Effects of Second Messengers

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1 Effects of Second Messengers Inositol trisphosphate Diacylglycerol Opens Calcium Channels Binding to IP 3 -gated Channel Cooperative binding Activates Protein Kinase C is required Phosphorylation of many target proteins 1

2 The domain structures of protein kinase C isoforms Binds Diacylglycerol Interaction with phospholipids Pseudosubstrate Sequence Resemble the substrate sequence Contains A-R-K-G-A-L-R-Q-K Substrate Sequence X-R-X-X-(S,T)-Hyd-R-X Binds to the Enzyme s Active Site. 2

3 Termination of IP3 Signal IP3 is a Short-Lived Messenger Lithium Ions, Used to treat some psychological disorders Inhibits IP 3 recycling 3

4 Why? A large difference in concentration 0.1μM mm Why? Ability to bind protein tightly 4

5 Useful Tools in Studying the role of Calcium Ionophores Introducing Calcium into the cell Calcium Chelators Decreasing Calcium Concentration Fluorescent Chelators Measuring Calcium Concentration Calcium Binding Proteins Mediate the effects of Calcium Many proteins Calmodulin, Troponin C, Parvalbumin Similar structures Rich in Asp and Glu Several α helical segments Binding site is formed by Helix Loop Helix 5

6 Helix E Calmodulin Found in almost all eukaryotic cells Consists of two globular regions Connected by flexible region Each contains 2 EF hands Four binding sites. 6

7 Calmodulin changes conformation upon binding to Calcium 7

8 2+ Calmodulin binds to Ca 4 which results in change in conformation ( Moving some hydrophobic residues from the inside to the outside of the domains) Calcium-Calmodulin Complex can Bind to a large Number of Enzymes,Pumps and Target proteins including Calmodulin-dependant Protein Kinase ATP ase Pump 8

9 Transporter In sarcoplasmic reticulum 80% of the membrane proteins 10 membrane spanning helices move against a large concentration gradient 2 / ATP Signal Transduction through Tyrosine Kinase Hormone Binding Dimerization of the receptor Auto phosphorylation of the receptor Phosphorylation of the target proteins 9

10 Some Hormones that use Tyrosine Kinase Growth Hormone Insulin Epidermal Growth Factor Platelet-derived growth Factor Growth Hormone Monomeric Protein 217 Amino Acids Compact Four-helix Bundle 10

11 Growth Hormone Receptor 638 A.Acid Membrane Spanning Protein Extracellular Domain 250 A.A Single Membrane-Spanning Helix Intracellular Domain 350 A.A Monomeric when not bound to hormone Dimeric when bound to hormone 11

12 Binding of one molecule of growth hormone Dimerization of the receptor Growth Hormone Each Intracellular Domain is associated with Janus a protein kinase called Janus Kinase 2 protein kinase-like protein kinase Interaction with membrane Binds peptides that contain Phosphotyrosine 12

13 Receptor dimerization brings two JAKs together Each Phosphorylates key residues on the other Activated JAK 2 can Phosphorylate other substrates STAT 5 Signal transducer and activators of transcription Regulator of transcription STAT5 Phosphorylation Dimerization Binding to specific DNA sites 13

14 STAT is phosphorylated on a tyrosine residue near the carboxyl terminus Phosphorylated tyr binds to SH2 domain of another STAT 5 molecule Activated JAK 2 can Phosphorylate other substrates (cont.) Phosphorylation of the Receptor Association with JAK 2 Association with other proteins in the signal transduction pathway 14

15 Signal-Transduction Cascades - 3 Tyrosine kinase and receptor dimerization Signaling Pathways and Cancer Receptor dimerization brings two JAKs together Each Phosphorylates key residues on the other 15

16 Tyrosine Kinase is Part of some receptors Epidermal Growth Factor Receptor Monomeric (inactive) EGF binding Dimerization Cross Phosphorylation Activation Insulin Receptor Dimer of 2 αβ pairs Insulin Binding Activation of the Kinase Do these receptors transfer information across the membrane in the same way? 16

17 Is dimerization alone sufficient for activation of EGF receptor? Synthesis of a gene that encoded a chemeric receptor Extracellular (insulin) Intracellular (EGF) EGF receptor and insulin receptor use a common mechanism for transmitting information across membrane Epidermal growth factor signaling pathway Grb-2 binds phosphorylated EGF receptor Binding to a protein called Sos Activation of Ras GDP GTP Activation of specific Protein Kinases 17

18 Ras is a member of small G proteins family Monomeric Exist in two forms GDP bound «-----» GTP bound Smaller than G proteins Have GTPase activity Many similarities in structure and mechanism with G α Include several groups or subfamilies Major role in growth, differentiation, cellular transport, motility etc SH2 18

19 Defects in Signaling Pathways Can Lead to Cancer and Other Diseases Cancer, is characterized by uncontrolled or inappropriate cell growth, Can be caused by certain viruses Can be associated with defects in signaltransduction proteins; failure of signal transduction process Rous sarcoma virus (in Chicken) carries a gene called v-src Oncogene Encode a tyrosine kinase protein Similar protein is found in cells called c-src Small differences in the amino acid sequences between the proteins 19

20 Impaired GTPase activity can lead to cancer in human Mammalian cells contain 3 Ras proteins Mutation Loss of ability to hydrolyze GTP Ras is locked in ON position continuous stimulation of growth Cholera and Whooping Cough Are Due to Altered G-Protein Activity The cholera toxin: protein composed of two functional units B subunit: binds to GM1 gangliosides of the intestinal epithelium A catalytic subunit: enters the cell. A subunit catalyzes the covalent modification of a Gα s protein: Attachment of an ADP-ribose to an arginine residue. Stabilization of the GTP-bound form of Gα s, The active G protein, activates protein kinase A. Openining of chloride channels Excessive loss of NaCl and the loss of large amounts of water into the intestine. 20

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