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Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -Binding Proteins

Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -Binding Proteins. Laboratory of Physiology KULeuven Leuven, Belgium. LGCC. VGCC. SOCC (TRP). MSCC. R. NCX. Second messenger. IP 3 R. RYR. NCX. Mitochondria. PMCA. Others??. IP 3 R. ER/SR. PMR1. SERCA. Uniporter. Golgi.

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Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -Binding Proteins

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  1. Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding Proteins Laboratory of Physiology KULeuven Leuven, Belgium

  2. LGCC VGCC SOCC (TRP) MSCC R NCX Second messenger IP3R RYR NCX Mitochondria PMCA Others?? IP3R ER/SR PMR1 SERCA Uniporter Golgi Buffers

  3. Agonists IP3 Ca2+ Plasma membrane associated: Homer-mGluR TRPs; RhoA-TRPC1 G 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 Intraluminal proteins: Chromogranins; Calnexin, Calreticulin IP3R I, II, III ER

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

  5. Effect of calmodulin on IP3-induced Ca2+ release A7r5 cells 70% IP3R1 Control CaM Control CaM HBE cells 90% IP3R3 A7r5 HBE

  6. Calmodulin and apocalmodulin Sienaert, I,. Nadif Kasri, N, Vanlingen S., Parys J. B., Callewaert G., Missiaen, L., and De Smedt, H. Localisation and Characterisation of a calmodulin/ apocalmodulin binding domain in the N-terminal part of th etype 1 inositol1,4,5- trisphosphate receptor Biochem. J. 365, 269-277, 2002

  7. Lbs-1: IP3 binding core (226-604) kDa kDa 62 62 – – 49 49 – – 38 38 – – 28 28 – – 17 17 – – 14 14 – – 6 6 – – (Adkins et al., 2000) NH2 CaM CYT Ca2+CaM COOH ER Recombinant ligand-binding domain of IP3R1 (LBS-1) 1 581 Lbs-1 1-225 W226 581 Lbs-1 1-225

  8. Calmodulin effect on IP3 binding 581 1 Lbs-1His W226 581 Lbs-1 1-225His 0.3 [3H]IP3 binding (%) 0.2 EC50= 1.7 µM B/F Control Ca2+ CaM Ca2+/CaM CaM1234 Ca2+/CaM1234 0.1 0.0 0 5 10 Bound (nM) CaM inhibits IP3 binding in Ca2+ -independent way

  9. 1 581 GST-fusion protein pull down of CaM1234 Lbs-1 W226 581 Lbs-1 1-225 pGST GST-Cyt1GST-Cyt2 309 159 1 50 M free Ca2+ Cyt1Cyt2 CaM1234 1 mM EGTA CaM1234 Localisation of a calmodulin-binding site

  10. Detailed localisation using peptides 159 1 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ (site1) A C D E B F Ca2+ EGTA A B C D E F CaM A B C D E F CaM Ca2+ independent CaM-binding site in the N-terminal region

  11. Calmodulin binding sites on IP3R1 Cytosol R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD CaM W1577A (Zhang et al, 2001; Nosyreva et al, 2002) R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA 31 25 13 18 Endoplasmic reticulum Ca2+/CaM

  12. CaM CaM 1234 Control CaM IICR is inhibited by CaM and CaM1234 ATP 0.5 µM 1 µM 100 µM 600 Control 400 Ca2+i (nM) 200 0 0 250 500 750 1000 Time (s)

  13. Summary • CaM inhibts IICR only in the presence of Ca2+ • Two CaM-binding sites on the IP3R Ca2+ dependent in the regulatory domain Ca2+ independent in the N-terminus

  14. Regulation of calcium release by neuronal Calcium Binding Proteins (CaBP) Regulation of Calcium release by neuronal Calcium Binding Proteins (CaBP) N. Nadif Kasri, H. Llewelyn Roderick, J.B. Parys, L. Missiaen, H. De Smedt and M. Bootman In preparation

  15. Inhibitory Activatory ?(Yang et al., 2002) Adapted from Haeseleer et al., 2000 CaM or other CaM-like Ca2+ sensor proteins ?

  16. IP3 binding core (226-581) NH2 GST GST 1-604 GST 1-225 GST 226-604 CYT sCaBP COOH ER CaBP binds to the IP3R

  17. CaBP binds to a similar region of the IP3R as CaM 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

  18. 1.0 Band intensity 0.5 0.0 0 2 4 6 8 10 Peptide B: CaBP Binding of CaBP to the IP3R is calcium independent Ratio of CaBP: peptide B CaBP CaBP 1/10 1/1 1/2 1/3 1/4 1/5 1/6 1/8 + Calcium + EGTA

  19. 100% 100 78 ± 1.9 % 68 ± 6.9 % 75 [3H]IP3 Binding (% vs control) 39 ± 4.7% 50 25 0 sCaBP1 Ca2+ Ca2+/ sCaBP1 Lbs-1 CaBP inhibits IP3 binding to the InsP3R 4 µg Lbs-1 10 µM sCaBP1 5 µM Ca2+

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

  21. EF1 EF2 EF3 EF4 100 % responsive cells 50 Control CaBP134 0 0.5 1 100 ATP (mM) CaBP inhibits IP3 induced calcium release independent of calcium binding 0.5 1 100 µM ATP 1000 800 600 Ca2+i (nM) 400 200 0 0 1500 500 100 Time (s)

  22. SCaBP1 CaBP overexpression inhibits InsP3ester induced calcium release: CaBP acts directly on the InsP3R 10 mM InsP3 ester 250 200 Control 150 Ca2+i (nM) 100 50 0 0 200 400 600 800 1000 1200 time (s)

  23. Summary • CaBP binds on the N-terminal CaM-binding site in a Ca2+-independent way • CaBP inhibits IP3 binding • CaBP inhibits IICR • CaBP activity is Ca2+-independent

  24. CaM important for inter-or intramolecular interactions? CaM antagonists Suramin Inhibits CaM binding CaM-binding peptides (MLCK) Removing CaM from its binding site

  25. Control 100 µM suramin IP3R1 + Suramin + Suramin CaM-Seph CaM-Seph Seph Ca 2+ Seph EGTA Input Control 1-225 10 µM CaM Ca 2+ EGTA Suramin interacts with the CaM-binding sites on the IP3R1

  26. IP3 MLCK peptide inhibits IICR High CaM affinity binding properties

  27. Properties of the inhibition of IICR by MLCK peptide

  28. 225-604 ?? sCaBP-1 AdPhos Control CaM CaM/CaBP GST IP3 Suramin 1-225

  29. Summary • Suramin interacts with the CaM-binding sites on the IP3R • Suramin inhibits IICR by lowering the IC50 for activation by IP3 • MLCK peptide inhibits IICR by lowering Vmax By removing endogenous CaM? By disrupting intermolecular protein-protein interaction?

  30. Conclusions • Ca2+ is the major regulator of the IP3R • CaM inhibts IICR only in the presence of Ca2+ • Two CaM-binding sites on the IP3R Ca2+ dependent in the regulatory domain Ca2+ independent in the N-terminus • CaM can play an important role in intramolecular protein-protein interactions within IP3R, as removing endogenous CaM inhibits IICR

  31. Humbert De Smedt Geert Callewaert Ludwig Missiaen Jan B. Parys Ilse Sienaert Karolina Szlufcik Geert Bultynck Sarah Vanlingen Lab. of Physiology K.U.L., Belgium Brabaham Institute, Cambridge, UK Llewelyn Roderick Martin Bootman Baylor College of Medicine, Houston, Texas Andreas Jeromin

  32. A new Calmodulin-dependent CICR mode in A7r5 cells N. Nadif Kasri, I. Sienaert, Jan B. Parys, G. Callewaert, L. Missiaen, A. Jeromin and H. De Smedt A novel Ca2+-induced Ca2+-release mechanism in A7r5 cells regulated by calmodulin-like proteins J. Biol. Chem, 2003 in the press

  33. Culture medium Saponin 45Ca2+ TG SDS 45Ca2+-efflux technique on permeabilized A7r5 cells Fractional loss

  34. Ca2+ release vs control Control Heparin 2-APB XeC RuRed Ryanodine CICR is not mediated via IP3R or RyR Ca2+ -induced Ca2+ Release in A7r5 cells

  35. Characteristics of the CICR mode Ca2+ dependence: EC50 = 700 nM Hill = 1.9 Mg2+ inhibition: EC50= 0.6 mM ATP stimulation: EC50= 320 µM

  36. Effects of CaM, CaM1 and CaM1234 40 30 20 control 10 0 0 10 20 Time (min) control CaM1 CaM CaM1234

  37. Calmodulin Calmodulin1234 Long CaBP1 Short CaBP1 NCS NCS - - 1/Frequenin 1/Frequenin NCS - 1/FrequeninE120Q Calmodulin Calmodulin Calmodulin1 Calmodulin1 Calmodulin1234 Calmodulin1234 Long CaBP1 Long CaBP1 Short CaBP1 Short CaBP1 NCS NCS - - 1/Frequenin 1/Frequenin NCS NCS - - 1/FrequeninE120Q 1/FrequeninE120Q

  38. Preincubation with a CaM-binding peptide inhibits CICR control RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

  39. CaM but not CaM1234 can restore CICR Preincubation with RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

  40. CaM but not CaM1234 can restore CICR CaM1234 Preincubation with RyR CaM-BS (peptide aa 3614-3643) CaM Ca2+ (3 µM)

  41. Summary • Novel CICR mechanism in A7r5 cells IC50: 700 nM Activated by ATP Inhibited by MgCl2 • CICR mechanism is regulated by CaM

  42. PIP2 PLC PLC IP3 224 576 Gα 2749 2275

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