Cell communication 1: cell signaling and signal transduction. 3-7-2016

Can single cell live alone without any kind of communication with others or environment?

For bacteria, Communicate with environment Chemotaxis: to detect nutrients and avoid toxicic substance Communicate with other bacteria Quorum sensing (來偷聽細菌們的對話吧！)

For multicellular organism, Communicate with environment Sensory system: see, smell, hearing, taste, touch etc. Communicate with others Chemical , visual , language and words. Communicate within Development : from single fertilized egg to adult body. Transport system: circulation, digestion, secretion. Neural system: fast communication, learning and memory. Endocrine system: homeostasis, growth and reproduction. Immune system: defense against non-self. Failure of communication: cancer and aging.

Elements of Communication Signal What it is? who send? how to produce? and when to send? Receiver Receptor: specificity and sensitivity Localization Control: amount and sensitivity Signal interpretation Signal transduction: amplify, diversify and integration Responses Cell contend dependent

Cell: integration of various signals and to make proper response 16_06_extracellular_sig.jpg 16_06_extracellular_sig.jpg

Cell communication within our body 16_03_signal_various.jpg 16_03_signal_various.jpg

Generation of body pattern 16_04_Contact_depend.jpg 16_04_Contact_depend.jpg

Path of signal transduction discovery 16_08_cascades.jpg

16_07_change_behavior.jpg 16_07_change_behavior.jpg

Biochemical study of hormonal action: how hormone activates liver/muscle phosphorylase to break down glycogen?

n n-1 Glycogen phosphorylase a* ATP Glycogen phosphorylase b Glc-1-P → Glc-6-P → Glycolysis Glycogen P n n-1 Glycogen phosphorylase a* P Phosphatase ATP 肝糖磷解脢 (GP) 可以對肝糖進行磷解反應，由肝糖長鏈的端點開始，把葡萄糖一個個切下來，並且加上磷酸成為 Glc-1-P，後者可以進入糖解作用以產生能量。 平常 GP 是以較不活躍的 b 型存在著，要用其專用的激脢 (GP kinase) 進行磷酸化，才成為具有活性的 a 型。而此激脢要受到另一種激脢 (protein kinase A, PKA) 的磷酸化才會有活性，才能去磷酸化 GP；而 PKA 又要受到 cAMP 的活化後，才有活性。 cAMP 是一個重要的信息傳導分子，當荷爾蒙 glucagon 與目標細胞接合後，就在胞膜的接受體上誘發胞膜內側的 G protein，後者會活化 ATP cyclase 使得 ATP 產生 cAMP。因此這一條路徑就把外界荷爾蒙所傳來的信息，經由 cAMP 傳到 PKA，再傳到 GP kinase 後，即可活化 GP 開始執行細胞任務，把肝糖分解成單糖，以供細胞或人體所需。 上述磷酸化只是 GP 的一種調節方式而已，是屬於共價鍵式的可逆修飾，因為磷酸化後的 GP 也可以用磷酸脢 (phosphatase) 去除磷酸，以恢復其非活性型 b。另外，GP 活性也受到其代謝物的影響，例如當血糖中有太多的單糖或能量分子，顯示不需要再水解肝糖；此單糖或能量分子 (如 Glc-6-P, ATP) 可以直接與 GP 結合，以迅速降低其活性。 因此，GP 除了磷酸化之外，其活性還可以受到 AMP, Glc-6-P 等小分子所調節；這些小分子都是結合在其活性區以外的地方，是有異於活性區的位置，因此特稱之為異位脢 。 Glycogen phosphorylase b (inactive)

How hormone (glucogon or epinephrine) activate hepatic enzyme to degrade glycogen? synthase Liver Pancreas Glycogen Glucose Glycogen phosphorylase Glucagon or Epinephrine Low blood sugar 血糖 太高或太低對人體都有不好的影響，因此體內有極為複雜的血糖調節系統，主角是合成肝糖的肝糖合成脢 (glycogen synthase, GS)，以及降解肝糖以生成葡萄糖的肝糖磷解脢 (glycogen phosphorylase, GP)。 此二酵素分別受到荷爾蒙胰島素 (insulin) 及胰增糖素 (glucagon) 的影響，人體便可利用控制荷爾蒙的濃度來調節血糖濃度。 這兩種荷爾蒙分子到達目標細胞時，細胞膜上有此荷爾蒙的接受體，當兩者接合之後信息便可傳入細胞內，引發酵素活性，進行所需要的糖類代謝。 這些信息是如何傳導給最終的酵素 GS 及 GP？ 這即是最近極為蓬勃的信息傳導，以及典型的酵素調控機制。以下就以肝糖磷解脢為主要對象及實例，說明酵素如何以磷酸化、信息傳導分子以及迴饋控制的方式，進行其活性的調節。 肝糖磷解脢具有以上各種調控機制，很少有酵素如此密集地被調控著，因為肝糖磷解脢在糖類的利用實在是太重要。 以下我們先瞭解肝糖磷解脢的分子構造及生化性質。 stress

Sutherland EW et al J. Biol. Chem. (1956) Effect of epinephrine on phosphorylase activity in dog liver slices. Sutherland EW et al J. Biol. Chem. (1956)

EFFECT OF EPINEPHRINE AND GLUCAGON ON THE REACTIVATION OF PHOSPHORYLASE IN LIVER HOMOGENATES J. Biol. Chem. 224: 463 – 475; 1957

Effect of boiled E/G treated membrane extract on activation of phosphorylase in the test tube

Adenylate Cyclase (AC) cAMP is converted from ATP by membrane bound enzyme Adenylate Cyclase (AC) AC in different tissues can only be activated by specific hormone (liver/glucagon; ACTH/ adrenal gland). Hormone may need interact with its specific receptor first!

The second messengers concept ? ? The second messengers concept Sutherland, E. W. Pharmac. Rev., 18, 145 (1966).

How cAMP activates glycogen phosphorylase through phosphorylaion? Isolation of phosphorylase kinase; Is phosphorylase kinase a direct targert of cAMP? NO! its activation also need to be phosphorylated – another kinase? EG Krebs isolated phosphorylase kinase kinase and demonstrated that is the direct target of cAMP! EH Fisher isolated protein phosphatase which reverse the phosphorylation reaction. Krebs and Fisher establish that protein phosphorylation is a reversible control mechanism of many enzymes and proteins!

p.428

A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. A protein phosphatase catalyzes removal of the Pi by hydrolysis.

Epinephrine cascade. Epinephrine triggers a series of reactions in hepatocytes in which catalysts activate catalysts, resulting in great amplification of the signal. Binding of a small number of molecules of epinephrine to specific β-adrenergic receptors on the cell surface activates adenylyl cyclase. The numbers of molecules shown are simply to illustrate amplification and are probably gross underestimates.

? ? How adenyl cyclase is activated? By hormone receptor directly? Or Need some other activators?

GTP dependent transmembrane signaling?!

GORDON M. TOMKINS found the cytocidal effect of cyclic AMP on clonal S49 lymphoma cells. Proc. Natl. Acad. Sci. U.S.A. 70, 76-79 (1973) Add Epinephrine Beta adrenergic receptor Activation of AC Increase cAMP Activate PKA Cell die!

What’s wrong about this cAMP resistant cells? Defect in regulatory subunit? Defect in catalytic subunit? Defect in down stream targets of kinase? What else?

Gillman et al were able to select another S49 cell variant - (cyc-) that appeared to have normal receptors and adenylyl cyclase but that failed to generate a cyclic AMP signal in response to appropriate hormones (b-adrenergic agonists or prostaglandins). Proc. Natl. Acad. Sci. U.S.A. 74, 2016 – 2020 (1977)

Solubilize membrane from wild type cells hormone adenylyl cyclase unc unc hormone Hormone No cAMP cAMP

G protein coupled receptor: 7 trans-membrane protein G proteins dissociate into two signaling proteins when activated 16_17_Gprot_dissociate.jpg

16_20_second_messeng.jpg Adenylyl cyclase ATP Cyclic AMP

None of them physiological relevant! If cAMP can play as an important intracellular second messager, how about other nucleotide? In 1975, FERID MURAD identified guanylate cyclase catalyzes the formation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) from GTP. Guanylate cyclase is activated by various agents including sodium azide, hydroxylamine, nitroglycerin, sodium nitrite etc. None of them physiological relevant!

The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine Furchgott RF, Zawadzki JF Nature 288, 373 – 376; 1980

Ignarro LJ, Byrns RE, Buga GM, Wood KS Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide. Circ Res. 1987;61:866–879.

How is the receptor mediated signaling desensitized?

How the signal is turned off ? Hormone association

Arachidonic acid present in membrane lipids are released for eicosanoid synthesis in the cell interior by phospholipase A2

Discovery of growth factor and its signal transduction pathway

Rita Levi-Montalcini Cancer Research 14: 49-54 (1954) 8-day chick embryo sensory ganglia cultured in the presence of fragments of mouse control or sarcoma 180 cells for 24 hrs. Rita Levi-Montalcini Cancer Research 14: 49-54 (1954)

What is biochemical nature of NGF? Protein or Nucleic Acid? A soluble factors secreted from mouse sarcoma 180 cells which can promote nerve growth from sensory ganglia (Nerve Growth Factor, NGF) What is biochemical nature of NGF? Protein or Nucleic Acid? To remove nucleic acid by digesting with snake venom. Snake venom itself contains NGF activity!

Cohen S. Proc. Nat. Acad. Sci. USA 46: 302-311 (1960) Mouse salivary gland extract (NGF) were injected into newborn mice. Some unexpected; non-NGF related phenomenon were observed! Cohen S. Proc. Nat. Acad. Sci. USA 46: 302-311 (1960)

J. Biol. Chem. 1962

Cohen S. J. Biol. Chem. (1973)

Characterization of growth factor receptors Label ligand with radioactive isotope Binding assay Determining specificity of the ligand binding Scatcard analysis Km verse total receptor number How signal of the growth factor receptor transduced?

Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro Nature 276, 409 - 410 (23 November 1978)

EGF stimulates protein phosphorylation in the membrane fraction of target cells (PNAS 1978)

Extrinic signals

How the signal is turned off ? a-subunit of G protein has GTPase activity. Cholera toxin blocks a-subunit’s GTPase activity through ADP-ribosylation of a-subunit. Then ?