I. Calcium in Cell Signaling. II. Nitric Oxide and Cyclic GMP in Cell Signaling

Transcription

1 I. Calcium in Cell Signaling II. Nitric Oxide and Cyclic GMP in Cell Signaling

2 Review G protein PKA

3 Review

4 I. Calcium signaling

5 Ca 2+ distribution -Extracellular: around 1 mm -Intracellular: free Ca2+: around 0.1 microm Ca 2+ stores -ER -Sarcoplasmic reticulum -Mitochondria Ca 2+ storage proteins -Calreticulin -Calsequestrin

6 Sources of intracellular Calcium Opening of the intracellular ligand-gated Ca 2+ channels lnsp 3 receptor and ryanodin receptor is regulated by -lnsp 3 -cadp-ribose -NAADP -Ca 2+ Ion channels involved in Ca 2+ influx -Ligand gated -Voltage gated -Mechanically operated -Store operated

7 Katsuhiko Mikoshiba discovered the IP3 receptor as a P400 decreased in the ataxic mutant mice and established it was a Ca2+ channel. Ins (1,4,5)-P3 receptor Calcium channel

8 Ryanodin receptor -abundant in the sarcoplasmic reticulum (the smooth ER of muscle) -Structurally related to lnsp 3 receptor -Opening induced by low Ca 2+ and by cadp-ribose or by NAADP -Closure induced by high Ca 2+ Ryanodine is a poisonous alkaloid found in a South American plant. It was originally used as an insectiside. The compound has extremely high affinity to the ryanodine receptor, a group of calcium channels found in skeletal and heart muscle cells. It binds with such high affinity to the receptor that it was used as a label for the first purification of that class of ion channels and gave its name to it.

9 Reactions of ADP-ribosyl cyclase. Structure of NADP,NAADP and cadp-ribose phosphate(cadprp). ADP-ribosyl cyclase, in base exchange mode, can catalyze replacement of the nicotinamide group of NADP(yellow) with nicotinic acid to generate NAADP. ADP-ribosyl cyclase can also catalyze cyclization of NADP to cadprp.

10 Tools for Ca 2+ release- major pathways for mobilizing Ca 2+ from internal stores. 1) Ca 2+ induced Ca 2+ release from ryanodine receptors (RyR) caused by the influx of Ca 2+ through voltage-or ligand-gated channels on the outer cell membrane. This release may be also triggered by direct interaction of the channel with RyR. (2) PLC/lnsP 3 evoked release of Ca 2+ from lnsp 3 receptors or ryanodine receptor. (3) cadp-ribose (cadpr)-evoked Ca 2+ release. (4) NAADP-evoked Ca 2+ release. 5, Ca 2+ release evoked by sphingosine. (6) Ca 2+ release from mitochondria

11 Tetrameric Ca 2+ channels and control Ca 2+ release (a) A change in the membrane potential (Δv) induces a conformational change in the dihydropyridine receptor of skeletal muscle; this is transmitted as a signal to the structurally coupled ryanodin receptor. Opening of the Ca 2+ channel takes place and efflux of Ca 2+ from the sarcoplasmic reticulum into the cytosol occurs. (b) In cardiac muscle, the release of Ca 2+ takes place by a Ca 2+- induced mechanism. A potential change Δv induces opening of voltage-gated Ca 2+ channels. Ca 2+ passes through, which serves as the trigger for release of Ca 2+ from Ca 2+ storage organelles by binding to ryanodin receptors on the surface of the storage organelles (c) membrane-associated signaling pathways are activated by ligands and lead, via activated receptor and PCL to formation of lnsp 3 and to release of Ca 2+ from storage organelles.

12 Paths for increase and reduction of cytosolic Ca 2+ concentration Influx of Ca 2+ from the extracellular space takes place via Ca 2+ channels; the open state of there is controlled by binding of ligand L or by a change in the membrane potential (Δv). According to the type of ion channel, the ligand may bind from the cytosolic or the extracellular side to the ion channel protein. The entering Ca 2+ binds to lnsp 3 receptors on the membrane of Ca 2+ storage organelles and induces, together with lnsp 3, their opening. Ca 2+ flows out of the storage organelle into the cytosol via the ion channel of the lnsp 3 receptor. Transport of Ca 2+ back into the storage organelles takes place with the help of ATP-dependent Ca 2+ transporters.

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14 Approxiamte levels of free Ca 2+ and Mg 2+ Ca 2+ Mg 2+ Extracellular fluid 1-3 mm 1 mm Intracellular (resting) nm mm EDTA (ethylenediaminetetraacetic acid) Binds to all divalent cations EGTA (ethylene glycol tetraacetic acid) BAPTA[1,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid]

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18 Inositol lipids and calcium signaling

19 Store-operated calcium channels (SOCCs) are calcium channels located in the plasma membranes of all non-excitable cells. SOCs are so called because they are activated by the ER stores depletion. These channels play important role in calcium entry into the cytoplasm from extracellular milieu. Transient receptor potential channels (TRP channels) are a group of ion channels located on the plasma membranes of numerous human and animal cell types. There are about 28 TRP channels that share some structural similarity to each other. Many of these channels mediate a variety of sensations like the sensations of pain, hotness, warmth or coldness, different kinds of tastes, pressure, and vision.

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21 Store-operated Ca 2+ channel (SOCC) and Store-operated Ca 2+ entry (SOCE) STIM1 activated SOCE. The current molecular concept of SOCE is STIM1-mediated activation of PM-SOCC (TRPC/Orai channels). Agonist-induced receptor (GPCR/RTK) activation results in PLC-mediated hydrolysis of PIP2, generating the diffusible and membrane-bound cellular messengers DAG (diacylglycerol) and IP 3 respectively. IP 3 binds to its receptor (IP 3 Rs) in the ER depleting the ER Ca 2+ stores. This leads to STIM1 oligomerization by interaction of mono- or dimeric STIM1 molecules. The STIM1 oligomeric-clusters thus formed are subsequently recruited to ER-PM juxtaposed sites allowing STIM1 to physically activate the TRPC and Orai channels to bring about Ca 2+ entry. This raises the (Ca 2+ ) cyt which results in store-operated Ca 2+ signaling and influences a variety of cellular functions. To complete the SOCE cycle, (Ca 2+ ) cyt is sequestered back to the ER by the SERCA pump and/or extruded to the cells exterior by PMCA. The membraneassociated lipid messenger - DAG also has the ability to activate select TRPC channels independent of ERstores and presumably STIM1 as well.

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27 Calcium-induced calcium release (CICR) describes a process whereby calcium is able to activate calcium release from intracellular Ca 2+ stores (e.g. endoplasmic reticulum and sarcoplasmic reticulum). Excitation-contraction coupling in cardiac muscle relies predominantly on CICR to activate Ca 2+ release from the sarcoplasmic reticulum. When an action potential depolarizes the cell membrane, voltage-gated Ca 2+ channels are activated. CICR occurs when the resulting Ca 2+ influx activates ryanodine receptors on the SR membrane, which causes more Ca 2+ to be released into the cytosol. In cardiac muscle, the result of CICR is observed as a spatio-temporally restricted calcium spark. The result of CICR across the cell causes the significant increase in cytosolic Ca 2+ that is important in activating muscle contraction.

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29 Luciferins are a class of light-emitting biological pigments found in organisms that causes bioluminescence. The term is used generically to refer to any light emitting molecule utilized by a luciferase. 1. Firefly: Firefly luciferin is responsible for the characteristic yellow light emission from fireflies. The chemistry was unusual as it was found that ATP was required for light emission. 2. Snail: Latia luciferin is chemically (E)-2-methyl-4-(2,6,6-trimethyl-1-cyclohex-1-yl)-1-buten-1-ol formate 3. Bacteria: Bacterial luciferin is consists of a long-chain aldehyde and a reduced riboflavin phosphate. 4. Coelenterazine: Coelenterazine is found in squid, fish, and shrimp. It is the prosthetic group in the protein aequorin responsible for the blue light emission. 5. Dinoflagellate : Dinoflagellate luciiferin is found in dinoflagellates, which are often responsible for the phenomenon of nighttime ocean phosphorescence. 6. Vargulin: Vargulin is found in certain in deep-sea fish. Coelenterazine Firefly luciferin

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31 Aequorin Aequorin (isolated from jellyfish) is composed of two distinct units, the apoprotein apoaequorin, with an approximate molecular weight of 22 kda, and the prosthetic group coelenterazine. In the presence of molecular oxygen the two components of aequorin reconstitute spontaneously, forming the functional protein. Researchers have located a number of EF-hand type regions in the structure of Aequorin that function as binding sites for Ca 2+ ions: when Ca 2+ occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO 2. As the excited coelenteramide relaxes to the ground state, blue light (469 nm) is emitted.

32 Coelenterazine coelenteramide

33 The Nobel Prize in Chemistry 2008 "for the discovery and development of the green fluorescent protein, GFP" GFP Osamu Shimomura Martin Chalfie Roger Y. Tsien In the 1960s and 1970s green fluorescent protein (GFP), along with the separate luminescent protein aequorin, was first purified from a jellyfish and its properties studied by Osamu Shimomura. The GFP is protein composed of 238 amino acids (26.9 kda), which exhibits bright green fluorescence when exposed to blue light. The tripeptide Ser65 Tyr66 Gly67 leads to chromophore formation

34 In GFP, the fluorophore originates from an internal Ser-Tyr-Gly sequence which is post-translationally modified to a 4-(phydroxybenzylidene)- imidazolidin-5-one structure

35 Calmodulin -Widespread Ca2+ sensor -Binds four Ca2+ ions via four EF hands -Organized in two EF domains -Flexible structure -Affinity for Ca2+ in the micromolar range -binding to target protein in different conformations possible

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37 Mechanisms of activation of target proteins by Ca 2+ /calmodulin (a) Binding of Ca 2+ /calmodulin relieves autoinhibition (CaMK, calcineurin). (b) Ca 2+ /calmodulin remodels the active site inducing an active conformation(anthrax adenylyl cyclase). (c) Ca 2+ / calmodulininduced dimerization of K + channels. AID=autoinhibition domain.

38 II. Nitric Oxide and Cyclic GMP in Cell Signaling Intracellular Messengers camp, IP 3, DAG, Ca 2+, cgmp, NO

39 The Nobel Prize in Physiology or Medicine 1998 Robert F Furchgott, SUNY Health Science Center Louis J Ignarro, UCLA School of Medicine Ferid Murad, University of Texas Medical School A New Principle Nitric Oxide, NO, is a short-lived, endogenously produced gas that acts as a signalling molecule in the body. Signal transmission by a gas, produced by one cell, which penetrates membranes and regulates the function of other cells is an entirely new principle for signalling in the human organism.

40 The Discovery of Nitric Oxide How synthesis of cgmp is modulated by a specific enzyme (the cytosolic isoenzyme of guanylyl cyclase or soluble guanylyl cyclase, sgc) was known as a consequence of the convergence of two very different lines of research. On the one hand, the group of Murad demonstrated in 1978 that the vasodilatory effects of nitroglycerine and other nitrates (that had been used for decades without knowing their mechanism of action) were mediated by a common degradation product that was very unstable, nitric oxide or NO. This compound was able to induce the activation of soluble guanylyl cyclase in the smooth muscle cells of blood vessels. On the other hand, Furchgott in 1980 observed that relaxation of blood vessels by acetylcholine required an intact endothelial layer. A compound released by endothelial cells and acting on smooth muscle cells was hypothesized and named endothelial derived relaxation factor (EDRF). During the following years it appeared that there were chemical similarities between nitric oxide and EDRF. Besides, both compounds exerted vasodilation by means of cyclic GMP synthesis. Finally, the groups of Furchgott and Ignarro independently proposed in 1986 that EDRF was really nitric oxide. Soon, the group of Moncada (1987) obtained the first results supporting that proposal. Moreover, they demonstrated that endothelial cells produced nitric oxide in sufficient amounts to explain the relaxation observed. So, the previous proposal was confirmed and, at the end, one of the pathways of cyclic GMP synthesis uncovered.

41 Guanylyl cyclases 1. Receptor guanylyl cyclase - Transmembrane proteins with a single TM helix -Activated by extracellular peptide ligands -Synthesize cgmp in an ATP-dependent manner 2. Cytoplasmic guanylyl cyclases -Contain a heme group -Activated by NO

42 Natriuretic peptide receptor (NPR) NPR is a dimeric TM receptor. The extracytosolic domain comprises the ligand-bindlng site and contains several disulfide bridges. The cytosolic part is composed of a kinase homology domain with multiple phosphorylation sites, an ATP-binding site of Unknown function and the catalytic guanylyl cyclase domain. Atrial natriuretic peptide (ANP) is a 28-amino acid peptide which is secreted by heart muscle cells and serves as a powerful vasodilator. ANP is released by muscle cells in the upper chambers (atria) of the heart response to high blood pressure. ANP acts to reduce the water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure. Vasodilation: Widening of blood vessels resulting from relaxation of smooth muscle cells.

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45 Nitric oxide synthase

46 nnos inos enos

47 Cofactors of NOS: FAD(flavin adenine dinucleotide) and FMN(flavin mononucleotide), Tetrahydrobiopterin, Heme. Substrates : Arginine, NADPH, O 2

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49 NO -Short-lived radical -Formed from arginine by NO synthase(nos) -Signals via covalent adducts with metal ions, SH groups, NH 2 groups of target proteins -Reacts with Me 2+ in metal ion centers -Reacts with O 2 and superoxide (O 2 )to yield No x products that form covalent adducts with biomolecules

50 NO reacts in biological systems with SH groups of glutathione (GSH), with SH groups of target proteins and with transition metals (Me). Furthermore, NO reacts with O 2 and with the superoxide anion O 2-. The products of this reaction, NO 2 /N 2 O 3 and peroxynitrite(oono) react further by nitrosylation of nucleophilic centers to yield S-nitroso (SNO), N-nitroso or nitrated compounds. Thiol and tyrosine as targets of NO Reactions of NO in biological systems. Reversible Irreversible

51 Metal ion centers as targets of NO 1. Guanylyl cyclase -Produces cgmp from GTP -Activated by NO binding to heme group -cgmp activates protein kinases and opens ion channels 2. Hemoglobin -NO binds to heme groups and/or Cys93 of the β-chain. This improves oxygen delivery in the periphery -NO binding is linked to O 2 tension -O 2 regulates delivery of NO for blood vessel relaxation

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53 enos

54 Cyclic nucleotide phosphodiesterase The Cyclic nucleotide phosphodiesterase (PDE) comprise a group of 11 isoforms that degrade the phosphodiester in the camp and cgmp. These multiple forms of phosphodiesterase were initially isolated from rat brain in the early 1970s. The potential for selective PDE to be used as therapeutic agents was predicted as early as This prediction has now come to pass in a variety of fields (e.g. Viagra as a PDE5 inhibitor). camp cgmp

55 Nitric oxide (NO) is produced by many cells in the body; however, its production by vascular endothelium is particularly important in the regulation of blood flow. Because of its importance in vascular function, abnormal production of NO, as occurs in different disease states, can adversely affect blood flow and other vascular functions. Under normal, basal conditions in blood vessels, NO is continually being produced by enos. The activity of enos is calcium and calmodulin dependent. There are two basic pathways for the stimulation of enos, both of which involve release of calcium ions from ER. First, shearing forces acting on the vascular endothelium generated by blood flow causes a release of calcium and subsequent enos activation. Therefore, increases in blood flow stimulate NO formation (flow-dependent NO formation). Second, endothelial receptors for a variety of ligands stimulate calcium release and subsequent NO production (receptor-stimulated NO formation). Included are receptors for acetylcholine, bradykinin, substance-p, adenosine, and many others vasoactive substances. In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980 he and others identified this substance as being NO. The other isoform of endothelial NOS is inos. It differs, in part, from enos in that its activation is calcium independent. Under normal, basal conditions, the activity of inos is very low. The activity of inos is stimulated during inflammation by bacterial endotoxins (e.g., lipopolysaccharide) and cytokines such as tumor necrosis factor (TNF) and interleukins. During inflammation, the amount of NO produced by inos may be a 1,000-fold greater than that produced by enos.

56 When NO forms, it has a half-life of only a few seconds, in large part because superoxide anion has a high affinity for NO (both molecules have an unpaired electron making them highly reactive). Therefore, superoxide anion reduces NO bioavailability. NO also avidly binds to the heme moiety of hemoglobin (in red blood cells) and the heme moiety of the enzyme guanylyl cyclase, which is found in vascular smooth muscle cells and most other cells of the body. Therefore, when NO is formed by vascular endothelium, it rapidly diffuses into the blood where it binds to hemoglobin and subsequently broken down. It also diffuses into the vascular smooth muscle cells adjacent to the endothelium where it binds to and activates guanylyl cyclase. This enzyme catalyzes the cyclization of GTP to cgmp, which serves as a second messenger for many important cellular functions, particularly for signaling smooth muscle relaxation. Cyclic GMP induces smooth muscle relaxation by multiple mechanisms including (1) increased intracellular cgmp, which inhibits calcium entry into the cell, and decreases intracellular calcium concentration. (2) activated K + channels, which leads to hyperpolarization and relaxation. (3) stimulation of a cgmp-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation. Because of the central role of cgmp in NO-mediated vasodilation, drugs (e.g., Viagra ) that inhibit the breakdown of cgmp (cgmp-dependent phosphodiesterase inhibitors) are used to enhance NO-mediated vasodilation, particularly in penile erectile tissue in the treatment of erectile dysfunction. Increased cgmp also has an important anti-platelet, anti-aggregatory effect.

57 Cyclic GMP is produced from GTP both by soluble (sgc) and particulate (pgc) guanylyl cyclases (also known as guanyl cyclases). Cyclic GMP is degraded by another group of enzymes known as phosphodiesterases (PDE). After degradation to GMP, ATP as supplier of metabolic energy is required to obtain again the initial compound: GTP. Many of the effects of cyclic GMP are mediated by the protein kinase G (PKG). In the diagram it is also shown that sgc is activated by nitric oxide (NO) and pgc by natriuretic peptides (NPs).

58 The NO signaling module at the cell membrane of neurons Binding of glycine and/or glutamate to the NR1 and NR2B subunits of the NMDA receptor triggers-together with an electrical stimulus- the influx of Ca 2+ into the cell. This binds to calmodulin and activates nnos. A spatial confinement of the reactions is achieved via the adaptor protein PSD95 that binds NMDR and nnos(via its PDZ2 domain). The NO produced by Ca 2+ /calmodulin-activated nnos may be transported out of the cell and may react with SH groups of the NMDR subunits leading to closure of the NMDR ion channel. Furthermore, NO may activate guanylyl cyclases and the regulatory GTPase Dexras that is associated with nnos via the adaptor protein Capon. NMDA (N-methyl-D-aspartate) receptor, a glutamate receptor, is the predominant molecular device for controlling synaptic plasticity and memory function. Activation of NMDA receptors results in the opening of an ion channel that is nonselective to cations.

59 NO targets (Cyclic nucleotide-gated ion channel)