Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms.
We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP(3)R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP(3)-binding domain and bound IP(3) and adenophostin A with high affinity. Ca(2+) and calmodulin were previously found to maximally inhibit IP(3) binding to lbs-1 by 42+/-6 and 43+/-6% respectively, and with an IC(50) of approx. 200 nM and 3 microM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca(2+) inhibited IP(3) binding to lbs-3 with an IC(50) of approx. 700 nM (37+/-4% inhibition at 5 microM Ca(2+)), while IP(3) binding to lbs-2 was not affected by increasing [Ca(2+)] from 100 nM to 25 microM. Calmodulin (10 microM) inhibited IP(3) binding to lbs-3 by 37+/-4%, while IP(3) binding to lbs-2 was inhibited by only 11+/-2%. The inhibition of IP(3) binding to lbs-3 by calmodulin was dose-dependent (IC(50) approximately 2 microM). We conclude that the IP(3)-binding domains of the various IP(3)R isoforms differ in binding characteristics for IP(3) and adenophostin A, and are differentially modulated by Ca(2+) and calmodulin, suggesting that the various IP(3)R isoforms can have different intracellular functions.
Vanlingen
S
S
Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium. Sara.Vanlingen@med.kuleven.ac.be
Sipma
H
H
De Smet
P
P
Callewaert
G
G
Missiaen
L
L
De Smedt
H
H
Parys
J B
JB
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006224:Cricetinae; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015220:Calcium Channels; D002147:Calmodulin; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium