Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B.
Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.
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National Creative Research Initiative Center for Secretory Granule Research, Korea Advanced Institute of Science and Technology, Yu Sung Gu, Dae Jeon, Korea 305-701. shyoo@kaist.ac.kr
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Kang
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Park
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D019556:COS Cells; D015220:Calcium Channels / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D053379:Chromogranin A; D002864:Chromogranins / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D007150:Immunohistochemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D015220:Calcium Channels; D053379:Chromogranin A; D002864:Chromogranins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear