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J Biol Chem, Vol. 275, Issue 4, 2305-2311, January 28, 2000
From the Department of Pathology and Cell Biology, Thomas
Jefferson University School of Medicine,
Philadelphia, Pennsylvania 19107
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
acids1314-1905) 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.
To whom correspondence should be addressed: Dept. of Pathology and
Cell Biology, Thomas Jefferson University, Room 230A JAH, 1020 Locust
St., Philadelphia PA 19107. Tel.: 215-503-1221; Fax: 215-923-6813;
E-mail: Suresh.Joseph@mail.tju.edu.
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