The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules.
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.
Yi
Zhaohong
Z
Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
Yokota
Hiromi
H
Torii
Seiji
S
Aoki
Takeo
T
Hosaka
Masahiro
M
Zhao
Shengli
S
Takata
Kuniaki
K
Takeuchi
Toshiyuki
T
Izumi
Tetsuro
T
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D015870:Gene Expression; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D007515:Islets of Langerhans / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000075745:rab27 GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D007328:Insulin; D046911:Macromolecular Substances; C512779:SYTL4 protein, human; C122015:Sytl4 protein, mouse; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein