Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K ؉ -induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

A novel rabphilin-3-like gene, granuphilin, has been identified in pancreatic beta cells by comparing genes expressed in pancreatic alpha and beta cell lines using mRNA differential display. The domain structure of the protein products of the granuphilin gene contains an amino-terminal zinc-finger motif and carboxyl-terminal C 2 -domains, similar to that of the rabphilin-3 gene. There are two isoforms: the larger isoform, granuphilin-a, has two C 2 -domains, whereas the smaller one, granuphilin-b, contains only the first C 2 -domain. Granuphilin is specifically expressed in pancreatic beta cells and the pituitary gland, but not in pancreatic alpha cells, the adrenal gland, or other major organs such as the brain. A portion of granuphilin associates with insulin-containing dense-core granules, but not with synaptic-like microvesicles in beta cells. Thus, its distribution pattern presents a striking contrast with that of rabphilin-3, which associates with small synaptic vesicles in neurons. The first C 2 -domain of granuphilin binds phospholipids in a Ca 2؉ -independent manner, whereas the second one does not. These distinctive characteristics of granuphilin suggest that it is not a simple counterpart of rabphilin-3 in endocrine cells and that it has a unique role in the regulated exocytosis of densecore granules in endocrine tissues.

A possible connection between the effects of aluminum (Al) on the growth of tea plants and the active oxygen species scavenging system in root tips of intact tea plants and suspension-cultured tea cells was examined. Intact tea plants were treated with or without Al in a modified Hoagland solution, while suspension-cultured tea cells were treated with or without Al in a simple salt solution containing 3% sucrose and 0.2 mM calcium. Compared with the control treatments without Al, the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) increased by Al both in roots of intact plants and cultured cells. The level of peroxidation of membrane lipids, as well as the activity of wall-bound peroxidas, the content of lignin and wall-bound phenols, however, reduced by the treatment with Al either in tea roots or in cultured tea cells. The results indicated that Al-induced increase in the activities of antioxidant enzymes, resulting in increased membrane integrity and delayed lignification and aging, can be considered as a possible reason for the stimulatory effects of Al on the growth of the tea plants and this is irrespect of the presence of other micronutrients and their interaction with Al.Abbreviations: APX -ascorbate peroxidase; CAT -catalase; CPO -covalently bound peroxidase; IPOionically bound peroxidase; MDA -malondialdehyde; PAL -phenylalanine ammonia-lyase; PO -peroxidase; ROS -reactive oxygen species; SOD -superoxide dismutase; SPO -soluble peroxidase.

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The receptor-type protein tyrosine kinases in murine pancreatic islets were screened to identify possible growth/differentiation factors in pancreatic beta-cells. The analysis revealed that insulin receptor-related receptor (IRR) is highly expressed in the islets as well as in several highly differentiated beta-cell lines derived from transgenic mice. Islets predominantly contain IRR as uncleaved proreceptors compared with IRR as processed forms in the beta-cell lines, suggesting that the activity of IRR is regulated on the level of processing proteases in vivo. To examine the IRR signaling pathway, a chimeric receptor consisting of the extracellular domain of insulin receptor and the intracellular domain of IRR was expressed in Chinese hamster ovary cells. The hybrid receptor is functional because insulin is capable of tyrosine-phosphorylating the catalytic domain in these cells. It also stimulates the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2, indicating that both proteins serve as substrates of IRR-protein tyrosine kinase in intact cells. The phenotype of the IRS-2 knockout mouse recently reported suggests that an IRS-2-mediated signaling pathway controls the compensatory increase in pancreatic beta-cell mass in insulin-resistant states. From our findings of the specific expression of IRR and its ability of signaling to IRS-2, we speculate that this receptor might play a role in the regulation of beta-cell mass.