CGA) And B Research Articles

The Functional Role of Chromogranins in Exocytosis

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with L-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.

Enrichment of the inositol 1,4,5-trisphosphate receptor/Ca2+ channels in secretory granules and essential roles of chromogranins

The high capacity, low affinity Ca2+ storage protein chromogranins are marker proteins of secretory granules that contain the most Ca2+ in secretory cells. Along with the abundantly expressed chromogranins, the IP3R/Ca2+ channels, the major intracellular Ca2+ channels, are also expressed in secretory granules the most. Chromogranins not only induce formation of secretory granules but also are suggested to produce the small IP3-sensitive nucleoplasmic Ca2+ store vesicles in the nucleus. Chromogranins A (CGA) and B (CGB) also directly bind the IP3Rs and activate the IP3R/Ca2+ channels at the intragranular pH 5.5. But at a near physiological pH 7.5 only CGB interacts with the IP3Rs due to stronger interaction of CGB for the IP3Rs, which is several orders of magnitude stronger than that of CGA, and activates the IP3R/Ca2+ channels. Therefore, the CGB-IP3R coupling is proposed to play key roles in the IP3-mediated Ca2+ signaling mechanisms in the cytoplasm through both secretory granules and the ER, and in the nucleus through the small IP3-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranin B is further suggested to participate in transcription control and to target secretory granule components, including the IP3Rs, to newly formed secretory granules. Defects in secretory granule-related functions are directly linked to major human diseases such as Alzheimer's disease, secretory cell cancers, cystic fibrosis, acute pancreatitis, and cardiac hypertrophy. Therefore, realization of secretory granules as the major intracellular Ca2+ storage and control organelle in secretory cells promises to open new horizon in understanding the Ca2+ storage, signaling, and control mechanisms throughout the biokingdom.

Presence of Secretogranin II and High-Capacity, Low-Affinity Ca2+ Storage Role in Nucleoplasmic Ca2+ Store Vesicles

Chromogranins and secretogranins have traditionally been known as marker proteins of secretory granules that contain the highest concentrations of cellular calcium, reaching approximately 40 mM. In addition, chromogranin B was also shown to exist in the nucleus, localizing in the putative inositol 1,4,5-trisphosphate (IP3)-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranins A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+ binding proteins, binding 30-90 mol of Ca2+/mol with dissociation constants (Kd) of 1.5-4 mM. Yet the Ca2+-binding property of secretogranins has not been studied. Here, we show the localization of secretogranin II (SgII) in the nucleus, more specifically, in the IP3-sensitive nucleoplasmic Ca2+ store vesicles along with CGB and the IP3 receptors. We have also determined the Ca2+-binding property of SgII and found that SgII binds 61 mol of Ca2+/mol (910 nmol Ca2+/mg) with a Kd of 3.0 mM at the intragranular pH 5.5 and 30 mol of Ca2+/mol (440 nmol Ca2+/mg) with a Kd of 2.2 mM at a near-physiological pH 7.5. Chromogranin B also bound 50 mol of Ca2+/mol (670 nmol Ca2+/mg) with a Kd of 3.1 mM at pH 7.5. Given the high-capacity, low-affinity Ca2+-binding property of SgII and its presence in the IP3-sensitive nucleoplasmic Ca2+ store vesicles, these results suggest that SgII may function in the storage and control of Ca2+ in the nucleus through its interaction with CGB in the nucleoplasmic vesicles.

Subcellular distribution of chromogranins A and B in bovine adrenal chromaffin cells

The major secretory granule proteins chromogranins A (CGA) and B (CGB) have recently been shown to play critical roles in inositol 1,4,5-trisphosphate-dependent intracellular Ca 2+ mobilizations. We determined here the subcellular distribution of CGA and CGB based on 3D-images of chromaffin cells, and found that ∼95% of cellular CGA was present in secretory granules while ∼5% was in the endoplasmic reticulum (ER), whereas ∼57% of cellular CGB was in secretory granules while ∼24% and ∼19% were in the ER and nucleus, respectively. These results suggest that chromogranins are at the center of intracellular Ca 2+ homeostasis in secretory cells.

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.

Chromogranin B-induced Secretory Granule Biogenesis: COMPARISON WITH THE SIMILAR ROLE OF CHROMOGRANIN A

The two major proteins of secretory granules of secretory cells, chromogranins A (CGA) and B (CGB), have previously been proposed to play key roles in secretory granule biogenesis. Recently, CGA was reported to play an on/off switch role for secretory granule biogenesis. In the present study we found CGB being more effective than CGA in inducing secretory granule formation in non-neuroendocrine NIH3T3 and COS-7 cells. The mean number of dense core granules formed/cell of CGA-transfected NIH3T3 cells was 2.51, whereas that of CGB-transfected cells was 4.02, indicating the formation of 60% more granules in the CGB-transfected cells. Similarly, there were 55% more dense core granules formed in the CGB-transfected COS-7 cells than in the CGA-transfected cells. Moreover, transfection of CGA- and CGB-short interfering RNA (siRNA) into neuroendocrine PC12 cells not only decreased the amount of CGA and CGB expressed but also reduced the number of secretory granules by 41 and 78%, respectively, further suggesting the importance of CGB expression in secretory granule formation.

