Application: GABAA Receptor is Pharmacological Target I. Barbiturates (e.g., phenobarbital): allosteric activators (i.e., enhance GABA binding) of GABAA receptor A. Utilized to treat seizures, insomnia, & anxiety II. Benzodiazepines (e.g., diazepam): allosteric activators of GABAA receptor A. Utilized to treat anxiety, panic disorder, alcohol withdrawal, muscle spasm, seizures, eclampsia, night terrors, & sleepwalking III. Ethanol: allosteric activator of GABAA receptor A. Responsible for its sedative effect IV. Flumazenil: allosteric inhibitor (i.e., inhibits GABA binding) of GABAA receptor A. Utilized to reverse general anesthesia & treat benzodiazepine overdose V. Propofol: allosteric activator of GABAA receptor A. Utilized to induce general anesthesia School of Osteopathic Medicine 114 Bone Remodeling I. Dynamic process occurring in bone tissue involving constant disassembly (bone resorption) & assembly (bone formation) A. Resorption mediated by osteoclasts (produce acid-enzyme mixture that dissolves bone) B. Bone formation mediated by osteoblasts (produce/secrete type I collagen) School of Osteopathic Medicine 115 Juxtacrine Signaling in Bone Remodeling I. Juxtacrine signaling is utilized to control bone remodeling A. Osteoblasts express RANKL (trimeric protein) in their plasma membrane; osteoclasts express RANK in their plasma membrane B. RANKL binding to RANK stimulates multiple signal transduction pathways in osteoclasts 1. Promotes their binding to bone & bone resorptive activity School of Osteopathic Medicine 116 Decoy Receptors in Bone Remodeling I. Osteoblasts produce & secrete osteoprotegerin (OPG) A. Acts as decoy receptor (possesses signaling molecule-binding domain but no signal transducing-activity) for RANKL 1. Upon binding to OPG, RANKL is incapable of binding RANK a. This inhibits osteoclast activation & bone resorption School of Osteopathic Medicine 117 School of Osteopathic Medicine 118 Clinical Application: Osteoporosis I. Most common metabolic bone disease in which low bone mass & structural deterioration lead to increased bone fragility A. Often results from disproportionate bone resorption II. 1 contributing factor is estrogen (female sex hormone produced by ovaries) deficiency A. Estrogen induces osteoblast secretion of OPG 1. Low estrogen levels in postmenopausal women lead to decreased OPG levels, promoting excessive osteoclast activity School of Osteopathic Medicine 119 Topic #10 Cellular Signaling Via Intracellular Receptors School of Osteopathic Medicine 120 Cytosolic Receptors: GR I. Glucocorticoid receptor A. Cytosolic protein that is bound & activated by glucocorticoids (GCs) 1. Steroid-hormones produced in adrenal gland a. Prototypical GC is cortisol B. Expressed in almost every body cell 1. Allows GCs to exert tremendous influence on body biochemistry & physiology C.Function as transcription factors that directly & indirectly regulate 10 – 20% of all genes School of Osteopathic Medicine 121 School of Osteopathic Medicine Changes in Energy Metabolism Changes in Carbohydrate, Lipid, & Protein Metabolism Changes in Inflammatory Response 122 Changes in Immune Response Clinical Application: GCs are Pharmacological Agents I. GCs are utilized as powerful therapeutic agents for treatment of numerous disease processes A. Particularly for disturbed adrenal function, inflammatory disorders, & immune disorders B. Because of widespread & powerful effects, they can induce serious adverse effects, particularly with long-term use School of Osteopathic Medicine 123 Nuclear Receptors: TR I. Thyroid hormone receptor A. Nuclear DNA-bound protein that is bound & activated by T3 (triiodothyronine) 1. Iodine-containing tyrosine-based hormone produced directly by thyroid gland & by metabolism of T4 (thyroxine; another thyroid hormone) in target cells B. Expressed in most body cells 1. Allows T3 to exert tremendous influence on body biochemistry & physiology C. Functions as transcription factor School of Osteopathic Medicine 124 School of Osteopathic Medicine 125 Clinical Application: Iodine Deficiency I. Dietary deficiency of iodine leads to development of simple goiter A. Lack of iodine prevents synthesis of T3 & T4 1. Pituitary gland produces excessive amounts of TSH to attempt to stimulate thyroid gland to uptake iodine (which is absent) & produce thyroid hormones (which it cannot do without iodine) a. Constant TSH stimulation causes enlargement of thyroid gland leading to a visible lump in neck (goiter) School of Osteopathic Medicine 126 Clinical Application: Levothyroxine I. Synthetic T4 utilized as pharmacological agent A. Used in therapy of hypothyroidism & myxedema coma (extreme, lifethreatening hypothyroidism), & to preserve organ function in brain-dead organ donors School of Osteopathic Medicine 127