| Budget Amount *help |
¥3,830,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Cholesterol-and glycosphingolipid-rich microdomains of biological membranes, collectively called lipid rafts, provide platforms for various functional molecules. In the present research project, we investigated the distribution and subcellular localization of the lipid raft-like microdomains in the rat anterior pituitary, and also examined the physiological roles of lipid raft-like domains and SgIII in the secretory granule biogenesis.
By using biotinylated cholera toxin b subunit (CTxB) as a probe for GM1-ganglioside-rich lipid microdomains, most of somatotropes and a limited subpopulation of gonadotropes in the rat anterior pituitary gland were labeled, whereas no significant labeling could be detected in mammotropes, thyrotropes, and corticotropes. Within pituitary somatotropes, the GM1-ganglioside-rich lipid rafts were predominantly localized on the secretory granule membrane, and were translocated to the cell surface shortly after the administration of GHRH. Judging from the findin
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g that the secretory granule membrane of all kinds of pituitary endocrine cells was labeled with filipin that form a complex specifically with cholesterol, lipid raft-like microdomains were significantly accumulated in general on the secretory membrane, although there is a certain heterogeneity in the composition of lipid rafts, depending on the cell types.
Since previous studies by us and others suggest that both SgIII and CPE could bind specifically to cholesterol-rich lipid microdomain(s) of secretory granule membranes, we next examined the possible interaction between SgIII and CPE on the lipid raft-like microdomains. SgIII and CPE were colocalized in the same secretory granules, occasionally neighbored together in the periphery of the secretory granule. Co-precipitation analyses demonstrated that SgIII and CPE form a complex in endocrine-derived AtT-20 cells, especially under the mildly acidic pH and high calcium condition that mimics the internal milieu at the TGN. Interestingly, the binding property of SgIII to cholesterol-rich lipid microdomain(s) was not altered even in the pituitary endocrine cells of mutant mice lacking CPE; SgIII was properly localized to secretory granules without CPE. These findings suggest that the lipid raft-like microdomain provides a platform, onto which SgIII and/or CPE, cooperatively or solely, tether(s) the aggregates of diverse secretory proteins. Less
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