Cloning: A human cDNA encoding for Rab31 was first isolated in 1996 (Chen et al. 1996, Bao et al. 1996). Chen et al. (1996) isolated a cDNA encoding for a novel human Rab protein from a human melanocyte cDNA library and human melanocytes. Since its sequence showed a significant homology to canine Rab22 it was named Rab22B. A BLAST analysis revealed that Rab22B shared 71% identity and 82% similarity with canine Rab22. Around the same time, Bao et al. (1996, 1997) identified a cDNA encoding for a previously unknown human Rab protein in human platelets. Because the accepted guideline for the use of the next available Rab numbers at that time was an identity less than 85%, the new protein was named Rab31 instead of Rab22B (Chen et al. 1996; Bao et al. 2002).
Rodriguez-Gabin et al. (2001) identified a Rab sequence (rRab22B) cloned from a rat oligodendrocyte cDNA library with a high identity to human Rab31. Since comparison of the rRab22B sequence with human Rab31 (on the protein level) showed 88% identity it has been suggested that rRab22b encodes the rat homolog of human Rab31. In view of the fact that a segment of the carboxy-terminal domain shows 9 differences among the last 18 residues, one must, however, consider that rRab22B (and also murine Rab22B which is 97.9% identical with rRab22B) still may encode a new member of the Rab family, especially, because the carboxy-terminal end is part of the motif responsible for the targeting of Rab proteins to specific membrane compartments (Rodriguez-Gabin et al. 2001). Despite of this notion the authors refer to rRab22B as Rab31 (Rodriguez-Gabin et al. 2009).

DNA size: 154,326, 7 exons (Fig. 1)

The coding sequence of human Rab31 corresponds to a 194 amino acid (aa) protein with a nominal molecular mass of 21.6 kDa and a pI of 6.7.
The crystal structure of human Rab31 bound to a non-hydrolyzable GTP analog, guanosine-5-(β γ)-imidotriphosphate (GppNHp) at 2.8 Å was described by Tempel et al. (2006) and is presented in Fig. 2A. Rab31 possesses a typically GTPase fold, composed of a six-stranded β-sheet core flanked by five α-helices. The binding site for guanine nucleotides and Mg²⁺ is well conserved among Rab proteins (Fig. 2B; aa 12-19, 60-64, and 118-121). The GTPase fold is followed by a hypervariable region, which harbors two cysteine residues to which geranylgeranyl moieties are covalently attached. Modification by geranylgeranylation is required for its proper subcellular localization, mainly the trans-Golgi network (Chua and Tang 2015).

Non-malignant tissue. Rab31 is expressed fairly ubiquitously in normal human tissue. At the transcript level, highest expression of Rab31 was found in testis, ovary, small intestine, brain, placenta, lung, adrenal gland, urinary bladder, endometrium, and smooth muscle (Chen et al. 1996; Bao et al. 2002; Human Protein Atlas: www.proteinatlas.org, Uhlén et al. 2015). High or medium protein expression levels of Rab31were detected by immunohistochemistry in 35 of 45 analyzed non-malignant tissue types (Human Protein Atlas).
Cancer tissue. Highest Rab31 immunoexpression was observed in malignant tissue of glioma, breast, and thyroid cancer as well as melanoma (Human Protein Atlas).

Most normal cells display weak to moderate cytoplasmic staining, in a few cases of peri-nuclear staining (Human Protein Atlas). Within the cytoplasm, Rab31 was shown to be localized mainly in the Golgi, the trans-Golgi network (TGN) and endocytic compartments (Rodriguez-Gabin et al. 2001).

The members of the Rab family of small GTPases regulate various steps of dynamic assembly and disassembly of multi-protein scaffolds, which are involved in vesicular traffic in both endocytic and secretory pathways. Rab31 regulates anterograde ("inside-out") and retrograde ("outside-in") membrane traffic between the Golgi/TGN and the plasma membrane and/or endosomes.
Rab proteins cycle between two alternate conformational states, inactive (GDP-bound) and active (GTP-bound) (Fig. 3). The activity of the Rab proteins is controlled by several regulatory factors: (i) Rab escort proteins (REPs), which transport newly synthesized Rab proteins to geranylgeranyl transferases for C-terminal prenylation enabling specific membrane association; (ii) guanine exchange factors (GEFs) which catalyze the exchange of GDP for GTP, turning the GTPase into the active state; (iii) GTPase activating proteins (GAPs), which promote efficient GTP hydrolysis resulting in an inactive state; (iv) GDP dissociation inhibitors (GDIs), which extracts GDP-Rab from the membrane; (v) GDI displacement factors (GDFs), which assist re-targeting and re-insertion of Rab into the appropriate membrane (Stenmark 2009; Hutagalung and Novick, 2011; Chua and Tang 2015).
Rab31 interacting proteins
GEFs.
Activation of Rab31 is mediated by several GEFs. Interaction of Rab31 with the different GEFs is mediated by the so-called Vps9 domain (Carney et al. 2006). Using pull-down assays, Lodhi et al. (2007) demonstrated that Gapex-5 (alternative name: GAPVD1, GTPase activating protein and Vps9 domain 1) directly interacts with Rab31. Another study (Kajiho et al. 2011) found that two members of the RIN family (RIN2, RIN3; Ras and Rab interactor) as well as ALS2 (amyotrophic lateral sclerosis 2) and ALS2CL (ALS2 C-terminal like), but not RIN1 or Rabex-5 (also known as RABGEF1) can act as GEFs for Rab31.
Cation-dependent mannose-6-phosphate-receptors (CD-M6PRs).
Rab31 is present in small tubulovesicular organelles trafficking from the Golgi/trans-Golgi network (TGN) to endosomes and is involved in the regulation of the formation these vesicles (Rodriguez-Gabin et al. 2001). Besides its role in the Golgi/TGN organization, Rab31 regulates the transport of CD-M6PRs from the TGN to endosomes (Rodriguez-Gabin et al. 2009). Here, RIN3 likely acts as a GEF to regulate Rab31 dependent CD-M6PR-transport (Kajiho et al. 2011). The M6PR system is one of the membrane transport pathways delivering newly synthesized proteins from the TGN to various endocytic compartments. Thus, this transport links biosynthetic to endocytic pathways and, therefore, is majorly involved in the biogenesis of endosomes, lysosomes, and the plasma membrane (Rodriguez-Gabin et al. 2010). Rab31 was also shown to interact with OCRL, a phosphatidylinositol 4,5-diphosphate 5-phosphatase (PI(4,5)P2 5-phosphatase) that regulates the levels of PI(4,5)P2 and PI(4)P, molecules involved in transport and Golgi/TGN organization. Co-localization of Rab31 and OCRL-1 in carriers transporting M6PRs as well as in endosomes may indicate that both proteins are also involved in the targeting and/or fusion of the carriers with endosomes (Rodriguez-Gabin et al. 2010).
EGF receptor (EGFR).
In addition to its function in the anterograde CD-M6PR-transport, Rab31 plays an important role in the regulation of retrograde EGFR trafficking. Direct interaction of Rab31 with internalized ligand-bound EGFR was demonstrated by co-immunoprecipitation and affinity pull-down assays (Ng et al. 2009). Silencing of Rab31 did not impact internalization of EGFR or its entry in early endosomes, but strongly affected EGFR-transition between early and late endosomes (Chua and Tang 2014). Furthermore, EEA1, a multi-domain tethering factor involved in the fusion of endosomes, also colocalizes with Rab31 suggesting that EEA1 is directly involved in Rab31/EGFR interaction and, by this, is important for Rab31-regulated trafficking of the EGFR between early and late endosomes. Interestingly, silencing of GAPVD1 (Gapex-5), a Rab GEF, reduced Rab31/EGFR interaction and abrogated Rab31-mediated enhancement of EGFR trafficking (Chua and Tang 2014).
Membrane curvature protein APPL2
Additionally, Rab31, together with other members of the Rab5 subfamily (Rab5a, Rab22a, Rab24, see Fig. 2B), has also been shown to bind the membrane-curving protein APPL2 (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein). APPL2 is associated with a distinct subpopulation of early endosomes that link cell surface signaling and endocytosis (King et al. 2012). In macrophages, Rab31 and its effector APPL2 are largely confined to phagosomal membranes displaying important roles in phagosome closure and FcγR signaling (Yeo et al. 2015)
Rab31 and glucose uptake.
In adipocytes, insulin-stimulated glucose uptake is mediated by the glucose transporter SLC2A4 (GLUT4). In the absence of insulin, Gapex-5 appears to function as a Rab31-GEF maintaining Rab31 in its active state which leads to intracellular retention of GLUT4. Upon insulin stimulation, Gapex-5 in complex with another protein, the membrane curvature protein TRIP10 (CIP4), translocates to the plasma membrane resulting in decreased activity of TGN-associated Rab31. In consequence, GLUT4 is translocated to the plasma membrane, where it enables increased glucose uptake into the cell (Lodhi et al. 2007). Recently, another Rab31-regulating protein, NGFR (the p75 neutrophin receptor (p75^NTR)), has been identified (Baeza-Raja et al. 2012). Similar to Gapex-5, p75^NTR supports retention of GLUT4 in intracellular vesicles, thus decreasing glucose uptake. Whereas Gapex-5 acts as a GEF, p75^NTR may function as a GDI displacement factor, which activates Rab31 by displacing it from its GDIs.
Factors regulating Rab expression
mRNA binding protein HuR.
The ubiquitously expressed mRNA binding protein ELAVL1 (HuR) is a member of the Hu/ELAV-family encompassing four members, HuR, ELAVL2 (HuB or HelN1), ELAVL3 (HuC), and ELAVL4 (HuD). It has been proposed to interact with a series of cancer-relevant genes, thereby stabilizing and translationally enhancing its mRNA targets and, furthermore, modulating their transport between the nucleus and the cytoplasm (Cataluce et al. 2010). In fact, overexpression and/or high cytoplasmic HuR levels (representing the functional pool) are associated with poor outcome in several types of cancer including breast cancer (Wang et al 2013, Heinonen et al 2005). In addition, increased cytoplasmic HuR expression was found to be linked to tamoxifen resistance of MCF7 breast cancer cells (Hostetter et al. 2008). Silencing of HuR leads to the downregulation of Rab31 protein in breast epithelial 184B5Me cells. Furthermore, HuR was demonstrated to directly bind to Rab31 mRNA both in 184B5Me and breast cancer MCF7 cells (Heinonen et al. 2011).
Mucin 1 C-terminal subunit (MUC1-C).
MUC1 (Mucin 1), which is overexpressed in most human breast cancers, is a heterodimeric transmembrane glycoprotein, generated by autocleavage of a single polypeptide (Kufe 2009; 2013). The extracellular N-terminal chain, MUC1-N, contains the O-glycosylated tandem repeats, which are typical for mucin family members. MUC1-N is linked to the cell surface via formation of a non-covalent complex with the C-terminal subunit, MUC1-C, comprising a short 58 aa extracellular region, a 28 aa transmembrane domain, and a 72 aa cytoplasmic tail. The cytoplasmic domain of MUC1-C interacts with various receptor tyrosine kinases such as EGFR and ERBB2 (HER2) and, by this, may modulate downstream signaling pathways (Kufe 2009; 2013). MUC1-C has been demonstrated to be internalized by clathrin-mediated endocytosis. Overexpression of mucin 1 in breast cancer is associated with targeting of the cytoplasmic MUC1-C to the nucleus, where it interacts with ESR1 (estrogen receptor α ERα) and stimulates ERα-mediated gene transcription. One of the target genes activated by the ERα/MUC1-C complex is RAB31, the promotor of which contains an ER-responsive element (Jin et al. 2012). Interestingly, Rab31 in turn stimulates upregulation of MUC1-C, likely by attenuating degradation of MUC1-C in lysosomes. In line with these results, Rab31 and MUC1-C are significantly co-expressed in ER-positive tissues of breast cancer patients (Jin et al. 2012). In a more recent study, MUC1-C was reported to block inhibitory effects of tamoxifen on ERα-mediated Rab31 promoter activity in breast cancer cells (Kharbanda et al. 2013).

Rab31 belongs to the large Rab protein family (66 human members) of the Ras superfamily of small GTPases. The Rabs can be subdivided into six supergroups, with human Rab31 belonging to group II encompassing nine human members: the quadrupled Rabs RAB5A, RAB5B, RAB5C, and RAB17; the duplicated RAB31 and RAB22A; the duplicated RAB24 and RAB20; as well as RAB21 (Klöpper et al. 2012). Rab31 displays 71.1% identity (86.1% similarity) with Rab22A, whereas for the other members of the superfamily the identity ranges from about 35-45% (with similarities between about 70-78%). An alignment of the nine members of the group II superfamily is given in Fig. 2B.