Assay and functional interactions of Rim2 with Rab3.
Rim was originally identified as a protein that contains a putative Rab3A-effector domain at the N-terminus, the same as rabphilin, and two forms of Rim, Rim1 and Rim2, have been reported in mammals. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs, and several alternative splicing events occur in the RBD1 of both Rims that result in the production of long forms and short forms of RBD. The short forms of Rim2 RBD are capable of interacting with Rab3A with high affinity in vitro, and it is recruited to dense-core vesicles (DCVs) in neuroendocrine PC12 cells through interaction with endogenous Rab3A, whereas the long forms of Rim2 RBD show dramatically reduced Rab3A-binding activity in vitro (more than a 50-fold decrease in affinity compared with the short forms of Rim2 RBD), and it is mainly present in the cytoplasm and nucleus. Expression of the shortest form of Rim2 RBD, but not its Rab3A binding-defective mutant (E36A/R37S), promotes high-KCl-dependent neuropeptide Y secretion from PC12 cells, suggesting that the Rim2 containing the short forms of RBD functions as a Rab3A effector during DCV exocytosis. In this Chapter, I describe several assay methods that have been used to determine the physiological significance of the alternative splicing event in the RBD1 of Rim2, including assays for the in vitro interaction between Rim2 RBD and Rab3A and for the localization of Rim2-RBD on DCVs in PC12 cells.
Mitsunori
M
Fukuda
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding; D012326:RNA Splicing; D014162:Transfection; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism*
C475524:Rim2 protein, mouse; D020692:rab3 GTP-Binding Proteins