Rab3A and Rab27A cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells

Abstract

Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense-core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense-core vesicles. We found that four Rabs (Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense-core vesicles in PC12 cells, and that three of them (Rab3A, Rab27A, Rab33A) are endogenously expressed on dense-core vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane.

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

“…However, the molecular mechanism by which Rab32/38 regulate tyrosinase and/or Tyrp1 transport remains completely unknown, and no specific effector molecules for Rab32/38 have ever been identified in melanocytes. Because Rab32 can compensate for the dysfunction of Rab38 in melan-chocolate (cht) cell lines (Wasmeier et al, 2006), a putative effector molecule(s) for Rab38 in melanocytes must interact with both Rab32 and Rab38, the same as rabphilin interacts with both Rab3A and Rab27A Fukuda, 2003;Fukuda et al, 2004), both of which regulate docking of hormone granules to the plasma membrane in neuroendocrine PC12 cells (Tsuboi and Fukuda, 2006). Wang et al (2008) very recently reported that vacuolar protein sorting 9 (VPS9)-ankyrinrepeat protein (Varp; official gene symbol in National Center for Biotechnology Information: Ankrd27), originally identified as a guanine nucleotide exchange factor (GEF) for Rab21 (Zhang et al, 2006), interacted with Rab38 in COS-7 cells by overexpression study.…”

Rab27a and Rab27b are involved in stimulation-dependent RANKL release from secretory lysosomes in osteoblastic cells

“…Rab proteins in their active form (GTP-bound form) are carried on the surfaces of cargo vesicles and function as markers for the transport and fusion of these vesicles with the acceptor membrane (17) by associating with effector molecules that mediate their interaction with motor proteins, (18) phospholipids, or soluble NSF attachment protein receptor (SNARE) components. (19) Rab27a, which is expressed in many types of secretory organs, is reported to be involved in secretion from lysosomal organelles such as secretory granules in insulin-secreting cells, (20) dense granules in platelets, (21) dense-core vesicles in neuroendocrine cells, (22) melanosomes in melanocytes, (23) and lytic granules in cytotoxic T-lymphocytes (CTLs). (24) Several Rab27a effector molecules also have been identified.…”

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis

“…A handful of laboratories have addressed these issues by investigating the behavior of both Rabs in parallel. In one study, for instance, in PC12 cells, both overexpressed and endogenous Rab3A and Rab27A localized to densecore vesicles (17). Silencing each Rab individually reduced the number of vesicles docked at the plasma membrane and exocytotic events.…”

“…Silencing each Rab individually reduced the number of vesicles docked at the plasma membrane and exocytotic events. Both phenomena were further reduced by silencing Rab3A and Rab27A simultaneously, which led to the conclusion that these small GTPases cooperate to regulate the docking of dense-core vesicles to the plasma membrane (17). Another study conducted in the same neuroendocrine cell line and published a year later found that overexpressed Rab3A cycles continuously between the cytosol and membranes, and that this cycling is not coupled to exocytosis (18).…”