1. Mapping of Calmodulin binding sites on the IP3R1 N. Nadif Kasri; I. Sienaert, S. Vanlingen, J.B. Parys, G. Callewaert, L. Missiaen and H. De Smedt Laboratory of Physiology, K.U.Leuven Campus Gasthuisberg, 3000 Belgium Introduction Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the inositol 1,4,5-trisphosphate receptor (IP 3 R). CaM inhibits IP 3 binding in both the presence and absence of Ca 2+ and IP 3 - induced Ca 2+ release (IICR) in the presence of Ca 2+. Aim In this study we further charactarized the different calmodulin binding sites on the IP 3 R and tried to correlate them to functional observations of calmodulin on the IP 3 R.We therefore used recombinant CaM and CaM1234, a Ca 2+ insensitive mutant. Figure 2. Ca 2+ -dependent and independent binding of GST-Cyt1( N-terminal 159 amino acid region of IP 3 R1) to dansyl-CaM. The emission ( ex was 340 nm) spectra of 40 nM dansyl-CaM (dCaM) were measured in 2 ml buffer containing 100 µM EGTA. (A) Spectra were measured after sequential addition of 0.5 mM Ca 2+ and 100 nM fusion protein (GST- Cyt1). (B) and (C) Spectra of Ca 2+ -independent binding of fusion protein to dCaM were measured after sequential addition of 0.5 mM Ca 2+, 1 mM EGTA and 100 nM fusion protein GST-Cyt1 or pGST, respectively. (D) Ca 2+ - dependent and Ca 2+ -independent binding curve of GST-Cyt1 to dCaM. Fluorescence change was measured at 500 nm after excitation at 340 nm. F is the fluorescence observed after addition of fusion protein, and F 0 the initial fluorescence of dCaM alone. Curve fitting was done by Microcal TM Origin Version 6.0. (Northampton, MA) and yielded a K d value of ~ 0.4  M. GST-Cyt1 ( N-terminal 159 amino acid region of IP 3 R1) was able to interact with dansyl-CaM in both the presence and absence of Ca 2+. The K d yielded 400 nM Conclusion In this study we show the presence of two complex calmodulin binding on the IP3R1. 1) In the N-terminal part we show the presence of a discontinuous Ca 2+ independent calmodulin binding site (49-81, 106-128) that could be resposible for the inhibition of IP 3 binding. 2) In the regulatory domain we show that the known Ca 2+ /CaM binding site also contains a apocalmodulin binding site, showing that this CaM binding site is much more complex. 3) It is conceivable that multiple calmodulin binding sites act together on the IP3R. Figure 3. Detailed analysis of CaM-binding properties of the N- terminal 1-159 amino acid region of IP 3 R1. (A) Map showing positions of synthetic peptides (A-F) used for binding experiments relative to the N-terminal 159 amino acid region of IP 3 R1. Partial consensus domains for CaM binding are indicated. (B) The increase in dCaM fluorescence emission at = 500 nm upon addition of 1  M peptide(A-F) in the presence or absence of Ca 2+. Data for each peptide are shown as mean  S.D. (n = 3). (C) The Ca 2+ -dependent CaM-binding curve of peptide B to dCaM, data in the presence of 50  M free Ca 2+ were fitted to a bindingcurve with K d  0.1  M. (D) The Ca 2+ - dependent and -independent CaM-binding curve of peptide E to dCaM; data in the presence of 1 mM EGTA are fitted to a binding curve with K d  1  M; in the presence of 50  M free Ca 2+ the estimated K d value was  1.5  M. Further analysis of the N-terminal 159 aa of the IP3R1 show that two amino stretches, peptide B and E were able to bind to dansyl- CaM. A B C E D F 1 159 1-5-10 1-5-8-14 70% IQ (site1) 76% IQ 53% IQ A F/F 0 500 B Fraction dCaM bound [Peptide B] (  M) K d  0.1 µM C 1 mM EGTA 50 µM free Ca 2+ [Peptide E] (µM) K d  1 µM Fraction dCaM bound D Figure 4. The ability of Ca 2+ CaM and apoCaM to bind to IP 3 R1 peptides (A-F) (A) and (B) A representative 15 % nondenaturing gel of 3.5  M CaM in the presence of each of the IP 3 R1 peptides (A-F) (105  M) in 200  M free Ca 2+ (A) and in 1 mM EGTA (B) stained with Sypro Orange TM. CaM bound to the peptide diminishes the intensity of the CaM band. Lane 1, peptide A (S16-N48); lane 2, peptide B (P49-N81); lane 3, peptide C (Y66-K91); lane 4, peptide D (D97-L123); lane 5, peptide E (E106-S128); lane 6, peptide F (I121-L151) and lane7, CaM alone. (C) Densitometric analysis of the CaM (3.5  M) bands by ImageQuant 4.2 [25] in the presence of a 30–fold excess peptide for three independent experiments in the presence of either 200  M free Ca 2+ (black bars) or 1 mM EGTA (gray bars). The vertical axis denotes the intensity loss of the CaM bands after interaction with peptide as compared to CaM alone. Band shift experiments show the ability of Ca 2+ CaM and apoCaM to bind to IP 3 R1 peptides B and E. Peptide 1- B/Bo A B C D E F CaM AB C Figure 1 The effect of Ca 2+, CaM and CaM1234 on binding to Lbs-1His, and Lbs-1His  1-225 (A) 3  H  IP 3 binding to IP 3 -binding proteins purified on Ni-NTA (Qiagen) (Lbs-1His and Lbs-1His  1-225) was measured in the presence and absence of Ca 2+ (5 µM) and/or CaM/CaM1234 (10µM) and was expressed as the percentage in absence of these modulators (control). Binding was measured at pH 7.0 in the presence of 1 mM EGTA and 3.5 nM  3 H  IP 3. Data are expressed as the means  S.E. of at least three experiments, consisting of independent triplicates. (B) 3  H  IP 3 binding to purified Lbs-1His ( ) and Lbs-1His  1-225 ( ) in the presence of indicated concentrations of CaM1234 was expressed as a percentage of the binding measured in Ca 2+ -free buffer (1 mM EGTA, pH 7.0) without CaM1234. Curve fitting was done by Microcal TM Origin Version 6.0. (Northampton, MA) and yielded a EC 50 value of  1.7  M for Lbs-1His. (C) A scatchard analysis of IP 3 binding to Lbs-1His in the presence and absence of CaM is presented. Affinity purified Lbs-1His (1.5  g) was incubated with 3.5 nM [ 3 H]IP 3 at pH 7.0 and increasing concentrations of unlabeled IP 3 in the absence (  ) or presence (  ) of 10  M CaM. CaM and CaM1234 inhibit IP 3 binding in both the presence and absence of Ca 2+. Ca 2+ CaM1234 10 µM CaM1234 Ca 2+ CaM 10 µM apoCaM 5 µM Ca 2+ control [ 3 H]IP 3 binding (%) Lbs-1His Lbs-1  1-225His 1 581 W226581 0510 0.0 0.1 0.2 0.3 B/F Bound (nM ) EC 50 = 1.7µM A B C Results Figure 5. Detailed analysis of CaM-binding properties of regulatory domain 1499-1649 amino acid region of IP 3 R1. (A) Map showing positions of synthetic peptides (G-J) used for binding experiments relative to the 1499- 1649 amino acid region of IP 3 R1. Partial consensus domains for CaM binding are indicated. (B) The I ncrease in dCaM fluorescence emission at = 500 nm upon addition of 1  M peptide (A-F) in the presence or absence of Ca 2+. CaM binding site of the regulatory domain of the IP 3 R1 contains both, a Ca 2+ -independent and a Ca 2+ -independent CaM binding site. ldsqvnnlflkshnivqkta ldsqvnnlflkshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaarrdsvlaasrd 14991649 72% 1-5-8-14 60 % IQ 76 % IQ 1-5-8-14 G H I J Figure 6.The effect of CaM and CaM1234 on the IP 3 - induced Ca 2+ release in permeabilized A7r5 cells. The IP 3 induced Ca 2+ release in efflux medium containing 6 mM BAPTA was calculated as the difference between the Ca 2+ release in the presence and that in the absence of IP 3. Ca 2+ release was induced by 1 µM IP3 in the absence or presence of CaM or CaM1234 at different free [Ca 2+ ] Ca 2+ /CaM is required for inhibitory effects on IP3 induced Ca 2+ release (IICR), CaM1234 does not inhibit IICR. 13 18 31 25 Endoplasmic reticulum Cytosol CaM ?? R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca 2+ /CaM Fig.7 Overview of the CaM binding sites on the IP3R. N-terminal: a discontinuous Ca 2+ independent CaM binding site (49-81, 106-128). Amino acids 49-81 are highly conserved among the three isoforms. Regulatory domain: Complex site consisting of a high affinity Ca 2+ dependent site and a low affinity Ca 2+ independent site. No conserved amino acids between type 1 and 3 IP 3 R.