Abstract
The secretory granules of neuroendocrine cells which contain large amounts of Ca(2+) and chromogranins have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)). Moreover, chromogranin A (CGA) has been shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R). To determine whether the IP(3)Rs interact directly with chromogranins A and B (CGB), two major proteins of the secretory granules, we have used purified IP(3)R from bovine cerebellum in the interaction study with CGA and CGB, and have shown that chromogranins A and B directly interact with the IP(3)R at the intravesicular pH 5.5. Immunogold cytochemical study using the IP(3)R and CGA antibodies indicated that IP(3)R-labeled gold particles were localized in the periphery of the secretory granules, indicating the presence of the IP(3)Rs on the secretory granule membrane. To determine whether the IP(3)R and chromogranins A and B are physically linked in the cells, bovine type 1 IP(3)R (IP(3)R-1) and CGA or CGB are co-transfected into COS-7 cells and co-immunoprecipitation was carried out. Immunoprecipitation of the cell extracts demonstrated the presence of CGA-IP(3)R-1 and CGB-IP(3)R-1 complexes, respectively, indicating the complex formation between the IP(3)R and chromogranins A and B in native state.
Highlights
The secretory granule of adrenal medullary chromaffin cells has been shown to release Ca2ϩ in response to IP3 1 [1], and this observation has been extended to the secretory granule of zymogen-secreting pancreatic acinar cells [2], demonstrating the IP3-sensitive intracellular Ca2ϩ store role of secretory granules
To determine whether the IP3 receptor (IP3R) interacts with chromogranin A (CGA) or chromogranin B (CGB) directly, the IP3R was immuno-affinity-purified from bovine cerebellum using the affinity-purified anti-peptide IP3R antibody [14] (Fig. 1A)
CGA or CGB, Glutathione S-Transferase (GST)-fusion forms of CGA and CGB were expressed in E. coli BL21, and purified to study their interactions with the IP3R
Summary
The secretory granule of adrenal medullary chromaffin cells has been shown to release Ca2ϩ in response to IP3 1 [1], and this observation has been extended to the secretory granule of zymogen-secreting pancreatic acinar cells [2], demonstrating the IP3-sensitive intracellular Ca2ϩ store role of secretory granules. The IP3-sensitive Ca2ϩ store role of secretory granules of bovine adrenal medullary chromaffin cells was attributed to the presence of high capacity, low affinity Ca2ϩ storage protein CGA, binding 30 –50 mol of Ca2ϩ/mol, inside the secretory granule [1, 4].
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