Functional analysis of Rab27a effector granuphilin in insulin exocytosis.
Granuphilin is specifically expressed on dense-core granules in a defined set of secretory cells such as insulin-producing pancreatic beta-cells. It preferentially binds the GTP-bound form of Rab27a and regulates the exocytosis of secretory granules. Furthermore, granuphilin directly interacts with syntaxin-la, the plasma-membrane-anchored SNARE protein, and with Munc18-1, a Sec1/Munc18 protein. We previously reported evidence that granuphilin mediates the docking of secretory granules onto the plasma membrane through these protein-protein interactions. This chapter details the methods and protocols we use to analyze the function of granuphilin with particular attention to the assays for detecting the expression, protein interactions, and effects on exocytosis of secretory granules in pancreatic beta-cells and their derivative cell lines.
Tetsuro
T
Izumi
Hiroshi
H
Gomi
Seiji
S
Torii
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D050827:Syntaxin 1 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D000075745:rab27 GTP-Binding Proteins
D007328:Insulin; C512779:SYTL4 protein, human; D050827:Syntaxin 1; C122015:Sytl4 protein, mouse; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins