(2) G Protein-Coupled Receptors
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Chapter 15 Cellular Signal Transduction The biochemistry and molecular biology department of CMU When environment changes: Monad——responds directly. Multicellular organisms——signal through elaborate system of intercellular or intracellular communication，and consequently regulate functions of organisms. Signaling molecule Receptor of target cell Signal transduction Intracellular molecule biological effect §1 Signaling Molecules Signaling molecules • Signaling molecules, which are released by signal-producing cells, reach and transfer biological signals to their target cells to initiate specific cellular responses. • Extracellular molecules • Intracellular molecules 1. Extracellular molecules protein & peptides: Hormone, cytokine AA & its derivatives: Gly, Glu, adrenaline, thyroxine Steroid: Sex Hormone, glucocorticosteroid Fatty acid derivatives: prostaglandin (1) Paracrine signaling (local chemical mediators) • Secreted by common cells. • Reach neighboring target cells by passive diffusion. • Time of action is short. • Such as GF, PG (2) Endocrine signal • • • • Secreted by endocrine cells. Reach target cells by blood circulation. Time of action is long. Such as insulin, thyroxine, adrenalin (3) Synaptic signal (neurotransmitters) • Secreted by neuronal cells. • Reach another neuron by synaptic gap. • Time of action is short. • Such as Acetylcholine (Ach), noradrenaline (4) Gaseous signal • Simple structure, half life is short and active in chemistry . • Such as NO, CO. GAS MOLECULE (5) Autocrine signal • Act back to their own cells. • Such as GF, cytokine, interferon, interleukin. 2. Intracellular molecule • Ca2+ ions • DG, ceramide • IP3 lipid derivatives carbohydrate derivatives • cAMP cGMP nucleotides • Ras, JAK, Raf proteins Second messenger: Small molecules synthesized in cells in response to an external signal are the second messengers, which are responsible for intracellular signal transduction. Such as Ca2+, DG, Cer, IP3, cAMP, cGMP Third messengers: Third messengers are the molecules which transmit message from outside to inside of nucleous or from inside to outside of nucleous, also called DNA binding protein. Proteins and peptides: Hormones, cytokines Effect by membrane Amino acid derivatives: receptors Catecholamines Fatty acid derivatives: Extracellular molecules Prostaglandins Effect by intracellular receptors Signal molecules Intracellular molecules Steroid hormones, Thyroxine, VD3 cAMP, cGMP, IP3, DG, Ca2+ §2 Receptor Receptor Receptors are specific membrane proteins, which are able to recognize and bind to corresponding ligand molecules, become activated, and transduce signal to next signaling molecules. Glycoprotein or Lipoprotein ligand A small molecule that binds specifically to a larger one; for example, a hormone is the ligand for its specific protein receptor. • Membrane receptors membrane Glycoprotein • Intracellular receptors Cytosol or nuclei DNA binding protein 1. membrane receptors (1) Ligand-gate ion channels type (cyclic receptor) ligand→receptor→ion channel open or close (2) G Protein-Coupled Receptors (serpentine R) 1) 7-helices transmembrane receptor Oligosaccharide unit Cytosolic side 2) G protein (Guanylate binding protein) • G protein refers to any protein which binds to GDP or GTP and act as signal transduction. • G proteins consist of three different subunits (, , -subunit). • -subunit carries GTPase activity, binding and hydrolysis of GTP. 3) Classes of G protein Gs→ s→AC→cAMP↑ Gi→ i→AC→cAMP↓ Gq→ q →PI-PLC→IP3+DAG Go→ o→ion channel Gt→ t →cGMP PDE→cGMP→ Rhodopsin Glucagon -adrenaline →s →AC↑ ACTH -adrenaline angiotensin Ⅱ acetylcholine(M2 M4) GF release inhibitory factor →i→AC↓ Cholera toxin Ribosylation of Arg of G G s -ATP ATPase Gs -ADP AC cAMP Cl- + H2O HCO3- Cavity of intestine diarrhea Pertussis toxin i -ADP-ribosylation Gi AC↑ cAMP↑ allergy of histamine 4) Effect proteins of G protein AC cGMP PDE (phosphodiesterase） PLC PLA2 Channel protein cAMP ATP 5) Pathway of G protein linked receptor H R G protein Es secondary messeger Protein kinase Phophorylation of Es or functional protein Biological effect (3) Single transmembrane α-helix receptor • Tyrosine protein kinase Receptor (catalytic receptor) IGF－R, EGF－R • Non tyrosine protein kinase Receptor Growth Hormone R, interferon R Tyrosine protein kinase Receptor or receptor tyrosine kinase (RTK) Cys-rich domain Immunoglobulin -like domain EGFR IGF-1R PDGFR FGFR Insulin Cytosol Intracellular insulin effects Non-receptor tyrosine kinase (NRTK) Domain of Downstream molecules of TPKR SH2 domain (Scr homology 2 domain) SH3 domain PH domain (pleckstrin homology domain) (4) Guanylate cyclase (GC) receptor Membrane receptor –ANP Soluble receptor – NO, CO 2. Intracellular receptor (transcription regulated receptor) Intracellular R is trans-acting elememt cis-acting element gene expression Localized in the cytosol and/or in the nucleus. ligand: Steroid H, VD3, Thyroxine 3. Properties of binding of H and R • highly specificity • highly affinity • saturation • reversible binding • special function model 4. Control of receptor activity • Phosphorylation or dephosphorylation of R • Phospholipid of membrane • Enzyme catalyzed hydrolysis • G protein regulation 5. Function of receptor (1) Recognize the special ligand (2) Binding to special ligand (3) Signal transduction biological effect §3 Pathway of Signal Transduction Signal transduction mediated by membrane receptor • cAMP dependent-protein kinase A pathway • cGMP dependent PKG pathway • Ca2+ dependent PK pathway • Tyrosine protein Kinase pathway • NF-κB pathway • TGF- βpathway 1. cAMP dependent-protein kinase A pathway H R G protein AC cAMP PKA Phosphorylation of Es or functional proteins Biological effects (1) cAMP metabolism ATP AC 2+ PDE cAMP 2+ Mg Mg PPi H2O PDE： Phosphodiesterase AC： Adenylate cyclase 5'-AMP NH2 N N O O P O O O P O O P O CH2 O ATP H H O O N N H H OH OH AC PPi NH2 NH2 N N N N O cAMP H H O P O O H H 2O H O O PDE CH2 O OH N N P N N O 5'-AMP CH2 O H O H H H OH OH (2) Mechanism of cAMP effect Activate cAMP-dependent protein kinase (PKA). (3) PKA effect Phosphorylate specifically Ser/Thr residues in several proteins （1）Regulation of metabolism （2）Regulation of gene expression hormons: glucagon, epinephrine inactive AC active AC ATP cAMP ATP inactive PKA phosphorylase b kinase Pi active PKA P ADP ATP phosphorylase b kinase ADP ATP ADP P glycogen synthase (active) P glycogen synthase (inactive) phosphorylase b phosphorylase a Pi Pi inhibitor-1 (inactive) H2O ATP H2 O protein phosphatase-1 inhibitor-1 (active) P H2O CRE : cAMP response element (TGACGTCA) CREB: CRE binding protein CRE DNA Transcription mRNA CREB PKA Ｐ CREB PKA Ｐ 2. Ca2+ dependent PK pathway (1) Ca2+ -DAG -dependent PKC pathway H R PIP2 PLC G protein IP3 ER DG Ca2+ PS PKC Phosphorylation of Es or functional proteins Biological effects [Ca 2+]i 0.01-1 mol/L（10-7 mol/L ） [Ca 2+]o 2.5mmol/L（10-3 mol/L ） 5000~10000× 1） Function of DG and IP3 IP3 + R→open of Ca2＋ channel →[Ca2＋]↑ DG PS, Ca2＋ PKC ↑ 2）Function of PKC • regulation of metabolism PKC →Ser/Thr-P of R, enzyme, Protein of Mb. • Gene expression Early response: PKC Trans-acting factor-P Immediate early genes Third messenger Late response: PKC Third messenger-P Activate genes Cell proliferation (2) Ca2＋-CaM dependent protein kinase pathway H R G protein PLC IP3 Ca2+ CaM CaMK Phosphorylation of Es or functional proteins Biological effects Calmodulin (CaM）： Ca2＋ binding protein 4 Ca2＋+ CaM → Ca2＋- CaM ↓ CaM kinase↑ ↓ Ser/Thr - P ↓ Ca2＋ pump, AC ↑ GC ↑ Es (glycogen synthase, phosphorylase kinase) 3. cGMP-dependent PKG pathway ANP NO, CO Receptorlinked GC cGMP Soluble GC PKG Phosphorylation of Es or functional proteins Biological effects (1) cGMP GTP GC PDE cGMP 2+ Mg PPi 2+ 5'-GMP Mg H2O GC: Guanylate cyclase (2) Function of PKG Ser/Thr- P of protein and E ANP（atrial natriuretic peptides） ↓ GC NO ↓ cGMP ↓ PKG ↓ Vascular dilatation peptide hormones neurotransmitters AFP G AC GC G PL GTP ATP cAMP neurotransmitters hypothalamic pituitrin Ca2+ PKA PIP2(PC) DG IP3 cGMP cell memberane Ca2+-CaM PKG PKC CaM-PK phosphorylation of enzymes or proteins trans-acting factor cis-acting element nucleus expression P effects 4. Tyrosine-protein kinase pathway (TPK) • TPK receptor is related to proliferation, differentiation, dissociation, carcinomatous change. • TPK： receptor TPK：Mb. non receptor TPK：cytosol (1) Receptor TPK－Ras－MAPK pathway • GRB2, SOS, Ras, Raf • Small G protein: Ras • MAPK (mitogen-activated protein kinase)： MAPK、MAPKK、MAPKKK EGF, PDGF receptor TPK ÊÜÌå ÐÍTPK GRB2－P SOS－P Ras－GTP Raf－P nucleus trans-acting factor －P expression MAPKK－P MAPK－P ϸ °û Ĥ (2) JAKs－STAT pathway ligand non TPK receptor JAKs STAT gene expression • JAKs • STAT: Signal transductors and activator of transcription interferons Plasma membrane ÖÊĤ JAK P£- £- P P 84 113 P 91 STAT complex 113 91 84 P 48 inactive STAT Nuclear membrane ºËĤ P 113 P 84 91 P 48 gene »ùÒò Interferons response ¸ÉÈÅËØ Ó¦´ð Ôª¼þ element transcription Interferon Dimerization of Receptor Autophosphorylation of JAK Phosphorylation of STAT Nuclear translocation of STAT Interferon response element Expression of gene 5. Nuclear factor-κB pathway 6. TGF-βpathway Intracellular receptor (DNA transcription regulated receptor) • Steroid H, VD3, Thyroxine • Cytosolic R: glycocorticosteroid H • Nuclear R: thyroxine, estrogen, androgen, progesterone Serum binding protein With bound bormone
