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Regulation and cellular function of the inositol trisphosphate receptor

Regulation and cellular function of the inositol trisphosphate receptor. H. De Smedt K.U.Leuven. Intracellular Ca 2+ homeostasis. Tetrameric structure 3 Genes Regulation by Ca 2+. (Clapham, 1995). Localization of IP 3 R1 and IP 3 R3 in A7r5 cells. IP 3 R1. IP 3 R3. Cytoplasm.

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Regulation and cellular function of the inositol trisphosphate receptor

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  1. Regulation and cellular function of the inositol trisphosphate receptor H. De Smedt K.U.Leuven

  2. Intracellular Ca2+ homeostasis Tetrameric structure 3 Genes Regulation by Ca2+ (Clapham, 1995)

  3. Localization of IP3R1 and IP3R3 in A7r5 cells IP3R1 IP3R3 Cytoplasm Perinuclear region

  4. IP3R redistribution is dependent on vesicle trafficking Agonist( ) IP3Rredistribution

  5. Agonists IP3 Ca2+ Plasma membrane associated: Homer-mGluR TRPs; RhoA-TRPC1 G; RACK-1 Cytoskeletal proteins: Actin; MyosinII Ankyrin; Tallin; Vinculin 4.1N Kinases and phosphatases: PKA; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1 Cytosolic proteins: Calmodulin; CaBP IRBIT CARP HAP1A-Htt Intraluminal proteins: Chromogranins; Calnexin IP3R I, II, III

  6. 3D reconstruction of the IP3R1(Hamada et al., 2003) Bar: 10 nm

  7. CaBP CaM CARP mGluR 1a, 5 Shank IP3R RyRCa2+ channels HOMER IRBIT TRP RACK1 Gβ CASK and/orsyndecan-2 AKAP9 Cyt c PP1 PKA PP1 4.1N Spectrin-actincytoskeleton HAP1A/Htt ANKYRIN B CHROMOGRANIN A & B SIG-1R Monomeric type 1 IP3R (IP3R1) N IP3 C

  8. IICR is dependent on luminal and cytosolic Ca2+

  9. Regulation of the IP3R by Ca2+ Bell-shaped IICR Structure of the IP3R Bezprozvanny et al., 1991 Hamada et al., 2002 Square Windmill Ca2+ is the primary modulator of its own release

  10. IP3-binding domain Coupling domain Channel domain Ca2+ (Sienaert et al., 1999) CaM SPA technique: Adkins et al., 2000 Location of Ca2+ and CaM-binding domains

  11. P IP3 CaM ?(Adkins et al) CaCaM P P 31 25 Ca2+ sensor P 18 Monomeric structure of the IP3R Cytosol ATP NH2 COOH ATP ATP 13 Endoplasmic reticulum

  12. The N-terminal Ca2+ -independent calmodulin-binding site on the inositol trisphosphate receptor is responsible for calmodulin inhibition, even though this inhibition requires Ca2+Mol. Pharmacol. 66, 276-284, 2004 Laboratory of Physiology, KULeuven, Belgium Nadif Kasri N, Bultynck G, Smyth J, Szlufcik K, Parys JB, Callewaert G, Missiaen L, Fissore RA, Mikoshiba K, & De Smedt H.

  13. IP3R I II III + CaM [IP3] (µM) Regulation of the IP3Rs by CaM IICR • CaM • (Michikawa et al., 1999) IP3R I II III + CaM (Missiaen et al., 1999) 300 [Ca2+] (nM) Inhibition of IICR Ca2+-dependent IP3- dependent

  14. Calmodulin-binding sites on IP3R1 W1577A (Zhang et al, 2001; Nosyreva et al, 2002) R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA 31 25 18 Cytosol CaM?? (Adkins et al,2000) CaCaM 13 Endoplasmic reticulum

  15. Calmodulin effects on IP3 binding N 226 581 C IP3 binding core 100 80 [3H]IP3 binding (%) 60 40 + CaM 0.3 20 0.2 B/F 0.1 Control Ca2+ CaM CaM1234 Ca2+/CaM1234 Ca2+/CaM 0.0 0 5 10 Bound (nM) 1 581 IP3 binding core suppressor CaM inhibits IP3 binding in a Ca2+ -independent way

  16. Detailed localisation using peptides 159 1 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D E B F A B C D E F CaM Ca2+ EGTA A B C D E F CaM Discontinue Ca2+-independent CaM-binding site in the N-terminal region

  17. 1 581 IP3 binding core suppressor Both sites essential B E ∆ B ∆ E control 100 80 60 [3H]IP3 binding (%) vs control 40 20 0 CaM + + - - - + Both CaM-binding sites are essential for inhibiting IP3 binding

  18. The N-terminal Ca2+ -independent CaM-binding site may be functionally important, but… CaM1234? 31 25 18 Ca2+ -indep CaM Cytosol R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca2+ R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA CaCaM ER 13 Functional CaM effects are Ca2+-dependent!

  19. 600 CaM 400 Ca2+i (nM) 200 CaM1234 0 0 250 500 750 1000 IICR is inhibited by CaM and CaM1234 (1) Intact COS cells 0.5 µM 1 µM 100 µM ATP Control Time (s) Decreased amount of responsive cells Increased latency

  20. 200 nM IP3 IICR is inhibited by CaM and CaM1234 (2) 45Ca2+ flux Permeabilized cells (%) 200 nM IP3 CaM 50 Ca2+ release 200 nM IP3 CaM1234 0 0.1 1 [Ca2+ ] (µM) • CaM is not the Ca2+ sensor for the IP3R

  21. Regulation of Inositol trisphosphate Receptor activity by neuronal Ca2+ -binding proteinsEMBO J. 23, 312-321, 2004 Laboratory of Physiology, KULeuven, Belgium & Babraham Institute, Cambridge, UK Nadif Kasri N, Holmes AM, Bultynck G, Parys JB, Bootman MD, Rietdorf K, Missiaen L, McDonald F, De Smedt H, Conway SJ, Holmes AB, Berridge MJ & Roderick HL

  22. Inhibitory Activatory ?(Yang et al., 2002) Adapted from Haeseleer et al., 2000 CaM or other CaM-like Ca2+ sensor proteins ? Interaction with neuronal Calcium Binding Proteins (CaBP)

  23. CaBP1 binds to the N-terminal part of the IP3R aa 1-225 IP3 binding core (226-581) NH2 GST GST 1-581 GST 1-225 GST 226-581 CYT CaBP1 ? CaBP1 COOH ER CaM Ca2+CaM

  24. CaBP1 binds to a similar region as CaM independently of Ca2+ A) 159 CaM CaM 1 A C D E B F B) + Ca2+ -Ca2+/ EGTA sCaBP1 C D F A B E C D F A B E sCaBP1

  25. 45Ca2+ flux 100µM 1µM 0.5µM ATP 1000 800 Ca2+i (nM) 600 400 Control sCaBP 200 lCaBP 0 0 200 400 600 800 1000 Time (s) Both long and short CaBPs inhibit IP3-induced calcium release in COS-1 cells

  26. EF1 EF2 EF3 EF4 Control CaBP1134 CaBP1 inhibits IICR independent of Ca2+ binding YFP 0.5 1 100 µM ATP 1000 CaBP1 activity is regulated by: 800 Myristoylation 600 Ca2+i (nM) Phosphorylation 400 200 0 0 1500 500 1000 Time (s)

  27. CaBP1 does not affect Ca2+ release through RyRs RyRs are not modulated by CaBPs: In neurons: 10µM CCh 1mM Caf 0.5mM Caf 2mM Caf RyR IP3R CaM CaBP1 sCaBP1 300 ) M 200 n ( i ] + 2 a C [ 100 Control 0 0 500 1000 1500 2000 Time (s)

  28. CaM or CaBP Synergetic effect of Ca2+ and Calmodulin on IP3R conformation?

  29. To summarise… Ca2+ Other regulators ? Ca2+ kinases phosphatases Closed « square » Inactive Open « windmill » IICR 300 [Ca2+] (nM)

  30. Regulation of the phosphorylation of the Inositol trisphosphate Receptor by protein kinase CBBRC 319, 888-893, 2004 Laboratory of Physiology, KULeuven, Belgium Vermassen E, Fissore RA, Nadif Kasri N, Vanderheyden V, Callewaert G, Missiaen L, Parys JB & De Smedt H

  31. Regulation via Ca2+-dependent phosphorylations ? Ca2+ -indep CaM P IP3 CaCaM P P What could be the function Of the CaCaM-binding site? 31 25 Ca2+ sensor P 18 Cytosol ATP NH2 COOH ATP ATP 13 Endoplasmic reticulum

  32. Effects of IP3R phosphorylation IP3R1: - PKA - PKG - PKC - CaMKII - Fyn and Lyn - Rho kinase - cdc2/cyclin B - ERK IP3R2: - PKA - Rho kinase IP3R3: - PKA - Rho kinase stimulates IICR ? stimulates IICR? ? stimulates IICR stimulates IICR IP3 binding ↑ ? ? no effect on IICR? ? no effect on IICR?

  33. Autoradiography Rbt475 Common Ab In vitro phosphorylation of IP3R1 and IP3R3 by PKC Catalytic subunit of PKC (, , ) IP3Rs purified from Sf9 microsomes on sucrose density gradient and/or CaM-sepharose column → highly purified fraction. IP3R1 32P phospho (Ser) PKC Ab IP3R1 is phosphorylated by PKC IP3R3 is phosphorylated to a lesser degree

  34. Anti-(1829-1848) Determination of the PKC phosphorylation site(s) →controlled proteolysis with -chymotrypsin after in vitro phosph. IP3R1 Yoshikawa et. al., J. Biol. Chem., 1999, 274, 316-327 5 fragments

  35. Determination of the PKC phosphorylation site(s) 0.2 µg/ml CT 0.4 µg/ml CT Control 273 kDa 273 kDa 225 kDa 210 kDa 225 kDa 210 kDa 130 kDa 130 kDa Blot stripped and reprobed 40 kDa 40 kDa Phosphorylated fragments anti-(1829-1848) Ab

  36. m/z Mr1 (Da) Mr2 (Da) Measured Mr (Da) Theoretical Mr (Da) Deviation (Da) Amino acid sequence S1852 433.2 864.4 784.4119 79.9881 79.9663 -0.021 1852SEKFFK1857 Mass Spectroscopy IVa 40k

  37. CaM S1852 IP3R1 S1832 CaCaM IP3R3 CaM Mass spectrometry IP3R1 and IP3R3

  38. Differential regulation of the PKC phosphorylation by Ca2+ + Ca2+ (50 µM) - Ca2+ IP3R1 IP3R3 IC50 ≤ 2 µM Ca2+ inhibits PKC phosphorylation of IP3R1

  39. Differential regulation of the PKC phosphorylation by Calmodulin CaM CaM1234 CaM1234 CaCaM but not CaM1234 inhibits the phosphorylation of IP3R1

  40. ACTIVATION INHIBITION PKC CaCaM PKC Ca2+ Ca2+ Potential physiological role ofPKC-mediated phosphorylation of IP3R1 apoCaM

  41. Caspase-3-induced truncation of Type 1 Inositol trisphosphate Receptor accelerates apoptotic cell death and induces Inositol trisphosphate-independent calcium release during ApoptosisJ. Biol. Chem. (2004) in the press Laboratory of Physiology, KULeuven, Belgium Assefa Z, Bultynck G, Szlufcik K, Nadif Kasri N, Vermassen E, Goris J, Missiaen L, Callewaert G, Parys JB & De Smedt H

  42. Truncation of IP3R1 Ca2+ -indep CaM  1-225 P Caspase-3 cleavage IP3 CaCaM P P Cytochrome C binding 31 25 Ca2+ sensor P 18 Cytosol ATP NH2 KKDDEVDRDA KKDIEVARDA COOH ATP ATP 13 Endoplasmic reticulum

  43. IP3R1 constructs: N cytoplasm C ER lumen N C N C - the constructs were stably expressed in DT40 cells that lack all three IP3Rs.

  44. Del(1-225)-IP3R1 is no longer functional

  45. Cleavage of IP3R1 invitro and in cells undergoing apoptosis

  46. Caspase-3 mediated cleavage of IP3R1leads to an increase in ... Caspase-3 activity Apoptosis

  47. Secondary necrosis in STS-treated cells

  48. Apoptosis-related rise in [Ca2+]i requires caspase cleavage of IP3R1 Fluo-3 Fura red WT-IP3R1 Fluo-3 Fura red IP3R1Dcasp (D1-225)IP3R1 IP3R-KO fluorescence intensity (relative units) (D1-1891)IP3R1 fluorescence intensity (relative units)

  49. Caspase-3 activation precedes the increase in [Ca2+]i WT-IP3R1

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