title: Localization and function of a calmodulin-apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor.
abstract: 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 d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2 microM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49-81 and 106-128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.
authors.affiliation: Laboratorium voor Fysiologie, Katholieke Universiteit Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium. Ile.Sienaert@med.kuleuven.ac.be
authors.forename: Ilse
authors.initials: I
authors.lastname: Sienaert
authors.forename: Nael
authors.initials: N
authors.lastname: Nadif Kasri
authors.forename: Sara
authors.initials: S
authors.lastname: Vanlingen
authors.forename: Jan B
authors.initials: JB
authors.lastname: Parys
authors.forename: Geert
authors.initials: G
authors.lastname: Callewaert
authors.forename: Ludwig
authors.initials: L
authors.lastname: Missiaen
authors.forename: Humbert
authors.initials: H
authors.lastname: de Smedt
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins; D002147:Calmodulin / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008840:Microfilament Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: C112986:AIF1 protein, human; D001059:Apoproteins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008024:Ligands; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins