Search

Inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major intracellular Ca(2+) channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP(3)R and the regulators, and whether it involves conformational changes of the lumenal regions of IP(3)R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca(2+), on the L3-1 lumenal loop region of IP(3)R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca(2+) showed little effect on the L3-1 conformation, suggesting that the regulation of Ca(2+) is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys(34) and Cys(42). A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys(34)-Cys(42) region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys(34) and Cys(42) may act together as a reduction sensor, and that Cys(65) may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca(2+) can regulate IP(3)R through totally different mechanisms: Ca(2+) by the indirect effect and the redox potential by direct action causing conformational changes.

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.

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.

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.

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)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006). To examine the functional aspect of this coupling, we measured the IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules.

The two major proteins of the secretory vesicles of neuroendocrine cells, chromogranin A (CGA) and chromogranin B (CGB), have been shown to undergo pH- and Ca2+-dependent conformational changes and aggregation and have been suggested to play essential roles during secretory vesicle biogenesis in the trans-Golgi network. CGA has been shown to exist primarily in a tetrameric state at pH 5.5 and primarily in a dimeric state at pH 7.5, and CGB has been shown to exist in a monomeric state at both pH 5.5 and pH 7.5. Using purified CGA and CGB, it recently has been shown that CGA interacts with CGB at pH 5.5 (Yoo, S. H.(1996) J. Biol. Chem. 271, 1558-1565). In expanding this investigation, we have studied the temperature dependence of the pH-dependent interaction of CGA and CGB by analytical ultracentrifugation and found that two molecules of CGA bound to two molecules of CGB at pH 5.5 with DeltaG0 values of -43.6 kcal/mol in the absence of Ca2+ at 37 degrees C and -40.3 kcal/mol in the presence of 0.1 mM Ca2+. However, one molecule of CGA bound to one molecule of CGB at pH 7.5 with DeltaG0 values of -13.6 kcal/mol in the absence of Ca2+ at 37 degrees C. The magnitude of DeltaG0 values increased with increasing temperatures at both pH values. However, the values for enthalpy and entropy changes decreased with increasing temperatures in both pH levels, suggesting formation of more ordered structures. In the absence of Ca2+ at pH 5. 5, the heterotetramerization reaction at 37 degrees C was entropically driven, whereas in the presence of Ca2+ (0.1 mM) the heterotetramerization was virtually an enthalpic reaction. On the other hand, the heterodimer formation in the absence of Ca2+ at pH 7. 5 showed large negative enthalpy and entropy changes at 37 degrees C, indicating an enthalpic interaction compensated by entropic changes. In view of the interaction of tetrameric CGA with tetrameric inositol 1,4,5-trisphosphate (IP3) receptor and the existence of heterotetrameric IP3 receptor in the cell, the heterotetramer formation by CGA and CGB not only raises the possibility of interaction between the heterotetrameric chromogranin and heterotetrameric IP3 receptor but also appears to reflect their important roles in the cell.