The chromogranins: their roles in secretion from neuroendocrine cells and as markers for neuroendocrine neoplasia.

Chromogranins are the major components of the secretory granules of most neuroendocrine cells. Within the secretory pathway, chromogranins are involved in granulogenesis, and in sorting and processing of secretory protein cargo prior to secretion. Once secreted, they have hormonal, autocrine, and paracrine activities. The chromogranin family includes chromogranins A (CgA) and B (CgB) and secretogranin II (SgII, once called chromogranin C). The related "granins" NESP55, 7B2, secretogranin III/1B 1075 (SgIII), and secretogranin IV/HISL-19 antigen (SgIV), are also sometimes included when considering the chromogranins. While it is useful to consider the granin proteins as a family with many common features, it is also necessary to examine the distinct features and properties of individual members of the granin family to understand fully their functions, employ them efficiently as tissue, serum, and urinary markers for neuroendocrine neoplasia, and develop an evolutionary-biologic perspective on their contribution to mammalian physiology. Recent advances in chromogranin research include establishing the role of CgA in granulogenesis and the role of CgB in nuclear transcription; new biologic activities for CgA-, CgB-, and SgII-derived peptides; and new marker functions for granins and their proteolytically processed products in endocrine neoplasias.

Inositol 1,4,5‐Trisphosphate Receptor/Ca2+ Channel Modulatory Role of Chromogranins A and B

The secretory granules function as the major IP(3)-sensitive intracellular Ca(2+) store of secretory cells. Recently it was found that the secretory granules contain three isoforms of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and high-capacity, low-affinity Ca(2+) storage proteins chromogranins A (CgA) and B (CgB). The IP(3)R/Ca(2+) channel was shown to directly interact with CgA and CgB at the intragranular pH 5.5, and this coupling led to modulation of the IP(3)R/Ca(2+) channel activity by the coupled chromogranins. These results provide the molecular structural basis of the IP(3)-mediated Ca(2+) release mechanism of secretory granules.

Localization of the Secretory Granule Marker Protein Chromogranin B in the Nucleus: POTENTIAL ROLE IN TRANSCRIPTION CONTROL

Chromogranins A (CGA) and B (CGB) are two major Ca(2+) storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per microm(2) of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per microm(2) of the secretory granules. Considering that CGB is estimated to exist in the 0.1-0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20-40 microm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5-5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60-75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus.

Purification, pH-Dependent Conformational Change, Aggregation, and Secretory Granule Membrane Binding Property of Secretogranin II (Chromogranin C)

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.

Chromogranin A as a Calcium-Binding Precursor for a Multitude of Regulatory Peptides for the Immune, Endocrine and Metabolic Systems

A multitude of regulatory peptides derive from the main members of the granin family, i.e. the chromogranins A (CGA) and B (CGB) and secretogranin II. These proteins are co-secreted from the diffuse neuroendocrine system with a wide range of neurotransmitters and peptide hormones upon adequate stimuli. The recent developments provide ample evidence of widely different effects and targets for the CGA derived peptides, implicated in the regulation of plasma calcium, plasma glucose, adrenomedullary catecholamine release and vascular contractility, as well as in the innate immunity. This review aims to attract the attention of endocrinologists and immunologists to this novel family of regulatory peptides, notably those derived from CGA.The main emphasis has been placed on functional aspects of the precursor protein and the biologically active peptides, such as the vasostatins I and II, pancreastatin and catestatin. The majority of properties so far assigned to this calcium binding prohormone and its peptide derivatives fits into patterns of inhibitory effects, of postulated relevance not only for homeostatic processes, but also in the inflammatory response and the early defence against invading microorganisms.

Interaction of Chromogranin B and the Near N-terminal Region of Chromogranin B with an Intraluminal Loop Peptide of the Inositol 1,4,5-Trisphosphate Receptor

Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP(3)R) with chromogranins A (CGA) and B (CGB), two major Ca(2+) storage proteins of secretory granules that have been shown to be IP(3)-sensitive intracellular Ca(2+) store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP(3)R with both intact CGB and the conserved near N-terminal region of CGB. The interaction studies carried out with CGB and glutathione S-transferase fusion proteins of intraluminal loop regions of bovine type 1 IP(3)R showed that CGB interacts with intraluminal loop 3-2 (the second loop formed between transmembrane regions 5 and 6) of the IP(3)R at both pH 5.5 and 7.5. Analytical ultracentrifugation studies also indicated that CGB interacts with the same intraluminal loop region of the IP(3)R and the interaction was much stronger than that between CGA and the loop. Moreover, the conserved near N-terminal region of CGB also interacted with the intraluminal loop region of the IP(3)R. The CGB interaction with the IP(3)R intraluminal loop peptide at pH 7.5 showed a DeltaG(0) value of -8.1 kcal/mol at 37 degrees C for a 1:1 stoichiometry, indicating a K(d) of approximately 1.9 micrometer. These results give insight into the molecular organization of the IP(3)-sensitive Ca(2+) store.

Coupling of the Inositol 1,4,5-Trisphosphate Receptor and Chromogranins A and B in Secretory Granules

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.