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Jan B. Parys K.U.Leuven

Modulation de la structure, localisation et fonction des récepteurs pour l’inositol trisphosphate: le rôle des interactions protéiques. Jan B. Parys K.U.Leuven. Orsay, 7 février 2003. Intracellular Ca 2+ homeostasis. (Clapham, 1995). Structure of the IP 3 R – the simplified view. Ca 2+.

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Jan B. Parys K.U.Leuven

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  1. Modulation de la structure, localisation et fonction des récepteurs pourl’inositol trisphosphate: le rôle des interactions protéiques. Jan B. Parys K.U.Leuven Orsay, 7 février 2003

  2. Intracellular Ca2+ homeostasis (Clapham, 1995)

  3. Structure of the IP3R – the simplified view Ca2+ Cytoplasm N C Lumen of ER Lumen of ER • 4 subunits = 1 functional IP3R (homo- or heterotetrameric) • each subunit can be divided in three parts: 1. an IP3-binding domain (about 600 aa) • 2. a coupling domain (about 1500 aa) • 3. a channel domain (about 600 aa)

  4. Protein interactions to consider: • Interactions of the monomers to form a tetramer • Interactions of monomer/tetramer with associated proteins - regulatory proteins  CaM - activatory proteins  CaBP1 - kinases / phosphatases  PKA, PP1 - anchor proteins  homer - determinants of the IP3R localization  talin, ankyrin, 4.1N, … - proteins regulated by the IP3R  trp-channels ALL THESE INTERACTIONS MAY BE MODULATED, DEPENDING ON THE PHYSIOLOGICAL SITUATION

  5. Inter- and intramolecular interactions with the N-terminal region of the IP3R(aa 1-225) • Dynamics concerning the intracellular localization of the IP3R

  6. Inter- and intramolecular interactions with the N-terminal region of the IP3R(aa 1-225)

  7. Why the N-terminal 225 amino acids of the IP3R1? • Aa 1-225 act as a suppressor of IP3 binding activity (Yoshikawa et al., 1999). • Homer proposed to couple to aa 48-55 (Tu et al., 1998). • CaM interacts with aa 1-159 (Adkins et al., 2000).

  8. What istherelationbetweenCaMandIP3R?

  9. Effects of CaM on Ca2+ release: Effects of CaM on IP3 binding: What istherelationbetweenCaMandIP3R? 200 μM Ca2++10 μM CaM +20 μM CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) Lbs-1 Lbs-2 Lbs-3 CaM (μM) Sf9 (Cardy & Taylor, 1998) Lbs-domains (Vanlingen et al., 2000)

  10. Effects of CaM on Ca2+ release: Effects of CaM on IP3 binding: What istherelationbetweenCaMandIP3R? 200 μM Ca2++10 μM CaM +20 μM CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) Lbs-1 Lbs-2 Lbs-3 CaM (μM) Sf9 (Cardy & Taylor, 1998) Lbs-domains (Vanlingen et al., 2000)

  11. CaM on Ca2+ release: INHIBITORY, Ca2+-DEPENDENT CaM on IP3 binding: INHIBITORY, Ca2+-INDEPENDENT What istherelationbetweenCaMandIP3R? 200 μM Ca2++10 μM CaM +20 μM CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) Lbs-1 Lbs-2 Lbs-3 CaM (μM) Sf9 (Cardy & Taylor, 1998) Lbs-domains (Vanlingen et al., 2000)

  12. 1 581 Lbs-1 226 581 Lbs-1 1-225 Effects of CaMon IP3 binding +HIS +HIS 100 80 60 [3H]IP3 binding (%) [3H]IP3 binding (%) 40 20 0 0.1 1 10 CaM1234 (μM) Ca2+ Control ApoCaM Ca2+ CaM CaM1234 Ca2+ CaM1234

  13. 1 581 Lbs-1 226 581 Lbs-1 1-225 Effects of CaMon IP3 binding +HIS +HIS 100 80 60 [3H]IP3 binding (%) [3H]IP3 binding (%) IC50~ 2 μM 40 20 0 0.1 1 10 CaM1234 (μM) Ca2+ Control ApoCaM Ca2+ CaM CaM1234 Ca2+ CaM1234

  14. 1 581 Lbs-1 226 581 Lbs-1 1-225 Cyt1 Cyt2 Localisation of the N-terminal CaM-binding site +HIS +HIS +GST 1 159 154 309 Ca2+ CaM1234 EGTA

  15. 1 581 Lbs-1 226 581 Lbs-1 1-225 Cyt1 Cyt2 Localisation of the N-terminal CaM-binding site +HIS +HIS +GST 1 159 154 309 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D B E F 1.0 2+ 200 µM free Ca 0.8 • Band-shift experiments on non-denaturing gels • Interaction with dansyl-CaM 1 mM EGTA 0.6 0.4 0.2 0.0 -0.2 A B C D E F

  16. 1 581 Lbs-1 226 581 Lbs-1 1-225 Cyt1 Cyt2 Localisation of the N-terminal CaM-binding site +HIS +HIS +GST 1 159 154 309 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D B E F 1.0 200 µM Ca2+1mM EGTA Kd 0.1 μM Kd 1 μM 0.8 0.6 0.4 Intensity loss (1-B/Bo) 0.2 0.0 -0.2 A B C D E F

  17. Inhibitory Activatory ?(Yang et al., 2002) CaM or CaBP1 ?

  18. CaM CaM 1 159 C A D E B F Binding of (activatory?) CaBP1to the same site? GST 1-604 GST 1-225 GST 226-604 GST CaBP1

  19. Binding of (activatory?) CaBP1to the same site GST 1-604 GST 1-225 GST 226-604 GST CaBP1 CaM CaM 1 159 C A D E B F CaBP1

  20. Ratio of CaBP: peptide B CaBP CaBP 1/1 1/2 1/3 1/4 1/5 1/6 1/8 1/10 Ca2+ EGTA 1.0 Ca2+ EGTA 0.5 Band intensity 0.0 0 2 4 6 8 10 Peptide B: CaBP

  21. 1225 The 1225 suppressor domain interacts withHomer1a GST-Homer1a GST

  22. Homer CaM CaBP1 N-terminal fragment of the IP3R CC

  23. Expression of IP3R11-225 inIP3R triple knockout cells [3H]IP3 binding (fmol/U IP3R) ko wt 1-225 IP3R1

  24. IP3R11-225 is not functionally active ko wt 1-225 IP3R1

  25. IP3 binding to IP3R11-225 is not sensitive to thimerosal ko wt 1-225 IP3R1 wt IP3R1 IP3R11-225 [3H]IP3 binding (fmol/U IP3R) [3H]IP3 binding (fmol/U IP3R) +BME +Thim. +BME +Thim.

  26. Can 1225(suppressor)interact with226604(IP3 binding domain) ? GST-226 604 GST-226 604 GST GST 1225

  27. Can 1225(suppressor)interact with226604(IP3 binding domain) ? GST-226 604 GST-226 604 GST GST 1225 wt IP3R1 IP3R11-225 [3H]IP3 binding (fmol/U IP3R) [3H]IP3 binding (fmol/U IP3R) +BME +Thim +BME +Thim

  28. The interactionbetween1225 &226604is sensitive tothimerosal BME Thim 1225 GST-226 604 GST-226 604 GST GST wt IP3R1 IP3R11-225 [3H]IP3 binding (fmol/U IP3R) [3H]IP3 binding (fmol/U IP3R) +BME +Thim +BME +Thim

  29. The 1225 suppressor domain interacts withthe IP3-binding core:role of Ca2+, calmodulin and CaBP1 Idem + CaBP1 GST-226 604 Idem + CaM Idem + AdA Idem + IP3 GST 1225

  30. 226604 1225 • IP3 • Ca2+ • CaM • CaBP1 • Homer • Ca2+ • Thim. ? Interactions between N- and C-termini of IP3R1 ? N C

  31. GST GST 1225 GST 226604 GST 1604 Flag3 c-myc = affinity matrix N C

  32. GST N GST 1225 GST 226604 C GST 1604 Flag3 c-myc Flag1 2170-2749~60 kDa Flag2 2253-2749~54 kDa Flag3 2170-2427~30 kDa Flag3 2170-2407~28 kDa Flag4 2407-2749~41 kDa Flag4 2560-2749~26 kDa TM1-6 TM1-4 TM(5)-6

  33. GST 226604 GST 226604 GST 1225 GST 1604 GST 1225 GST 1604 GST GST M SOL Flag1 BME Thim The interactions between N- and C-termini of the IP3R1 are not thimerosal sensitive

  34. GST 226604 GST 226604 GST 1225 GST 1225 GST 1604 GST 1604 SOL GST GST M The interactions between N- and C-termini of the IP3R1 are Ca2+ sensitive Flag1 EDTA Ca2+ 1mM 25 M

  35. GST 1225 GST 226604 FLAG2 FLAG3 FLAG4 D FLAG3 D FLAG4

  36. 226604 1225 • CaM • CaBP1 • Homer • Ca2+ • Thim. • Ca2+ Multiple interactions between N- and C-termini of IP3R1 • IP3 • Ca2+ N C

  37. CaM/CaBP1

  38. CaM/CaBP1 C C C C

  39. CaM/CaBP1 Oligom. C C C C Oligom. • Interactions between monomers ?

  40. IRBIT Homer CaM/CaBP1 C C C PP1 4.1N C Ankyrin • Interactions between monomers ? • Interactions with other proteins ? Regulation of localization ? Regulation of phosphorylation ?

  41. Inter- and intramolecular interactions with the N-terminal region of the IP3R(aa 1-225) • Dynamics concerning the intracellular localization of the IP3R

  42. Dynamics concerning the intracellular localization of the IP3R

  43. Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells IP3R1 IP3R3

  44. AVP > 1h ImipramineIP3-ester ThapsigarginCPA [Ca2+]cyt Redistribution of IP3R1 after prolonged stimulation Resting cells + AVP

  45. Perinuclear localizationafter AVP wash-out Redistribution during AVP treatment:time dependence CytoplasmicIP3R1localization Perinuclear IP3R1localization AVP treatment

  46. Structure of the endoplasmic reticulum ER-targeted EYFP PDI Resting cells +AVP

  47. SERCA localization and redistribution Resting cells +AVP

  48. Percentages of cells with: perinuclear IP3R1 cytoplasmic IP3R1 AVP AVP OAG OAG Control Control TG+Staur. TG+Staur. AVP+Staur. AVP+Staur. AVP+BIM AVP+BIM Role of PKC 100 80 60 Cells (%) 40 20 0

  49. Resting cells Percentages of cells with: perinuclear IP3R1 +AVP AVP Taxol Nocod. Control AVP+Taxol AVP+Nocod. Microtubular network Role of cytoskeleton 80 70 60 50 Cells (%) 40 30 20 or +OAG 10 0

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