The role of protein interactions in IP 3 R function

1. The role of protein interactions in IP3R function Effects of FKBP12 and Calmodulin.

2. Agonists IP3 Ca2+ Protein Interactions Cytosolic Calmodulin, FKBP12, CaBP1, IRAG Cytoskeletal proteins Talin, vinculin, -actin, ankyrin Plasma membrane TRP, Homer, G Luminal Chromogranin A, calnexin Modulators Phosphorylations Redox status (SH-reagents) ATP pH Mg2+ IP3R Monomer: Cytosolic: 2400 aa Luminal: 104 aa Isoforms: I, II, III homo/ hetero-tetramers

3. + FKBP12 FKBP12 and FKBP12.6 • 12-kDa FK506-binding protein (108 aa) high-affinity receptor for FK506 •  immunophilin protein family • peptidylprolyl isomerase activity cis/trans isomerization of XP residues • peptidomimicry : FK506 mimics the twisted-amide transition state of a peptidylprolyl bond

4. TEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIP IP3-binding modulation channel Interaction of FKBPs with intracellular Ca2+-release channels FKBP RyR1 IP3R1 high affinity RyR1-(FKBP12)4 high affinity ? Interaction Function stabilization cooperativity coupled gating stabilization scaffold for calcineurin Homologous to IP3R site Y2H: center of modulatory domain Binding site

5. Primary amino acid sequence of the FKBP12-binding site in RyR and IP3R isoforms

6. DT40 triple knock-out FK506-induced Ca2+ rise in permeabilized cells SH-SY5Y neuroblastoma EC50 ~ 50 µM

7. Effect of FK506 on SERCA IC50~20 µM FK506 inhibited Ca2+-uptake activity of the SERCA pump

8. NO EFFECT + FKBP12 + FKBP12 Effect of FK506 on intracellular Ca2+-release channels ? IP3R RyR neuroblastoma cells skeletal muscle cells FK506 enhanced Ca2+ release through the RyRs but not through the IP3Rs

9. Specific binding ~42 % Retention of RyR1 from TC by GST-FKBP12 affinity assay

10. Retention of other intracellular Ca2+-release channels by GST-FKBP12 affinity assay

11. Co-immunoprecipitation of FKBP12 with intracellular Ca2+-release channels RyR1 IP3R1

12. FKBP12 Mutational analysis of FKBP12-binding site in RyR3 IP3R1 VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP3R2 ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP3R3 (human) ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV (as in RyR1 or RyR3) RyR1 QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2 HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3 QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL RyR3/V2322L QTGKGEAIRIRSILRSLLPTEDLVGIISIPLKLPSL (as in IP3R1) RyR3/V2322I QTGKGEAIRIRSILRSLIPTEDLVGIISIPLKLPSL (as in RyR2) RyR3/V2322D QTGKGEAIRIRSILRSLDPTEDLVGIISIPLKLPSL RyR3/IP3R1 QTGKGEAIRIRSICNSLLPLDDIVGIISIPLKLPSL (as in IP3R1) IP3R1 VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP3R2 ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP3R3 (human) ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV (as in RyR1 or RyR3) RyR1 QAGKGEALRIRAILRSLVPLDDLVGIISLPLQIPTG RyR2 HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3 QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL RyR3/V2322L QTGKGEAIRIRSILRSLLPTEDLVGIISIPLKLPSL (as in IP3R1) RyR3/V2322I QTGKGEAIRIRSILRSLIPTEDLVGIISIPLKLPSL (as in RyR2) RyR3/V2322D QTGKGEAIRIRSILRSLDPTEDLVGIISIPLKLPSL (negative charge) RyR3/IP3R1 QTGKGEAIRIRSICNSLLPLDDIVGIISIPLKLPSL (as in IP3R1)

13. Mutational analysis of FKBP12-binding site in RyR3

14. RyRs IP3Rs mutants PSIPRED

15. DIFFERENT FKBP-BINDING PROPERTIES OF RyRS AND IP3RS • FKBP12-binding site of the IP3R is much less efficient than the homologous site in the RyR. • Higher-order structure may be important. • RyRs: stable interaction regulatory subunit • IP3Rs: weak or transient interaction chaperone function? effects on kinetics?

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

17. Cytosol R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD CaM ?? R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA 31 25 13 18 Endoplasmic reticulum Calmodulin binding sites on IP3R1 Ca2+/CaM

18. 581 Ca2+ N N IP3 C Recombinant ligand-binding domain of IP3R1 (LBS-1) 1 581 Lbs-1 W226 581 Lbs-1 1-225

19. 581 1 Lbs-1His W226 581 Lbs-1 1-225His EC50= 1.7µM [3H]IP3 binding (%) 0.3 B/F 0.2 0.1 control 5 µM Ca2+ Ca2+ CaM Ca2+ CaM1234 10 µM apoCaM 10 µM CaM1234 0.0 0 5 10 Bound (nM)

20. 1 581 Lbs-1 W226 581 Lbs-1 1-225 309 159 1 Cyt1 Cyt2 50 M free Ca2+ 1 mM EGTA Localisation of a Calmodulin-Binding Site GST-fusion protein pull down of CaM1234 GST GST-Cyt1 GST-Cyt2

21. 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 1.0 2+ 200 µM free Ca 0.8 1 mM EGTA 0.6 0.4 0.2 0.0 -0.2 A B C D E F CaM Detailed localisation using peptides C B/Bo - 1 EC50  1-1.5 µM

22. Effect of CaM1234 on Ca2+ release in A7r5 cellsCaM1234 could not replace CaM for the inhibition at high [Ca2+ ]

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

24. The N-terminal CaM-binding site is in a region involved in the interaction with the channel domain

25. Conclusions: N-terminal CaM-binding site • Involved in inhibition of IP3-bindingCaM effect is Ca2+ independent CaM1234 is equally effective Low affinity • Not involved in inhibition of IP3-induced Ca2+ releaseCa2+ /CaM required CaM1234 is not effective Low affinity • The N-terminal site may be involved in an indirect regulation via CaM or CaM-like proteins.

26. IP3-team: Leuven, Belgium Nael Nadif Kasri Ilse Sienaert Sara Vanlingen Geert Bultynck Patrick De smet Elke Vermassen Karolina Szlufcik Kristel Van Acker Ludwig Missiaen Jan Parys Geert Callewaert