1. Mapping of Calmodulin Binding Sites on the IP 3 R1 N. Nadif Kasri, I. Sienaert, 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 CaM- binding sites on the IP 3 R1 in search for their role in the functioning of the intact IP 3 R1. We therefore used recombinant CaM and CaM1234, a Ca 2+ -insensitive mutant. Conclusion In this study we show the presence of two complex CaM-binding sites on the IP 3 R1. 1) In the N-terminal part we show the presence of a discontinuous Ca 2+ - independent CaM-binding site (aa P49-N81and aa E106-S128) that might be responsible for the inhibition of IP 3 binding. 2) In the regulatory domain we show that CaM-binding consists of overlapping Ca 2+ -independent and Ca 2+ -dependent CaM-binding sequences, with the Ca 2+ - independent sequence (aa L1554-R1585) located N-terminal of the Ca 2+ - dependent CaM-binding sequence (aa 1564-R1585). 3) It is conceivable that simultaneous binding to multiple CaM-binding sites is required for proper function on the intact IP 3 R1. Figure 2. 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) CaM- binding curve of peptide E to dCaM in the presence or absence of Ca 2+ ; 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 IP 3 R1 shows that two amino acid stretches, peptide B and E were able to bind to dansyl-CaM in a Ca 2+ -independent way. Same conclusions could be drawn from the band-shift experiments(data not shown). 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 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 4.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 IP 3 in the absence or presence of 10 µM CaM or CaM1234 at different free [Ca 2+ ] Ca 2+ /CaM is required for inhibitory effects on IP 3 IICR while CaM1234 does not inhibit IICR in the same conditions as Ca 2+ /CaM. 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 Figure 5. Overview of the CaM binding sites on the IP 3 R. N-terminal: a discontinuous Ca 2+ -independent CaM binding site (P49-N81, E106-E128). Amino acids P49-N81 are highly conserved among the three isoforms. Regulatory domain: Complex site consisting of a high affinity Ca 2+ -dependent CaM-binding site and a low affinity Ca 2+ -independent CaM- binding site. No conserved amino acids between type 1 and 3 IP 3 R. Table 1. Different methods were used to assay both the Ca 2+ -dependent and Ca 2+ -independent CaM and/or CaM1234 interaction of IP 3 R1 fusion proteins GST-Cyt1 (aa 1-159) or GST-Cyt11 (aa 1499-1649). Figure 3. Detailed analysis of CaM-binding properties of the 1499-1649 amino acid region in the regulatory domain 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 Increase in dCaM fluorescence emission at = 500 nm upon addition of 1  M peptide (G-J) in the presence or absence of Ca 2+. (C) CaM-binding curve of peptide H to dCaM in the presence or absence of Ca 2+ ; data in the presence of 1 mM EGTA are fitted to a binding curve with K d  0.35 µM; in the presence of 50  M free Ca 2+ the estimated K d value was  0.5 µM. (D) The Ca 2+ -dependent CaM-binding curve of peptide I to dCaM, data in the presence of 50  M free Ca 2+ ; data are fitted to a binding curve with K d  0.125 µM. (E) The Ca 2+ -dependent CaM-binding curve of peptide J to dCaM, data in the presence of 50  M free Ca 2+ ; data are fitted to a binding curve with K d  0.075 µM. The CaM binding site of the regulatory domain of the IP 3 R1 contains both, a high affinity Ca 2+ -dependent and a low affinity (K d 0.5 µM) Ca 2+ -independent CaM binding sequence. Same conclusions could be drawn from the band-shift experiments(data not shown). ldsqvnnlflkshnivqkta ldsqvnnlflkshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaarrdsvlaasrd 14991649 72% 1-5-8-14 60 % IQ 76 % IQ 1-5-8-14 G H I J Fraction dCaM bound [Peptide H] (µM) 1 mM EGTA 50 µM free Ca 2+ K d ~ 0.35-0.5 µM Fraction dCaM bound [Peptide I] (µM) K d ~ 0.125 µM Fraction dCaM bound [Peptide J] (µM) K d ~ 0.075 µM A ED C B Different assays show the Ca 2+ -independent interaction of CaM with both GST-Cyt1 and GST-Cyt11