No abstract

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.

TSH is an important physiological regulator of growth and function in thyroid gland. The mechanism of action of TSH depends on interaction with its receptor coupled to heterotrimeric G proteins. We show here that TSH induces the phosphorylation of tyrosine in the intracellular kinases Janus kinase 1 (JAK1) and -2 (JAK2) in rat thyroid cells and in Chinese hamster ovary (CHO) cells transfected with human TSH receptor (TSHR). The JAK family substrates STAT3 (signal transducers and activators of transcription) are rapidly tyrosine phosphorylated in response to TSH. We also find that JAK1, JAK2, and STAT3 coprecipitate with the TSHR, indicating that the TSHR may be able to signal through the intracellular phosphorylation pathway used by the JAK-STAT cascade. TSH increases STAT3-mediated promoter activity and also induces endogenous SOCS-1 (suppressor of cytokine signaling-1) gene expression, a known target gene of STAT3. The expression of a dominant negative form of STAT3 completely inhibited TSH-mediated SOCS-1 expression. These findings suggest that the TSHR is able to signal through JAK/STAT3 pathways.

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 m 2 of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per m 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 M. 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.The secretory granules of neuroendocrine cells are loaded with hormones, neurotransmitters, and ions such as Ca 2ϩ , Mg 2ϩ , and Zn 2ϩ along with peptides and proteins of which chromogranins A and B are the most abundant (1-5). Chromogranins A and B are acidic proteins (1-5) with acidic residues constituting 25-30% of the amino acid residues (6 -12), and this high content of negatively charged amino acid residues is thought to be responsible for the high capacity, low affinity Ca 2ϩ binding property of chromogranins (13,14), binding 32-93 mol of Ca 2ϩ /mol (14, 15). The comparison of the amino acid sequences of CGA 1 (6 -8) and CGB (9 -12) shows little sequence homology except the two conserved regions, one near the N-terminal region bordered by two cysteine residues (residues 17-38 in bovine CGA and 16 -37 in bovine CGB) and the other the C-terminal region (residues 409 -431 in bovine CGA and 604 -626 in bovine CGB). Despite the differences in amino acid sequences, chromogranins A and B and secretogranin II (also called chromogranin C) were shown to aggregate in an acidic pH and high calcium environm...