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

(Clapham, 1995) Intracellular Ca 2+ homeostasis

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

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 IP 3 R localization  talin, ankyrin, 4.1N, … - proteins regulated by the IP 3 R  trp-channels Protein interactions to consider: ALL THESE INTERACTIONS MAY BE MODULATED, DEPENDING ON THE PHYSIOLOGICAL SITUATION

1) Inter- and intramolecular interactions with the N-terminal region of the IP 3 R (aa 1-225) 2) Dynamics concerning the intracellular localization of the IP 3 R

1) Inter- and intramolecular interactions with the N-terminal region of the IP 3 R (aa 1-225)

Why the N-terminal 225 amino acids of the IP 3 R1? Aa act as a suppressor of IP 3 binding activity (Yoshikawa et al., 1999). Homer proposed to couple to aa (Tu et al., 1998). CaM interacts with aa (Adkins et al., 2000).

What is the relation between CaM and IP 3 R ?

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) Effects of CaM on Ca 2+ release: Effects of CaM on IP 3 binding: CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs-3 What is the relation between CaM and IP 3 R ?

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) Effects of CaM on Ca 2+ release: Effects of CaM on IP 3 binding: CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs-3 What is the relation between CaM and IP 3 R ?

A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) CaM on Ca 2+ release: INHIBITORY, Ca 2+ -DEPENDENT CaM on IP 3 binding: INHIBITORY, Ca 2+ -INDEPENDENT CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs-3 What is the relation between CaM and IP 3 R ?

Ca 2+ CaM1234 CaM1234 Ca 2+ CaM ApoCaM Ca 2+ Control [ 3 H]IP 3 binding (%) [ 3 H]IP 3 binding (%) Lbs-1 Lbs-1  HIS Effects of CaM on IP 3 binding CaM1234 (μM)

Ca 2+ CaM1234 CaM1234 Ca 2+ CaM ApoCaM Ca 2+ Control [ 3 H]IP 3 binding (%) [ 3 H]IP 3 binding (%) Lbs-1 Lbs-1  HIS Effects of CaM on IP 3 binding CaM1234 (μM) IC 50 ~ 2 μM

Cyt1 Cyt2 Lbs-1 Lbs-1  HIS +GST Ca 2+ EGTA Localisation of the N-terminal CaM-binding site CaM1234

Cyt1 Cyt2 Lbs-1 Lbs-1  HIS +GST A B C E D F % IQ 76% IQ 53% IQ ABCDEF µM free Ca 2+ 1 mM EGTA 309 Band-shift experiments on non-denaturing gels Interaction with dansyl-CaM Localisation of the N-terminal CaM-binding site

Cyt1 Cyt2 Lbs-1 Lbs-1  HIS +GST A B C E D F % IQ 76% IQ 53% IQ ABCDEF µM Ca 2+ 1mM EGTA 309 Intensity loss (1-B/B o ) K d 0.1 μM K d 1 μM Localisation of the N-terminal CaM-binding site

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

CaBP1 GST GST GST Binding of (activatory?) CaBP1 to the same site? A B C E D F 1 CaM 159

CaBP1 GST GST GST Binding of (activatory?) CaBP1 to the same site A B C E D F 1 CaM 159 CaBP 1

CaBP 1/11/21/41/51/61/81/3 CaBP Ratio of CaBP: peptide B Band intensity Peptide B: CaBP Ca 2+ EGTA 1/10 Ca 2+ EGTA

GST GST-Homer1a The 1  225 suppressor domain interacts with Homer1a 1  225

N-terminal fragment of the IP 3 R CaM CaBP1 Homer CC

Expression of IP 3 R1  in IP 3 R triple knockout cells ko wt  IP 3 R1 [ 3 H]IP 3 binding (fmol/U IP 3 R)

IP 3 R1  is not functionally active ko wt  IP 3 R1

IP 3 binding to IP 3 R1  is not sensitive to thimerosal ko wt  IP 3 R1 [ 3 H]IP 3 binding (fmol/U IP 3 R) +BME +Thim. wt IP 3 R1 IP 3 R1  1-225

1  225 GST GST- 226  604 GST GST- 226  604 Can 1  225 (suppressor) interact with 226  604 (IP 3 binding domain) ?

[ 3 H]IP 3 binding (fmol/U IP 3 R) +BME +Thim 1  225 GST GST- 226  604 GST GST- 226  604 wt IP 3 R1 IP 3 R1  Can 1  225 (suppressor) interact with 226  604 (IP 3 binding domain) ?

[ 3 H]IP 3 binding (fmol/U IP 3 R) +BME +Thim 1  225 BME Thim GST GST- 226  604 GST GST- 226  604 wt IP 3 R1 IP 3 R1  The interaction between 1  225 & 226  604 is sensitive to thimerosal

1  225 GST Idem + IP 3 GST-226  604 Idem + AdA Idem + CaM Idem + CaBP1 The 1  225 suppressor domain interacts with the IP 3 -binding core: role of Ca 2+, calmodulin and CaBP1

1   604 CaM CaBP1 Homer Ca 2+ Thim. IP 3 Ca 2+ ? N C Interactions between N- and C-termini of IP 3 R1 ?

N C Flag 3 c-myc GST 1  225 GST 226  604 GST 1  604 GST = affinity matrix

N C GST 1  225 GST 226  604 GST 1  604 GST Flag 3 c-myc Flag ~60 kDa Flag ~54 kDa Flag ~30 kDa Flag3  ~28 kDa Flag ~41 kDa Flag4  ~26 kDa TM1-6 TM1-4 TM(5)-6

M SOL GST GST 1  225 GST GST 1  225GST 1  604 GST 226  604 GST 1  604 GST 226  604 Flag1 BME Thim The interactions between N- and C-termini of the IP 3 R1 are not thimerosal sensitive

M GST GST 1  225 GST GST 1  225 GST 1  604 GST 226  604 GST 1  604 GST 226  604 SOL Flag1 The interactions between N- and C-termini of the IP 3 R1 are Ca 2+ sensitive EDTA Ca 2+ 1mM 25  M

FLAG2 FLAG3 FLAG4 FLAG3  FLAG4 

Multiple interactions between N- and C-termini of IP 3 R1 1   604 CaM CaBP1 Homer Ca 2+ Thim. IP 3 Ca 2+ N C

CaM/CaBP1

CC C C

CC C C Interactions between monomers ? Oligom.

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

1) Inter- and intramolecular interactions with the N-terminal region of the IP 3 R (aa 1-225) 2) Dynamics concerning the intracellular localization of the IP 3 R

2) Dynamics concerning the intracellular localization of the IP 3 R

Localization of IP 3 R1 and IP 3 R3 in A7r5 smooth-muscle cells IP 3 R1IP 3 R3

Redistribution of IP 3 R1 after prolonged stimulation Resting cells+ AVP AVP > 1h Imipramine IP 3 -ester Thapsigargin CPA [Ca 2+ ] cyt

Redistribution during AVP treatment: time dependence Cytoplasmic IP 3 R1 localization Perinuclear IP 3 R1 localization AVP treatment Perinuclear localization after AVP wash-out

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

SERCA localization and redistribution Resting cells +AVP

Role of PKC perinuclear IP 3 R1 cytoplasmic IP 3 R1 Percentages of cells with: Cells (%) Control AVP AVP+Staur. TG+Staur. AVP+BIM OAG

Role of cytoskeleton perinuclear IP 3 R1 Percentages of cells with: Cells (%) Control AVP AVP+Nocod. AVP+Taxol Nocod. Taxol Resting cells +AVP Microtubular network or +OAG

Role of vesicle trafficking Brefeldin A (2 μg/ml, 2h)  complete IP 3 R1 redistribution (29 ± 2 % cells with perinuclear localization) Cooling (15°C, 2h)  inhibition of AVP-induced IP 3 R1 redistribution (76 ± 3 % cells with perinuclear localization)

Conclusions (part 1)  The first 225 amino acids of IP 3 R1 contain important regulatory sites, including for Ca 2+ -binding proteins as calmodulin and CaBP1, for homer as well as potentially for thimerosal.  Interactions occur between the N-terminal suppressor domain, the IP 3 -binding domain and the channel domain. These interactions are under control of Ca 2+, calmodulin/CaBP1 and thimerosal.  The role of additional interactions between monomers or with other proteins and the role of phosphorylation in this process will be investigated.

