Last lecture we started talking about other system,which is TRANSDUCTION OF NMORMAL SIGNAL.

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1 First there s no need to refer to the slides (numbered 3) it s all here.. :D Lets start the lec.. Last lecture we started talking about other system,which is TRANSDUCTION OF NMORMAL SIGNAL. -When hormonal signal binds to receptor it will trigger the replacing of GDP into GTP which in turn leads to dissociation of alpha subunit,alpha subunit will bind to phospholipase C leads to its activation which in turn leads for the cleavage of inistol 4,5 bisphosphate into inistol triphosphate(ip3) and diacylglycerol(dag). The role of IP3 is opening the Calcium channels on endoplasmic reticulum (E.R) which causes the efflux of Ca into the cytoplasm. -IP3 has a cooperative binding feature meaning that the binding of the first molecule of IP3 facilitates the binding of the second molecule of IP3 and so on And this feature causes the rapid activation of Ca channels, so the difference in time is little between the rest cell(low lvl of Ca in cytoplasm) and activated cell. -In the other hand DAG is hydrophobic molecule which remains in the plasma mem. And causes the activation of protein kinase C,this activation requires the binding of Ca that have been effluxed from the E.R. and in turn this protein kinase causes the phosphorylation of many different proteins. 1 P a g e

2 The domain structure of protein kinase C: -As phospholipase C has many Isoforms, protein kinase dose so -This Isoforms are found in alpha, beta and gamma subunits (all these subunits has protein kinase domain). -C2 domain which is also found in phospholipase C,it interacts with lipids. -C1A and C1B (these domains binds DAG) and the effect of DAG occurs in these subunits. -also there s pseudo-substrate, its resembles the substrate sequence, (proteins that are phsphorylated have the same sequence of this pseudo substrate). Like in the slide: X(amino acid)-arginine-x-x-(serine,thyronine)-hydrophobic amino acid-arginine If this sequence us found the serine or therionine will be phosphorylated. Pseudo-substrate sequence is almost similar but instead of serine or therionine there s Alanine (which dose not have OH group ) so when pseudo substrate bind to active site of protein kinase C no phosphorylation would occur. -In case of inactivated protein kinase pseudo substrate blocks the active site. -upon stimulation binding of C1A or C1B or even binding of C2 to phospholipids (this binding requires the Ca ions) every one of these bindings removes the pseudo substrate from the active site, as a consequence the active site become ready for interaction with target proteins and get phosphorylated. 2 P a g e

3 Termination of IP3 signal: How its terminated?? By dephosphorylation,the 3 groups of phosphate are removed one after the other, once 1 group is removed the IP3 is no longer activated. NOTE: even phsphorylation by 4 phosphate molecules (which becomes in these cases inistol tetraphosphate) is still inactive and it becomes dephosphorylated in deferent sequence. So we have two ways for inactivating the IP3: 1- Either by removing one group after the other 2- or by addition of of 4 th group of phosphate then removing them -because of its function (IP3) it requires the immediate activation and deactivation (that s why its short lived messenger). Clinically: Lithium ions is used in the past for treatment of some psychological disorders (by inhibiting the IP3 recycling,it keeps IP3 in its inactive from) Why Calcium is suitable as second messenger? Notice that phosphate and Ca. forms insoluble salts,so if Ca. or phosphate levels are increased precipitation would occur. Also if Ca. and phosphate gather up in the same place.precipitation occur. -as we know phosphate level inside of cell in found is high and logically Ca. levels must be low,but out side of cells Ca. is found in higher con. (in mill molar range ). 3 P a g e

4 -the difference in Ca. levels is 10,000 times more outside as compared to intracellular Ca. concentration, and when Ca, channels are opened it will rapidly influx the Ca. (Found in red in slide ). -any regulatory signal should be easily activated as well as easily terminated. So how its terminated?? By calcium pumps which continuously pump Ca. outside of cell, so the overall result is that we can get rapid increase in intra calcium levels and followed by rapid decrease,and this change in concentration can be used to modulate the effect of many enzymes. Sec. thing about Ca. is that it can make coordination complexes with many oxygen atoms. - In this polypeptide chain asparatate contains carboxyl group as well as glutamate but the main chain contains oxygen,so many oxygen atoms from different polypeptide chains can coordinate with Ca. So it s a distinctive feature about Ca. that it can from many chains with different molecules binding them together. Useful tools in studying the role of Ca. Biochemist use different ways in studying the role of Ca. inside of cells. -Ionophores: its an Ion channel (artificial channel) which can bind Ca. in the center of it,and they have hydrophobic periphery but inside it can bind Ca.,so if we add this Ionophores to cell membrane it can diffuse into cell throw mem. (because its hydrophobic from outside) and releasing the Ca. that have been bound to selectively outside then do the cycle again. 4 P a g e

5 -Calcium Chelators: they could be added to cell in order to decrease the Ca. concentration allowing scientist to observe the effects of decreasing Ca. con. After adding these chelators. -Fluorescent Chelators: these chelators bind Ca. also but they have fluorescent property, Whats fluorescent? They are composed of light that have different wave lengths,and if they are bound to Ca. they appear in certain color. The whole purpose is measuring how much Ca. is found inside cell, by binding it with fluorescent. Calcium binding proteins: -Ca. dose not act alone (its bounded to Ca. binding protein) and this protein mediate the action of Ca. when it binds to enzymes (causing the activation or deactivation). -there are many binding proteins including : Calmodulin (the most famous), Troponin C and Parvalbumin. They have similar structures,and they are rich in asp. And glu. Why? Because they contain carboxyl groups which can bind Ca. -they have several alpha segments (its fixed molecule but when it binds Ca. it causes conformational changes) -the binding site is formed of helix-loop-helix ( they found that all binding proteins are made in this sequence). -Ca. binds at the loop and could make large conformational changes between the 2 helices. in case of parvalbumin (helix E) is numbered 5 while (helix F) is numbered 6 because its made up of 6 helices.(see the slide attached in next page) Theses super-secondary structures are found in many binding proteins 5 P a g e

6 HELIX E HELIX F Clmodulin: -Found in all eukaryotic cells -consist of 2 globular regions (2 domains) -connected by flexible region -each region contains 2 EF hands (so 4 Ca. binding sites found in calmodulin).. -This structure resembles the amino acid sequence of calmodulin and consist of 149 a.a. -also it has 4 domains,which represents that Ca. can bind several A.A. together. -as we said calmodulin is rich in Asp. And Glu. (this is why Ca. can bind to calmodulin) Calmodulin changes upon binding: The inactive calmodulin undergoes changing in structure after Ca. binding which make it able to bind to inactive enzymes and making them active ones. (plz refer to slide for both pic. ) 6 P a g e

7 -calmodulin binds to Ca. which results in conformational changes. What happens actually here is that some hydrophobic residues move from inside to out side of the domain,so once Ca. binds it will cause minimal changes leading to expose the hydrophobic residues(or patches ) and the purpose is allowing other proteins to be bound to calmodulin throw these hydrophobic patches(only complementary onces). Hydrophobic patches or residues -so we conclude that large number of protiens can bind to calmodulin as a result of exposing the hydrophobic patches caused by Ca. binding. The most important one is : 1-Calmodulin dependet protien kinase: it can add phosphate groups to verity of target protiens. How its activeated? As name suggests when it bind to calmodulin -so the overall interactions: when theres and increase in Ca. level it will bind to calmodulin and in turn,calmodulin will bind to calmodulin binding protien and the latter will phosphorylate many target protiens (including it self,meaning it will phsphorylate many other calmodulin. 7 P a g e

8 Q:whats the diffrenece between calmodulin-dependent protiens (phosphorylated and nonphosphoryelated ones)?? -the phosphorylated form is partially active when Ca. level is low,so once Ca level increases it will lead to phosphorylation of calmodulin-dependent protien eventually and it will remains active(or phosphorylated) even after the Ca. level decreases. 2-Calcium dependept ATPase pump: This pump will be activated once Ca. level decrases which leads to pump Ca. ions and lowring the con. Again. So both of these(calmodulin-dependent protien and Calcium dependent ATPase pump) regulates the con. Of Ca. Calcium transporters: -they are abundant in sacroplasmic reticulum -80% of membrane protiens are Ca. transporters -10 mem. Spanning helices -Ca. move against large concentration gradient The movement of 2 Ca. ions requires one ATP. So if theres a depletion in in ATP, Ca. will remain inside of cell which in turn leads to cont. interaction between actin and myosin because of Ca. (..) Signal transduction throw tyrosine kinases: Tyrosine kinase (hormone binding) to cell receptor which leads to dimrization of the cell receptor,and then causes phosphorylation of receptor(its intracellular domain),finally phosphorylation of target protiens Hormones that uses trytosine kinase pathway: -Growth hormone -insulin -Epidermal Growth factor(e.g.f.) -Platelet derived growth factor(p.d.g.f) 8 P a g e

9 Growth hormone: -it s a monomeric protien -composed of 217 Amino acids -compact four helices bundel Growth hormone receptor: (composed of large protien) -composd of 638 Amino acids - singel membrane spanning protien -the receptor must be composed of Extra-cellular domain (composed approximatly of 250 AA) -the single peptide connects between the E.C domain and the intracellular one which therefore can transfere information when the hormone is bound. -monomeric when not bound to hormone,but dimeric when its bound Note: here theres a change in quaternary structure instead of teritary structure,because 2 molecules will be bounded at the same time. 9 P a g e

10 This is representation of he 3D structure of the receptor when bound to hormone -binding of one molecule of growth hormone will lead to dimerization of receptor,so one molecule of growth hormone will lead to dimer of bringing 2 receptors together. -Each intracellular domain is associated with : -protein kinase domain -protien kinaselike protein -SH2:it bind to phosphorylated peptides (peptides that contain phosphotyrosine) -ERM: its important for interaction with the membrane. 10 P a g e

11 Receptor dimerization brings two JAKs together: (bounded to intracellular domain) When dimerization occur they are brought together and each one will phsphorylate the other,also every one will phsphorylate the receptor itself. This all occur when dimerization takes place as a consequence of growth hormone binding Remember :the domains (in blue) are SH2 that binds phsphorylated tyrosine. -after that the activated JAK 2 can phosphorylate other substrate,meaning it phosphorylate it slef,the receptor and also phosphorylates other proteins,among these proteins is STAF 5 (which is signal transducer and activator of transcription),so this protein regulates transcription STAF 5 Phosphorylation,how? It will dimerize then binds to specific sites on DNA. -STAT is phosphorylated on a tyrosine residue near the carboxyl terminus - When tyrosine is phsphorylated it will bind to SH2 of another STAT 5 molecule. And when they are in this form they can bind to DNA for regulation of transcription. 11 P a g e

12 -tyrosine kinase is a part of some receptor. Now in case of growth hormone,jak2 is associated with intracellular domain of the receptor -some times the same receptor has the tyrosine kinase, and this is true for E.G.F -where its monomeric (its inactive ) -E.G.F binding will cause dimerization which leads to cross phosphorylation and then activation. -also Insulin receptor but in this case,insulin already a dimer of 2 alpha and beta pairs. Insulin binding will lead to activation of kinases. Is dimerization alone sufficient for activation of EGF receptor? For scientist to be able to answer this kind of questions,they synthesis a gene that encode a chemeric receptor,which has: -Insulin(on Extracellular domain) -E.G.F (on Intracellular domain) Notice that E.G.F domain become close to each other (although its not active) how it could be activated? by addition of insulin; when insulin is added the receptor is activated,so its not only dimerization that causes activation,also the addition of Insulin. -so in this experiment they concluded that both E.G.F and insulin receptor use a common mechanism for transmitting information across membrane. 12 P a g e

13 Epidermal growth factor signaling pathway: -Grb 2 (which is protein) binds phosphorylated E.G.F receptor. - Grb 2 protein acts as an adaptor allowing the E.G.F receptor for binding a protein called Sos, now this leads for activation of a third protein called RAS. This RAS protein contains GDP binding site and the activation leads to dissociation of this GDP and the association of GTP NOTE: RAS protein is called small G-protein and its only similar to alpha subunit of G- protein. So the activation of RAS will lead to activation of specific protein kinases. How this process is terminated? Since RAS is called (small G-protein) and just like G-protein it has GTPase activity, if this GTPase is impaired,the RAS will stay in active form,meaning that there s a continuous synthesis and transcription.(just like in cases of cancer) So RAS has a major role in growth, differentiation, cellular transport as well as motility. SH2 13 P a g e

14 Defects in signaling pathways can lead to cancer and other diseases. Whats cancer? simply we defined it as uncontrolled or inappropriate cell growth -it can be caused by certain viruses (these viruses act by replacing the original gene with defective one, this defective gene may not have a GTPase activity and might lead to cancer eventually) -failure of signal transduction pathway. Rous sarcoma virus: (first discovered in chickens) -caries a gene called V-Src -oncogene -encode a tyrosine kinase protein -similar protein found in cells called c-src - Small differences in the amino acid sequences between the proteins. Impaired GTPase activity can lead to cancer in human -Mammalian cells contain 3 Ras proteins mutation, which leads to loss of the ability to hydrolyze GTP,so RAS is locked in ON position which causes continuous stimulation of growth. Cholera and Whooping Cough Are Due to Altered G-Protein Activity The cholera toxin: protein composed of two functional Units 1- B subunit: binds to GM1 gangliosides of the intestinal epithelium 2- A catalytic subunit: enters the cell. - A subunit catalyzes the covalent modification of Gαs proteins 14 P a g e

15 - Attachment of an ADP-ribose to an arginine residue. - Stabilization of the GTP-bound form of Gαs, -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. NOTE: the doctor skipped the last 2 slides So I just copied them here. I tried to mention all the information that doctor said and explained, any missing info. That s my responsibility :P End of lecture. Good luck :D Done by: Hamza Zyad Salameh with special thanks for mohammed abuejhesheh 15 P a g e