Ashen (ash, Rab27a)

FUNCTION

Ontology

a) Biological process: protein transport, vesicle-mediated transport
b) Biological process: small GTPase mediated signal transduction
c) GTP binding
d) GTPase activity
e) Belongs to the small GTPase superfamily (Rab family).

Location

Membrane-bound.

Interaction

[RAB27A effectors]
RAB27A is a tissue-specific Rab that associates with lysosome-related organelles and secretory granules. Molecular activation involves a GDP to GTP exchange. RAB27A and its close paralog RAB27B interact with at least 10 different effectors, tentatively termed as exophilins or Slp/Slac2. Most of the effectors interact with RAB27 via a conserved RAB27-binding domain, which is present in melanophilin (the leaden gene product, also called Slac2-a) and Slp (synaptotagmin-like protein) proteins. The Slp homology domain (SHD) of Slp1~3 and Slac2-a/b (sequences lacking C2 domains, Slac2) specifically and directly binds the GTP-bound form of RAB27A both in vitro and in intact cells. The SHD of Slp1~3 and Slac2 functions as a RAB27A binding domain (Kuroda, et al (2002a)). The N-terminal SHD consists of two conserved alpha-helical regions (SHD1 and SHD2) that are often separated by two zinc finger motifs. SHD1 of Slac2-a/melanophilin alone is both necessary and sufficient for high affinity specific recognition of the GTP-bound form of RAB27A. By contrast, the zinc finger motifs and SHD2 seem to be important for stabilization of the structure of the SHD or higher affinity RAB27A binding (Fukuda, et al (2002a)). In addition, the SHD of Slp5 preferentially interacted with the GTP-bound form of RAB27A and marginally with RAB3A and RAB6A, both in vitro and in intact cells (Kuroda, et al (2002b)). However, Slp4-a interacts with the GDP-bound form of RAB27A (Fukuda, et al (2003a)).Slp2-a controls melanosome distribution in the cell periphery and regulates the morphology of melanocytes. Slp2-a is the most abundantly expressed of the Slp- and Slac2-family proteins in melanocytes and colocalizes with Rab27a on melanosomes. Knockdown of endogenous Slp2-a protein by small-interfering RNAs (siRNAs) markedly reduced the number of melanosomes in the cell periphery of mouse melanocytes ('peripheral dilution') (Kuroda, et al (2004)).

[RAB27A in melanocytes]
RAB27A directly associates in its GTP-bound form with mature melanosomes through its posttranslational C20 geranylgeranyl lipid tail. The RAB27A interacts with myosin-VIIa and myosin-Va via MyRIP (myosin and RAB interacting protein, also called Slac2-c) or melanophilin and mediates melanosome binding to actin. Slac2-a/melanophilin and Slac2-c/MyRIP are linker proteins between RAB27A and myosin Va. Slac2-a directly interacts with RAB27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively (Fukuda, et al (2002b)). Melanophilin is required with RAB27A to recruit myosin Va to melanosomes in melanocytes (Hume, et al). RAB27A binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin-Va. Melanophilin creates this link by binding to RAB27A in a GTP-dependent fashion through its amino terminus, and to myosin-Va through its carboxy terminus (Wu, et al). Melanophilin directly activates the actin-activated ATPase activity of myosin Va and thus its motor activity (Li, et al). Rab3GEP, previously isolated as a GEF for Rab3a, is identified as the non-redundant Rab27a GEF to activate Rab27a in melanocytes (Figueiredo, et al). GTP-hydrolysis leads to the inactivation of RAB27A and presumably to the dissociation of melanophilin and myosin Va. cAMP stimulates the expression of RAB27A and rapidly increases the interaction of the melanophilin/Slac2-a with actin, allowing the rapid accumulation of melanosomes in the actin-rich region of the dendrite extremities after the action of melanocyte-differentiating agent such as alpha-melanocyte-stimulating hormone (Passeron, et al). Knockdown of Slac2-a caused perinuclear aggregation of melanosomes alone without altering cell shape (Kuroda, et al (2004)). MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity, indicating that RAB27A is a new direct transcriptional target of MITF, and link MITF to melanosome transport(Chiaverini, et al).

[RAB27A in RPE]
Melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction (Gibbs, et al). A melanophilin homolog, MyRIP, mediates the formation of a tripartite complex with RAB27A and myosin VIIa in retinal pigment epithelium (RPE), to regulate the melanosome motility (El-Amraoui, et al; Futter, et al). However, the RAB27A/MyRIP protein complex does not appear to require recruitment of a myosin on the secretory granules in PC12 cells for function (Waselle, et al). Slac2-c functions as a functional myosin VIIa receptor rather than a myosin Va receptor in melanosome The Rab27A.Slac2-c.myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells (Kuroda, et al (2005)).

[RAB27A in pancreatic cells]
RAB27A acts in concert with RAB3 interacting proteins in most regulated secretory events (Tolmachova, et al). Granuphilin, whose domain structure is similar to that of the RAB3 effector protein rabphilin3, is localized on the membrane of insulin granules and specifically expressed in pancreatic islets and in pituitary tissue. Granuphilin preferentially binds to the GTP form of RAB27A. The RAB27A/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules (Yi, et al ) through a direct interaction with a t-SNARE plasma membrane syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a and plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a (Torii, et al ).In beta-cell line MIN6B1, miR124a increases the levels of SNAP25, Rab3A and Synapsin-1A and decreases those of Rab27A and Noc2. Inhibition of Rab27A expression is mediated by direct binding of miR124a to the 3'-untranslated region of Rab27A mRNA. In contrast, miR96 raises mRNA and protein levels of Granuphilin, a negative modulator of insulin exocytosis, and decreases the expression of Noc2 resulting in a decreased capacity of MIN6B1 cells to respond to secretagogues (Lovis, et al). Actin-bundling protein coronin 3 is a novel Rab27a effector that paradoxically bound guanosine diphosphate (GDP)-Rab27a in the pancreatic beta-cell line MIN6. Coronin 3 directly bound GDP-Rab27a through its beta-propeller structure (Kimura, et al). In contrast to another effector, granuphilin, in beta cells, a Rab27a/b effector, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells to promote the targeting of glucagon granules to the plasma membrane (Yu, et al). In insulin exocytosis, granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area (Kasai, et al (2008)). Rab27a exerts dual roles in glucose-mediated insulin granule exocytosis, facilitating refilling of releasable granule pools while also limiting the rate of release from these pools (Merrins, et al ).

[RAB27A in PC12 cells]
In PC12 cells, three Rab27A effectors (granuphilin, rabphilin and Noc2) exist. Granuphilin-a (also called Slp4-a) is specifically localized on dense-core vesicles in PC12 cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27a via the N-terminal Slp homology domain (SHD). Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane (Tsuboi, et al ). Bitesize is a granuphilin homolog and the only drosophila synaptotagmin-like protein. Mutations that affect bitesize have reduced cell size and number, resulting in smaller animals that develop slowly (Serano, et al ).Rabphilin and Noc2 interact with Rab3a/b/c/d, Rab8a, and Rab27a/b by cotransfection assay (Fukuda, et al (2003b)). Rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27a, not with Rab3a, in PC12 cells. The N-terminal Rab binding domain of rabphilin and Noc2 is proposed to be referred as "RBD27 (Rab binding domain for Rab27)" (Fukuda, et al (2004)).

[RAB27A in platelets]
RAB27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4. Munc13-4 directly bound to GTP-RAB27A and -RAB27B in vitro, but not other GTPases, and enhanced dense core granule secretion (Shirakawa, et al).

[RAB27A in CTLs]
Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. Both Rab27a and Munc13-4 are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant Rab27a (W73G) strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4 (Delta 608-611) failed to bind Rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. This observations suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and that the Rab27a/Munc13-4 complex may be an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion (Neeft, et al). Both Slp1 and Slp2-a are expressed and interact with Rab27a in CTL, by forming part of a docking complex, capturing secretory lysosomes at the immunological synapse,contributing to secretory lysosome exocytosis (Holt, et al). The hematopoietic form of Slp2a (Slp2a-hem) is a specific effector of the active form of Rab27a. Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs. Following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse (Menasche, et al (2008)).

[RAB27A in other cells]

(Granulocytes/neutrophils) Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes (Munafo, et al ). Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation (Herrero-Turrion, et al ). Rab27a effectors JFC1/Slp1 and Munc13-4 are components of the exocytic machinery of granulocytes. Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. JFC1-downregulated granulocytes have impaired MPO secretion. Interference with Rab27a or Munc13-4 but not with JFC1 impaired gelatinase B secretion in neutrophils. Munc13-4 localizes at secretory organelles in neutrophils and plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils (Brzezinska, et al ).

(Enothelial cells) Weibel-Palade body-like structures induced in HEK-293 cells by the expression of von Willebrand factor can recruit endogenous RAB27A. In the absence of von Willebrand Factor, RAB27A is not lysosome associated. Newly formed Weibel-Palade bodies lack RAB27A, which is acquired some hours after initial appearance of the cigar-shaped organelle (Hannah, et al). Rab27a and its effector MyRIP both are present on only mature WPB, and this rab/effector complex appears to anchor these WPB to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, indicating that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content (Nightingale, et al).

(Prostate cells) JFC1 [synaptotagmin-like protein (slp1)], a Rab27a- and PtdIns(3,4,5)P3-binding protein, regulates the androgen-dependent secretion of PSAP and PSA in human LNCaP prostate carcinoma cells. Rab27a and PI3K play a central role in the exocytosis of prostate-specific markers (Johnson, et al ).

(Acinar cells) Rab27 was detected in the apical plasma membrane (APM) and secretory granule membrane (SGM) fractions in acinar cells. Noc2/Rab27 complex is an important constituent of the early stages of IPR-stimulated amylase release (Imai, et al ).

[RAB27B]
RAB27A function can be compensated by a closely related protein, RAB27B. RAB27B is functionally redundant with RAB27A and the pathogenesis of Griscelli syndrome is determined by the relative expression of RAB27A and RAB27B in specialized cell types (Barral, et al ). Up-regulated RAB27B in melanocytes of the Griscelli patient can partially take over the function of Rab27a, which could explain the fact that this patient had an evenly pigmented skin and was able to tan (Westbroek, et al (2004)).

RAB27 shows some homology to Sec4p, which interacts with Myo2b. 8 proteins are shown to be associated with SEC4 in Yeast GRID.

RAB27A drosophila homolog CG14791/Rab27 interaction information in CuraGen interaction database.

Pathway

Involved in melanosome transport.

View the m_rabPathway from BioCarta. Rab27a is required for a late step in granule exocytosis (Haddad, et al ).