A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP 3 R) 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 Ca 2؉ -dependent manner as measured by CaMSepharose 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. [3][4][5]. The functional role of isoform diversity has not been established. The gene encoding the type-I isoform is subject to alternative splicing and gives rise to three splice variants that have been denoted as S1, S2, and S3 (6 -8). The S1 splice site is located in the ligand binding domain, whereas the S2 and S3 splice sites are in the regulatory domain. Functional studies comparing the S1(Ϫ) and S1(ϩ) forms of the type-I IP 3 R have not revealed any marked differences in ligand binding or channel function (9, 10), and the functional consequences of the S3 insertion/deletion have not been investigated. Both short and long forms of S1 and S3 appear to co-exist in the same tissues (7,8,11). In contrast, the expression of the S2 insert is more stringent with the S2 insert being present in neurons and absent from the type-I IP 3 Rs of peripheral tissues (7,12).The S2 splice site encodes a region of 40 amino acids and is located between serine 1589 and serine 1756. Both serine residues can be phosphorylated by protein kinase A (13), and serine 1756 in cerebellum IP 3 R is phosphorylated by G-kinase (14). In addition, binding sites for Ca 2ϩ (15), CaM (16), ATP (17), and FKBP-12 (18) are all found in the vicinity of the S2 splice site (see Fig. 1A). Hence, the presence or absence of the S2 splice site may modify regulation of type-I IP 3 Rs. For example, there is evidence that the deletion of the S2 region can alter the serine that is preferentially phosphorylated by protein kinase A (12). There is also experimental evidence to suggest that the effects of protein kinase A phosphorylation on IP 3 -gated channels in neuronal and peripheral tissues are different (reviewed in Joseph et al. (19) (S2(ϩ)) is also different from that observed in a number of tissues that express high amounts of the S2(Ϫ) form of the type-I IP 3 R, e.g. vas deferens (20). However, it is difficult to definitively attribute such differences solely to the presence of the alternatively spliced forms of the type-I IP 3 R, and the basis for these differences is not well understood. In the presen...

A prominent labelled polypeptide having the same mobility as type-I inositol trisphosphate receptor (IP $ R) was immunoprecipitated from WB-cell lysates by antibodies to calnexin, an ER integral membrane chaperone. The identity of this polypeptide was confirmed by re-immunoprecipitation of the radioactive polypeptides released from calnexin-antibody immunoprecipitates with type-I IP $ R antibody. The interaction of calnexin with newly synthesized type-I IP $ R was transient and inhibited by treatment of the cells with dithiothreitol or the glucosidase inhibitor N-methyldeoxynojirimicin. In similar experiments, there was no evidence for the binding of type-I IP $ R to calreticulin, an ER luminal chaperone. Calnexin (but not calreticulin) associated with newly synthesized FLAG (DYKDDDDK epitope)-tagged type-III IP $ R expressed in COS-7 cells. In order to further define the mechanism of interaction of nascent IP $ R with chaperones, we have utilized an in itro rabbit

No abstract