Conclusions (part 2)  Various IP 3 R isoforms can have a different intra- cellular distribution.  The localization of the IP 3 Rs is dynamic and change with the physiological status of the cell.  This redistribution is dependent on PKC activation and on the microtubular network and is probably mediated by vesicle trafficking. The results indicate a dynamic regulation of the structure and localization of the IP 3 R, which we believe to be functionally relevant. Future work will focus on the understanding of the physiological importance of the various interactions.

Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN - Jan B. PARYS IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Patrick DE SMET Rafael A. FISSORE Nael NADIF KASRI Joelle NSIMIRE CHABWINE Ilse SIENAERT Karolina SZLUFCIK Kristel VAN ACKER Sara VANLINGEN Marijke VAN MOORHEM Esther VENMANS Leen VERBERT Elke VERMASSEN

Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN - Jan B. PARYS IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Patrick DE SMET Rafael A. FISSORE Nael NADIF KASRI Joelle NSIMIRE CHABWINE Ilse SIENAERT Karolina SZLUFCIK Kristel VAN ACKER Sara VANLINGEN Marijke VAN MOORHEM Esther VENMANS Leen VERBERT Elke VERMASSEN

Geert CALLEWAERT - Humbert DE SMEDT - Ludwig MISSIAEN - Jan B. PARYS IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Patrick DE SMET Rafael A. FISSORE Nael NADIF KASRI Joelle NSIMIRE CHABWINE Ilse SIENAERT Karolina SZLUFCIK Kristel VAN ACKER Sara VANLINGEN Marijke VAN MOORHEM Esther VENMANS Leen VERBERT Elke VERMASSEN

IP 3 R1 IP 3 R2 IP 3 R3 RyR1 RyR2 RyR3 Intraluminal proteins Calreticulin Calsequestrin Chromogranins A and B Annexin VI ER membrane proteins Triadin Junctin Kinase anchor proteins Plasma membrane proteins Trp’s DHPR G-proteins Cytosolic proteins CaM CaBPs FKBP’s Sorcin S100 IRAG Cytoskeletal proteins Homer Ankyrin Talin Vinculin Alpha-actin Myosin II

FKBP12- binding site IP 3 Ca 2+ FKBP12 NH 2 COOH SI SII POREPORE SIII P P ATP IP 3 R1VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP 3 R2ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP 3 R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV RyR1QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL Cytosol Lumen of the store

Effects on RyR (Rodney et al., 2001) ApoCaM: Ca 2+ /CaM: ACTIVATOR OF RYR1INHIBITOR OF RYR1 Ca 2+

IP 3 Ca 2+ CaM NH 2 COOH SI SII CaM POREPORE SIII Cytosol Lumen of the store P P ATP CaM- binding sites High-affinity Ca 2+ -dependent Types 1 and 2 Low-affinity Ca 2+ -dependent Not in neuronal IP 3 R1 (SII + )

IP 3 R A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) Effects of CaM on Ca 2+ release: Effects of CaM on IP 3 binding: CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs-3

Discontinuous (aa and ). Low affinity. Ca 2+ independent. Involved in inhibition of IP 3 binding. Not involved in inhibition of IP 3 -induced Ca 2+ release. Other possible functions: Role in conformation of the IP 3 R ? Tethering of CaM ? Binding site for CaM-like proteins ?  CaBP-1? Protective effect ?  Proteolysis? Oxidative stress? N-terminal CaM-binding site of the IP 3 R