CGA1 is a member of the granin protein family and is stored in high concentrations in the large dense core secretory granules of most endocrine and neuroendocrine cells as well as in many nerve cells in the periphery and brain (1, 2). CGA, the first member of the granin family to be discovered (3-5), has a wide variety of functions, both extracellular and intracellular.As one of its extracellular functions, CGA acts as a prohormone, a protein that contains numerous sites for proteolytic processing. Following secretion, extracellular proteases cleave CGA, generating several peptide fragments with biological activity, including pancreastatin (6, 7), vasostatins I and II (8 -10), parastatin (11), catestatin (12), and chromacin (13). In healthy individuals, CGA and its peptide fragments are present in the circulatory system in low nanomolar quantities. However, in patients suffering from pheochromocytoma and other neuroendocrine tumors, concentrations are significantly higher (14). Elevated plasma levels of CGA are associated with a number of pathological conditions making the protein an ideal marker not only for neuroendocrine tumors but also for chronic heart failure and brain disorders such as Parkinson's and Alzheimer's diseases (15).Among its intracellular roles, CGA has been shown to interact with ATP, catecholamines, and Ca 2ϩ (16,17), to acidify the intravesicular medium and to sort proteins for the regulated secretory pathway via a range of protein-protein interactions (15). These sorting functions include aggregation with chromogranin B, complexing with dopamine ␤-hydroxylase, t-plasminogen activator, and binding secretory granule membrane constituents such as the InsP 3 R (15).In recent years, secretory granules of neuroendocrine cells have been identified as inositol (1,4,5)-trisphosphate (InsP 3 )-sensitive Ca 2ϩ stores (18 -20). In the granules CGA forms a tetramer and appears to bind four molecules of the intraluminal loop of the InsP 3 R at the intravesicular pH 5.5 (21-23). In vitro studies show that purified InsP 3 R interact directly with CGA at this pH and dissociate from it at pH 7.5, a pH encountered when exocytosis occurs (24). Co-transfection of InsP 3 R and CGA into COS-7 cells followed by co-immunoprecipitation demonstrates that these two proteins form a complex in vivo (24).We have investigated the functional aspect of this coupling via InsP 3 -mediated Ca 2ϩ release studies using InsP 3 R-reconstituted liposomes in the presence and absence of CGA. We have further characterized the molecular basis of this phenomenon at the single channel level using planar lipid bilayer studies. In the presence of CGA the open probability and mean open time of the InsP 3 R channel increases significantly. Hence, modulation of InsP 3 R channel activity by CGA appears to be an essential component in the control of intracellular Ca 2ϩ concentration in secretory granules. MATERIALS AND METHODS Purification of the InsP 3 ReceptorFor Flux Studies-The type I InsP 3 receptor was isolated from bovine cerebella as d...

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 cotransfected 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 experiements 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.The secretory granules of endocrine cells, neurons, and neuroendocrine cells contain many hormones, ions, peptides and proteins, including 40 mM Ca 2ϩ and 1-2 mM chromogranins A and B in addition to high concentrations of hormones (1-6). The secretory granule contents are secreted to the extracellular space and then into the bloodstream during exocytosis, which is initiated by a sudden increase of intracellular Ca 2ϩ concentration (7). In bovine adrenal medullary chromaffin cells the secretory granules occupy ϳ10% of the total cell volume (8), thereby storing a majority of the intracellular Ca 2ϩ of the cell in the secretory granules. Hence it appears inevitable for the secretory granules to participate in the control of intracellular Ca 2ϩ concentrations. Consistent with this notion, the secretory granules from adrenal medullary chromaffin cells (9), pancreatic acinar cells (10), and the goblet cells (11) concentrations but also in exocytotic processes. Despite the importance of the IP 3 -sensitive intracellular Ca 2ϩ store role of secretory granules, the study of IP 3 R/Ca 2ϩ channels in the secretory granules did not begin until the secretory granule Ca 2ϩ storage protein chromogranin A was shown to...

Secretogranin II (SgII) is one of the three major proteins, the other two being chromogranins A (CGA) and B (CGB), of secretory granules of neuroendocrine cells. The Ca(2+) storage proteins CGA and CGB not only are coupled to the IP(3) receptor (IP(3)R)/Ca(2+) channels that exist on the secretory granule membrane but also are known to play key roles in secretory granule biogenesis. Unlike the better studied CGA and CGB, secretogranin II has never been completely purified in the native state and studied. We have therefore purified SgII in native form from bovine adrenal medulla and subjected it to biochemical characterization. Secretogranin II consisted of largely beta-sheet and random coil structures with a low level of alpha-helicity. Like CGA and CGB, it also underwent pH-dependent conformational changes, showing 9.5% alpha-helicity at pH 7.5 and 17.0% alpha-helicity at pH 5.5. Secretogranin II also underwent acidic pH- and Ca(2+)-dependent aggregation, and it was approximately 8-fold more sensitive than CGA to Ca(2+) in its pH-dependent aggregation but was 8-fold less sensitive than CGB. Further, similar to CGA and CGB that had interacted with the secretory granule membrane at the intragranular pH 5.5, SgII also interacted with the secretory granule membrane at pH 5.5 and dissociated from it at near-physiological pH 7.5, implying similar roles of SgII in the cell as those of CGA and CGB. Secretogranin II hence appeared to actively participate in secretory granule biogenesis as has been proposed for CGA and CGB.