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Modulation of Inositol Trisphosphate Receptor Interactions and Their Role in Calcium Homeostasis

This paper explores the intricate structure and function of inositol trisphosphate receptors (IP3Rs) in regulating intracellular calcium dynamics. It highlights how IP3Rs, composed of multiple subunits, interact with various proteins, including regulatory proteins, kinases, and anchoring proteins, which modulate their activity and localization. The involvement of the N-terminal region in suppressing IP3 binding is discussed, along with the effects of calmodulin (CaM) on IP3R functionality. The findings provide insights into the physiological implications of IP3R modulation in cellular signaling processes.

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Modulation of Inositol Trisphosphate Receptor Interactions and Their Role in Calcium Homeostasis

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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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