Analysis of the role of Rab27 effector Slp4-a/Granuphilin-a in dense-core vesicle exocytosis.
Slp4-a/granuphilin-a is a member of the synaptotagmin-like protein (Slp) family and consists of an N-terminal Slp homology domain (SHD) and C-terminal tandem C2 domains. Slp4-a is specifically localized on secretory granules in some endocrine and exocrine cells through its SHD, and it attenuates Ca(2+)-dependent dense-core vesicle (DCV) exocytosis when transiently expressed in endocrine cells. Although the SHD of Slp4-a interacts with three distinct Rab species (Rab3A, Rab8A, and Rab27A) in vitro, in contrast to other Slp members, which only recognize Rab27 isoforms, Slp4-a functions as a Rab27A effector during DCV exocytosis under physiological conditions. This chapter describes various approaches that have been used to characterize the function of Slp4-a as a Rab27A effector, rather than a Rab3A or Rab8A effector, both in in vitro and in neuroendocrine PC12 cells. Specifically, the methods that have been used to analyze (1) the physical interaction between Slp4-a and Rab27A, including pull-down assay and cotransfection assay in COS-7 cells; (2) the localization of Slp4-a-Rab27A complex on DCVs in PC12 cells; and (3) the involvement of Slp4-a and Rab27A in DCV exocytosis by neuropeptide Y (NPY) cotransfection assay combined with site-directed mutagenesis are described.
Mitsunori
M
Fukuda
Eiko
E
Kanno
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000494:pharmacology; D016716:PC12 Cells; D051381:Rats; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000502:physiology*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D000075745:rab27 GTP-Binding Proteins
D009478:Neuropeptide Y; C512779:SYTL4 protein, human; C122015:Sytl4 protein, mouse; C512778:Sytl4 protein, rat; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C066694:RAB27A protein, human; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins