Assay of the Rab-binding specificity of rabphilin and Noc2: target molecules for Rab27.
Rabphilin and Noc2 were originally described as Rab3A effector proteins involved in the regulation of secretory vesicle exocytosis in neurons and certain endocrine cells. Both proteins share the conserved N-terminal Rab-binding domain (RBD) that consists of two alpha-helical regions separated by two zinc finger motifs. However, the RBD of rabphilin and Noc2 has been shown to bind Rab27A (the closest homologue of Rab3 isoforms) in preference to Rab3A, both in vitro and in vivo. Rabphilin and Noc2 are recruited to dense-core vesicles (DCVs) in neuroendocrine PC12 cells and regulate their exocytosis through interaction with Rab27A rather than with Rab3A. Rab3A-binding-defective mutants of rabphilin(E50A) and Noc2(E51A) retain the ability to target DCVs in PC12 cells, the same as the wild-type proteins, whereas Rab27A-binding-defective mutants of rabphilin(E50A/I54A) and Noc2(E51A/I55A) do not (i.e., they are present throughout the cytoplasm). Expression of the wild-type or the E50A mutant of rabphilin-RBD, but not the E50A/I54A mutant of rabphilin-RBD, in PC12 cells significantly attenuated DCV exocytosis monitored by high-KCl-stimulated neuropeptide Y secretion. In this chapter we describe various assay methods that have been used to characterize the RBD of rabphilin and Noc2 as "RBD27 (Rab-binding domain for Rab27)."
Mitsunori
M
Fukuda
Akitsugu
A
Yamamoto
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000378:metabolism; D016716:PC12 Cells; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D009478:Neuropeptide Y; D011506:Proteins; C484660:Rph3al protein, mouse; C109307:Rph3al protein, rat; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins