The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor.
We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.
Yamada
M
M
Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan.
Miyawaki
A
A
Saito
K
K
Nakajima
T
T
Yamamoto-Hino
M
M
Ryo
Y
Y
Furuichi
T
T
Mikoshiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins