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Signaling Overview3. Three major classes of surface receptors for signaling, cont.:C. Enzyme-linked receptors:1. Tyrosine kinase-linked receptors (TKRs).A. Overview of TKRs:1. Cell surface receptors that are directly linked to intracellular enzymes (kinases).2. Includes receptors for most growth factors (NGF, EGF. PDGF), insulin, and Src.3. Common structure: N terminal extracellular ligand-binding domain, single TM domain, cytosolic Cterminal domain with tyrosine kinase activity.4. Can be single polypeptide or dimer.Examples of tyrosine kinase-linked receptors (TKRs):66

Signaling Overview3. Three major classes of surface receptors for signaling, cont.:C. Enzyme-linked receptors, cont.:Pabio 552, J. R. Lingappa4/27/061. Tyrosine kinase-linked receptors (TKRs)B. Mechanism of activation of TKRs:i. ligand binding induces receptor dimerization (receptor crosslinking).ii. dimerization leads to autophosphorylation of the receptor (cross-phosphorylation).iii. phosphorylation increases kinase activity & also creates specific new binding sites.iv. proteins that bind to these new binding sites transmit intracellular signals.67

Pabio 552, J. R. Lingappa4/27/06How receptor tyrosine kinases work together with monomeric GTPases:68

Signaling Overview3. Three major classes of surface receptors for signaling, cont.:C. Enzyme-linked receptors, cont.:1. Tyrosine kinase-linked receptors (TKRs)B. Mechanism of activation of TKRs:4/27/06v. example: Src homology 2 (SH2) domains on other proteins bind to phosphotyrosinecontaining regions of TKRs resulting in:a. localization of SH2-containing proteins at plasma membrane.b. association of SH2-containing proteins with other proteins.c. phosphorylation of SH2-containing proteins.d. activation of enzymatic activity of SH2-containing proteins.69From Janeway, Immunobiology, 5th edition

Signaling Overview3. Three major classes of surface receptors for signaling, cont.:C. Enzyme-linked receptors, cont.:1. Tyrosine kinase-linked receptors (TKRs)C. Different ways that TKRs can be activated:i. Ligand dimerizationii. Monomeric ligand binds to a crosslinking proteiniii. Clustered monomeric cell-surface ligand70

Signaling Overview3. Three major classes of surface receptorsfor signaling, cont.:C. Enzyme-linked receptors, cont.:A. Overview1. N-term. extracell. ligand-binding domain, transmembα helix,C-term. cytosolic domain2. Cytosolic domain has no catalytic (kinase) activity3. Acts in conjuction with a non-receptor tyrosine kinasethat is activated as a result of ligand binding.4. Activation is similar to that of RTKs: ligand bindingcauses cross phosphorylation of associatedtyrosine kinases that phosphorylate the receptor,providing phosphotyrosine binding sites forrecruitment of proteins with SH2 domains.Pabio 552, J. R. LingappaCytokine receptors, as well as T and B cellreceptors, stimulate tyrosine kinases that are noncovalently associated with receptor.4/27/062. TKs non-covalently associated withreceptor (includes cytokine receptors, T & Bcell receptors) NRTKsFrom Janeway, Immunobiology, 5th edition 71

Signaling Overview3. Three major classes of surface receptors for signaling, cont.:C. Enzyme-linked receptors, cont.:C. Receptors can be linked to or associatedwith other enzymes, besides TKs, i.e.Protein-tyrosine phosphatases (removephosphates, thereby terminate signals initiatedby protein-tyrosine kinases).Serine/ threonine kinases, i.e. TGF-βGuanylyl cyclasesPabio 552, J. R. Lingappa1. Src family protein kinases - important for Band T cell signaling2. Janus kinases (JAK) - universally requiredfor signaling from cytokine receptors.4/27/06B. Two kinds of kinases associate with NRTKs:From Janeway, Immunobiology, 5th edition 72From Janeway, Immunobiology, 5th edition

Enzyme-linkedReceptor (theLeptin receptor)JAK JanusKinaseSTAT SignalTransducerand Activatorof Transcription

University of Jordan The receptor exists as a homodimer (two identical parts) Leptin binds to the extracellular part of the receptor This causes activation of the intracellular associated januskinas 2 This causes phosphorylation of signal transducer and activatorof transcription (STAT) proteins This then activates the transcription of target genes andsynthesis of proteins JAK 2 phosphorylation also activates several other enzymesystems that mediate some of the more rapid effects of leptin74Enzyme-linked receptor(the leptin receptor)

University of Jordan75Tyrosine Kinase

University of Jordan76Tyrosine Kinase Receptors:

Tyrosine Kinase Insulin receptor consists of 2 units that dimerizewhen they bind with insulin. Autophosphorylation occurs, increasing tyrosinekinase activity. Activates signaling molecules.University of Jordan Insulin binds to ligand–binding site on plasma membrane,activating enzymatic site in the cytoplasm. Stimulate glycogen, fat and protein synthesis. Stimulate insertion of GLUT-4 carrier proteins.77

University of JordanTyrosine Kinase78

University of Jordan79The Insulin Receptor & Mechanismsof Insulin Action

University of Jordan80

University of Jordan81Insulin Action on Cells:

Signaling Overview4. Second Messengers: for Hormones thatcan’t cross PMcAMP production:Pabio 552, J. R. Lingappai. Production:ATP converted to cAMP by adenylate cyclase (a largemultipass TM protein)Degraded by cAMP phosphodiesteraseii. Action:a. cAMP-dependent protein kinase (protein kinase A(PKA)).PKA is a tetramer of catalytic and regulatory subunitscAMP binding leads to dissociation of regulatorysubunits and release of catalytic subunits which thenphosphorylate target proteins in cytoplasm:4/27/06A. cAMP:From Janeway, Immunobiology, 5th edition 82

Signaling Overview4. Second Messengers, cont.:A. cAMP, cont.iii. Action:b. PKA enters the nucleus and phosphorylates CREB (CRE binding protein), which binds tothe cAMP response element (CRE), a regulatory DNA sequence associated with specificgenes. This results in activation of transcription of those genes.iv. Rapid turn on and rapid turn off of cAMP and activation by cAMP :Question: what turns off proteins activated by protein kinases?v. Amplification of signal at each step of signaling pathway - characteristic feature of signaltransduction.83

Signaling Overview4. Second Messengers, cont.:A. cAMP, cont.:Summary of how cAMP activates transcription:vi. Regulation of adenylate cyclase:Receptors that cause increase in cAMP do soby activating Gs, a stimulatory proteinthat activates adenylyl cylase.Adenylyl cyclase is turned off by Gi, aninhibitory protein.vii. Pathogens alter cAMP production:Cholera toxin active subunit catalyzestransfer of ADP ribose from intracellularNAD to the asubunit of Gs, causing it tobe continuously active, stimulatingadenylyl cyclase indefinitely. Thiscauses ion channels that exportchloride to produce a net effux of Cland water, leading to severe diarrheacharacteristic of cholera.B. cGMP:1. produced from GTP by guanylyl cyclase;2. activates cGMP-dependent kinases orother targets3. example: G-prot. Coupled rhodopsinphotoreceptor in rod cells of retina84

Phosphorylatesenzymes within thecell to producehormone’s effects. Modulates activity ofenzymes present inthe cell. Alters metabolism ofthe cell. cAMP inactivated byphosphodiesterase. Hydrolyzes cAMPto inactivefragments.University of JordanAdenylate Cyclase-cAMP85

86University of Jordan

University of Jordan87G-Protein-coupled Receptors

Signaling OverviewPabio 552, J. R. Lingappa1. Overview: Phosphotidylinositol 4,5bisphosphate (PIP2) triggers a 2armed signaling pathwaya. PIP2 is a minor PL in inner leaflet ofPM bilayer that is produced byphosphorylation of phosphatidylinositol and is involved in signalingb. Ligand binding to certain receptorsstimulates PIP2 hydrolysis byphospholipase C (PLC)c. This produces diacylglycerol (DAG)and inositol 1,4,5-phosphate (IP3),both of which are 2nd messengersd. PIP2 hydrolysis is activated by bothGPRs and TKRs via different formsof PLCe. PLC-β is stimulated by Gq proteinswhile PLC-γ has SH2 domains thatallow binding to activated tyrosinekinases4/27/064. Second Messengers, cont.:C.IP3 and DAG:88

University of Jordan89PhospholipaseC-Ca2

Signaling Overview4. Second Messengers, cont.:C. DAG and IP3, cont.:2. DAG: Remains associated with the PMa. Stimulates the Ca 2-dependent protein kinase C signaling pathway, which activates othertargets including the MAP kinase cascade (see below)b. Can also be cleaved to form another messenger, eicosanoids, which include prostaglandinsc. Tumor producing phorbol esters mimic DAG and thereby stimulate protein kinase C3. IP3: Small polar molecule released into cytosola. Stimulates Ca 2 release from intracellular stores. Question: where are these?b. Elevated Ca 2 alters activities of target proteins including kinases & phosphatases90

PLC- signaling pathwayPKCPhosphorylatesmany substrates,can activatekinase pathway,gene regulation

Signaling Overview4. Second Messengers, cont.:D. Ca 2 also acts as a second messengerCa 2 concentration kept low (10-7 M), rising locally due to transient signalingEffects of intracellular Ca 2 are mediated by the Ca 2 binding protein calmodulin.Ca 2 /calmodulin binds to target proteins, including protein kinases (Ca 2calmodulin-dependentkinases; CaM-kinases), adenylyl cyclases, and phosphodiesterases, causing change inconformation and activation of these proteins.92

University of Jordan93Epinephrine Can Act Through Two 2ndMessenger Systems

Ca2 diffuses into thecytoplasm. Ca2 binds tocalmodulin. Calmodulin activatesspecific protein kinaseenzymes. Alters themetabolism of thecell, producing thehormone’s effects.University of Jordan2 Ca - Calmodulin (continued)94

95Calmodulin (continued)University of Jordan2 Ca -

Guanylate cyclase (GC) receptorMembrane receptor –ANPSoluble receptor – NO, CO

Pabio 552, J. R. Lingappa4/27/06NO signaling97

Signaling Overview4. Second Messengers, cont.:E. PIP3:PIP2 phosphorylated by PI 3-kinase, resulting in PIP3, which is also a 2nd messenger.PI 3-kinase can be activated by GPRs or TKRs.One target of PIP3 is a protein-serine/threonine kinase called Akt, or protein kinase B,which becomes activated by a kinase called PDK1.PIP3 binds to Akt at the pleckstrin homology domain.Activation of Akt leads to regulation of target molecules, including BAD, which is pro-apoptotic andbecomes inactivated by phosphorylation.98

Signaling Overview5. Signaling Cascades, cont.:5 downstream kinases activated by different signaling cascades99

100Pabio 552, J. R. Lingappa4/27/06

University of Jordan101§ Receptors superfamilies:§Ionotropic receptors (ligand-gated channels)§Metabotropic receptors (G protein-coupled receptors)§Tyrosine KinaseAlmost all neurotransmitters discovered so far have morethan one kind of receptor -- called receptor subtypes.

Transmitter synthesized and stored2.Action Potential3.Depolarization: open voltage-gated Ca2 channels4.Ca2 enter cell5.Ca2 causes vesicles to fuse withmembrane6.Neurotransmitter released (exocytosis)7.Neurotransmitter binds to postsynapticreceptors8.Opening or closing of postsynapticchannels9.Postsynaptic current excites or inhibitspostsynaptic potential to changeexcitability of cell10.Retrieval of vesicles from plasmamembrane (endocytosis)University of Jordan1.102§ Neurotransmitter Release: exocytosis and endocytosis

Reuptake bytransporters(glial cells)103Transmitter Inactivation:reuptake and enzymatic breakdownEnzymaticbreakdownNeurotransmitter can be recycled in presynaptic terminalor can be broken down by enzymes within the cellUniversity of Jordan§

NT – Receptor BindingReceptors are large, dynamic proteins thatexist along and within the cell membrane.Ionotropic receptors: NT binding results in direct opening orclosure of specific ion channelsMetabotropic receptors: binding of NT initiates a sequence ofinternal molecular events which in turn open specific ionchannelsUniversity of JordanTwo Types of Post synaptic Receptors:104Dynamic – they can increase in number and avidity for theirneurotransmitter according to circumstances.