TY - JOUR
T1 - The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor
AU - Lin, Chi
AU - Widjaja, Johan
AU - Joseph, Suresh K.
PY - 2000/1/28
Y1 - 2000/1/28
N2 - A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP3R) isoform (type-I long, amino acids 1314-1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CAM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CAM in a Ca2+-dependent manner as measured by CAM- Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CAM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full- length cDNAs encoding type-I long, type-I short, and type-III IP3R isoforms were expressed in COS cells, and the Ca2+ sensitivity of [3H]IP3 binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca2+ inhibition (IC50 = 0.55 μM) than the type-I short (IC50 = 5.7 μM) or the type-III isoform (IC50 = 3 μM). In agreement with studies on the fusion proteins, the full-length type-I short bound more CAM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP3R. Although type-III IP3Rs did not bind directly to CAM-Sepharose, hetero-oligomers of type-I/III IP3Rs retained the ability to interact with CAM. We conclude that the deletion of the SII splice site in the type-I IP3R results in the differential regulation of the alternatively spliced isoforms by Ca2+, CAM, and protein kinase A.
AB - A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP3R) isoform (type-I long, amino acids 1314-1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693-1733 deleted) were expressed as glutathione S-transferase fusion proteins. These domains encompass a putative calmodulin (CAM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CAM in a Ca2+-dependent manner as measured by CAM- Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CAM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full- length cDNAs encoding type-I long, type-I short, and type-III IP3R isoforms were expressed in COS cells, and the Ca2+ sensitivity of [3H]IP3 binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca2+ inhibition (IC50 = 0.55 μM) than the type-I short (IC50 = 5.7 μM) or the type-III isoform (IC50 = 3 μM). In agreement with studies on the fusion proteins, the full-length type-I short bound more CAM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP3R. Although type-III IP3Rs did not bind directly to CAM-Sepharose, hetero-oligomers of type-I/III IP3Rs retained the ability to interact with CAM. We conclude that the deletion of the SII splice site in the type-I IP3R results in the differential regulation of the alternatively spliced isoforms by Ca2+, CAM, and protein kinase A.
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U2 - 10.1074/jbc.275.4.2305
DO - 10.1074/jbc.275.4.2305
M3 - Article
C2 - 10644679
AN - SCOPUS:0034723250
VL - 275
SP - 2305
EP - 2311
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
ER -