Advanced Drug Delivery Reviews 55 (2003) 1421–1437 www.elsevier.com/locate/addr

Rab and endocytic trafficking: potential targets for therapeutic intervention

Mary-Pat Stein, Jianbo Dong, Angela Wandinger-Ness*

Molecular Trafficking Laboratory, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA

Received 5 July 2003; accepted 30 July 2003

Abstract

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (), mental retardation, neuropathy (Charcot–Marie–Tooth), kidney disease (tuberous sclerosis), and blindness () arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway. D 2003 Elsevier B.V. All rights reserved.

Keywords: Small GTPases; Pigmentation and bleeding disorders; Neuropathy; Thyroid disease; Cancer

Contents

1. Introduction ...... 1422 1.1. Constitutive endocytosis and rab proteins ...... 1422 1.2. Maintenance of normal cell physiology ...... 1424 2. Regulation of rab protein function ...... 1424 2.1. Regulated expression levels ...... 1424 2.2. Regulated membrane association and localization ...... 1425 2.3. Regulated activation through nucleotide binding and hydrolysis ...... 1425 2.4. Regulated function through specific effectors ...... 1427 3. Altered rab proteins in disease...... 1429 3.1. Loss of function mutations in rab proteins, rab regulatory molecules, or rab effectors ...... 1429 3.2. Altered rab expression or activation in disease ...... 1431

* Corresponding author. University of New Mexico HSC, 2325 Camino de Salud NE, CRF 225, Albuquerque, NM 87131, USA. Tel.: +1- 505-272-1459; fax: +1-505-272-4193. E-mail address: [email protected] (A. Wandinger-Ness).

0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2003.07.009 1422 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437

4. Therapeutic targets ...... 1432 4.1. Modalities for stimulating rab protein expression and/or function ...... 1432 4.2. Modalities inhibiting rab protein expression and/or function ...... 1433 5. Concluding remarks ...... 1433 Acknowledgements ...... 1433 References ...... 1433

1. Introduction endocytic transport, providing the spatial and temporal control required for endocytic transport fidelity. Based Numerous human diseases can be attributed to on our current understanding, the rab proteins regulate alterations in endocytic trafficking. The rab GTPases individual transport steps by controlling cargo selec- and associated proteins are critical regulators of endo- tion [2] and modulating vesicle budding, directed cytic transport. Increasingly, rab proteins and their targeting, and fusion [3]. As detailed further below, effectors are found overexpressed or subject to loss of rab protein activity depends on their selective activa- function mutations in human disease. The alterations tion and ability to act as scaffolds for the recruitment of impact cellular physiology by perturbing the homeo- specific effectors in a spatially and temporally con- stasis of key cell surface receptors, lipid metabolism, trolled manner. Approximately 60 rab proteins are hormone processing, and specialized secretory path- encoded by the , with additional rab ways. Consequently, diverse diseases ranging from proteins possibly generated by alternative splicing [4]. mental retardation to cancer may be attributed to the A subset of 13 rab proteins is utilized on the endocytic derangement of the regulatory machinery governing endocytic membrane trafficking. This review begins Table 1 with an overview of rab protein function and regulation Endocytic rab proteins and highlights how these processes are disrupted in Rab Rab function References disease with the aim of identifying potential avenues Rab4 Localized on EEa and RE; regulates [84,85] for therapeutic intervention. sorting/recycling Rab5 Formation of CCV from PM; CCV–EE [41,42,86–88] 1.1. Constitutive endocytosis and rab proteins and EE–EE homotypic fusion; endosome motility Rab7 Required for EE–LE transport and [10,89,90] Endocytosis is a fundamental cellular process re- LE–Lys fusion quired for the regulated uptake and intracellular trans- Rab9 Endosome to TGN transport [12,91] port of macromolecules. There are numerous routes Rab11 Recycling through perinuclear RE; [92–95] whereby molecules may be internalized including re- exocytosis from TGN to PM; implicated in polarization of the Drosophila oocyte ceptor-mediated endocytosis via clathrin-coated vesi- Rab14 Implicated in endocytosis, lysosome [96,97] cles, caveolar uptake, macropinocytosis, and phagocy- fusion, and phagocytosis tosis (reviewed in Ref. [1]). The discussions in this Rab15 Implicated in EE sorting and perinuclear [6] review will center on receptor-mediated endocytosis. recycling Beginning with the initial steps of receptor seques- Rab17 Required for transcytosis in epithelial [98,99] cells tration and culminating with the delivery of cargo to Rab20 Implicated in endocytosis and recycling [100] specific intracellular destinations, endocytosis is a in epithelial cells highly regulated process. Following internalization, Rab22 Localized on EE and LE; implicated in [101,102] molecules in vesicular carriers derived from the plasma endocytosis membrane are transported to specific intracellular des- Rab25 Implicated in ARE recycling [93] Rab34 Implicated in macropinosome formation [103,104] tinations allowing for coordinate regulation of hor- and lysosome distribution mone and growth factor receptor signaling, sampling Rab39 Implicated in endocytosis [105] and presentation of antigens for immune recognition, a ARE, apical recycling endosome; CCV, clathrin-coated vesicle; and general cellular homeostasis. The small ras-related EE, early endosome; LE, late endosome; RE, recycling endosome; rab GTPases have emerged as important regulators of TGN, trans-Golgi network. M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1423 pathway (Table 1) and serves to determine the fate of transport to lysosomes for degradation. Recycled mol- endocytosed cargo. ecules can either be sorted into rab4 containing micro- Although many rab proteins have been implicated in domains within early endosomes that permit fast endocytic transport (Table 1), only a small subset has recycling to the plasma membrane [8] or be transported been extensively characterized (Fig. 1). Early endo- to perinuclear recycling endosomes where rab11 regu- cytic events are primarily regulated by rab5 and rab15. lates transport back to the plasma membrane [9]. Rab5 facilitates segregation of cargo into clathrin- Molecules destined for degradation are delivered in a coated pits and together with a number of identified rab7-dependent transport step from early to late endo- effector molecules, promotes cytoskeletal motility and somes [10]. Rab7 may also function downstream of late homotypic early endosome fusion (reviewed in Ref. endosomes facilitating transport to lysosomes in asso- [5]). In contrast, rab15 inhibits transport to early endo- ciation with its one identified effector protein, RILP somes, directly opposing the activity of rab5 [6,7]. [11]. An additional arm of the endocytic pathway Molecules that reach early endosomes are subsequently provides for rab9-mediated recycling of molecules sorted for recycling back to the plasma membrane or such as the mannose 6V-phosphate receptor (M6PR)

Fig. 1. Schematic diagram of the rab-regulated endocytic pathway. Molecules internalized by endocytosis are initially transported to early endosomal compartments that are characterized by the presence of rab5 and rab4. Sorting of molecules into rab4-containing microdomains within early endosomes results in immediate transport back to the plasma membrane while sorting into rab5-containing microdomains leads to transport either to perinuclear localized recycling compartments or toward lysosomes for degradation. Transport to perinuclear recycling endosomes is subsequently followed by rab11-mediated recycling to the plasma membrane. Transport from early endosomes to late endosomes is facilitated by rab7, and cycling of molecules from late endosomes to the TGN is mediated by rab9. The transport of molecules to lysosomes is mediated by rab7 and RILP. Finally, secretory lysosomes, such as those found in melanosomes, are delivered back to the plasma membrane in a calcium-regulated process. Abbreviations: CCP, clathrin-coated pits; CCV, clathrin-coated vesicle; EE, early endosome; LE, late endosome; LYS, lysosome; RE, recycling endosome; TGN, trans-Golgi network. 1424 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 from late endosomes to the TGN [12].Theserab 2. Regulation of rab protein function proteins are the most highly understood of the consti- tutively expressed endocytic rabs. Several additional Rab proteins direct vesicular transport by means of endocytic rab proteins, primarily localized to special- their localization to select intracellular compartments ized cells, have also been characterized: (trans- and through specific interactions with multiple effec- port of melanosomes), rab25 (apical recycling tor proteins. Downstream regulation of rab effector endosome to TGN), and rab17 (endosomal recycling proteins is in turn dependent on the tight regulation of in epithelia) [3]. Each endocytic rab protein character- the rab proteins themselves. Regulation occurs at ized to date functions in concert with a relatively multiple levels and in conjunction with numerous unique set of effector molecules. Therefore, continued regulatory proteins. Transcriptional and translational characterization of the less well-characterized rab pro- mechanisms control rab protein expression. Mem- teins is imperative to achieve a more complete under- brane localization is controlled through specific post- standing of the molecular mechanisms governing translational modification and membrane recruitment. endocytosis. Selective rab protein activation depends on nucleotide exchange and hydrolysis, and localized function 1.2. Maintenance of normal cell physiology occurs through interaction with unique effector pro- teins. Thus, the rab proteins govern intracellular Functional endocytic trafficking is central to normal vesicular trafficking by acting as localized scaffolding cellular physiology. For this reason, the manipulation platforms to exert temporal and spatial control of of constitutive or cell-type-specific rab proteins could transport. Below, the well-characterized regulatory provide a means for treating diseases resulting from events associated with rab protein expression and aberrant intracellular transport. For example, overex- functional control are presented. As the identification pression and delayed degradation of epidermal growth of factors that modulate rab function in vivo contin- factor receptors, in particular ErbB2, has been associ- ues, additional regulatory mechanisms will surely be ated with tumor formation and poor prognosis in breast revealed. cancer patients [13]. Targeting ErbB2 receptors to lysosomes might aid in reducing intracellular pools of 2.1. Regulated expression levels ErbB2 receptors resulting in decreased and reduced cell proliferation. In thyrocytes, increased Most of the endocytic rab proteins are constitutively overall levels and increased membrane-bound fractions expressed in all mammalian cells. Nevertheless, indi- of rab5a and rab7 have been linked to formation of vidual rab protein levels vary quantitatively between benign thyroid autonomous adenomas [14]. Altering cell types (reviewed in Ref. [19]). In addition, tissue- the amount and rate of thyroglobulin transport and specific expression of rab proteins in cells such as processing, and the subsequent basolateral secretion epithelia and neurons fulfill the need to regulate of T3 and T4 by reducing rab5a and rab7 expression, specialized transport processes in these polarized cells. might lead to a reduction in tumor formation. In The data imply that individual cells express a partic- Alzheimer’s patients, aberrant processing and transport ular repertoire of rab proteins to fulfill their cellular of amyloid precursor protein (APP) in axons leads to vesicular transport needs. the generation of Ah plaques and the progression of Altered rab expression in response to various neurodegenerative disease (reviewed in Ref. [15]). inflammatory stimuli and diseases has been reported Manipulating the transport of Ah-containing vesicles and suggests additional plasticity is exerted through may facilitate clearance of accumulated APP. Finally, the control of rab gene expression. Modulation of roles for rab proteins in G-protein-coupled receptor expression offers another level of control that might endocytosis, desensitization, and downregulation be manipulated for therapeutic purposes. Increased (reviewed in Ref. [16]) and in cholesterol and lipid expression of rab5a and rab7 occurs in response to metabolism and trafficking [17,18] demonstrate that cAMP [14], while rab5a can also be increased by rab proteins are critically important for a wide variety interferon gamma in macrophages [20]. Conversely, of normal cellular functions. intestinal epithelia treated with transforming growth M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1425 factor h, exhibited decreased rab11 expression [21]. (GDP-rab) complexes in the cytosol. Importantly, The possibility of regulating membrane trafficking rabGDI extracts GDP-rab from intracellular mem- through the control of rab protein expression is clearly branes and recycles GDP-rab back to donor mem- illustrated through these studies on inflammatory branes (Fig. 2, steps 11–13). Major conformational responses and may serve as a paradigm for other changes in rabGDI occur upon binding to membrane- disease situations with altered rab expression. Indeed, associated GDP-rab, resulting in the dissociation of the altered expression of rab proteins in diseases such as rabGDI:GDP-rab complex from acceptor membranes low-grade dysplasia associated with Barret’s esopha- [28]. Recycling of GDP-rab proteins to specific mem- gus (rab11) [22], cardiomyopathy (rab1, rab4, and brane compartments is mediated by compartment- rab6) [23], lung tumor progression (rab2) [24], pros- specific receptors that recognize the soluble rabG- tate and liver cancer (rab25) [25,26], and possibly DI:GDP-rab complex. The membrane-bound Hsp90 atherogenesis (rab7) [27], point to the importance of chaperone complex has been identified as a putative understanding RAB gene control and the identification rabGDI:rab receptor [29]. Alternatively, the rab GDI of factors affecting RAB expression. Such pathways displacement factor (GDF, discussed below) may also may then be targeted to selectively increase or decrease serve as a rabGDI:GDP-rab receptor. Upon binding to rab protein expression and achieve the desired modu- donor membrane receptors, rabGDI is released from lation of endocytic transport. GDP-rab, allowing recycling of rabGDI for additional rounds GDP-rab extraction and transport. 2.2. Regulated membrane association and localization Highly conserved sequences within REP and rabGDI form a general binding platform that facilitates Following protein synthesis, rab proteins interact rab protein recognition. Complementary conserved with several common rab regulatory molecules, which residues within all mammalian rab proteins enable result in posttranslational modification and membrane rab protein binding to these and other common regu- association of rab proteins. One or two cysteine latory molecules [30]. Since the conserved binding residues in the C-termini of all rab proteins are motifs within the regulatory molecules bind to a variety modified with isoprenoid moieties to allow for mem- of rab proteins, the disruption of multiple cellular brane localization. Modification occurs when newly functions by mutation would be predicted to have dire synthesized rab proteins first interact with rab escort consequences. However, due to the expression of proteins (REP) (Fig. 2, steps 1–4). Next, the rab:REP multiple isoforms of rab regulatory molecules with complex is specifically recognized by a rab geranyl- redundant function, mutation of REP1 results specifi- geranyl transferase (RabGGTase), which catalyzes the cally in retinal degeneration whereas loss of GDI addition of geranylgeranyl groups to C-terminal function results in severe mental retardation. Modulat- cycteine residues in the motifs CC, CXC, CXXX, ing endocytosis through the manipulation of common CCXX, or CCXXX (where X is any amino acid). The regulatory molecules is therefore unlikely to be a geranylgeranylated rab proteins are subsequently de- tractable therapeutic strategy. Deficits in these regula- livered to intracellular membranes in a complex with tory molecules should be considered candidates for REP, which maintains the modified rab protein in a . soluble form. Targeting of REP proteins to specific intracellular membranes may involve membrane 2.3. Regulated activation through nucleotide binding receptors, which have not yet been identified. Once and hydrolysis the geranylgeranylated rab protein is presented to the specified target membrane, REP is released and In addition to cycling between membrane compart- recycled to the cytosol for additional rounds of escort. ments, all rab proteins also cycle between GDP-bound REP proteins share with rab GDP (‘‘inactive’’) and GTP-bound (‘‘active’’) states (Fig. 2, dissociation inhibitors (rabGDI), another rab regula- steps 5–10). After extraction from membranes by tory protein. rabGDI, rab proteins either remain in a cytosolic Similar to REP, rabGDI binds GDP-bound rab complex associated with rabGDI or are delivered back proteins, producing soluble rabGDI:GDP-bound rab to donor membranes. Dissociation of rabGDI at the 1426 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437

Fig. 2. Regulation of rab membrane localization and nucleotide cycling. Newly synthesized GDP-bound rab proteins are bound by REP (1), which then presents the new rab proteins to rabGGTase (2). Following geranylgeranylation of rab protein C-termini, REP delivers the newly modified rab proteins to their appropriate donor membranes (3), dissociates and recycles for additional rounds of rab escort (4). Once released, rab proteins quickly insert into the donor membrane and undergo exchange of GTP for GDP with the aid of GEFs (5–7). Rab proteins may exist in a nonnucleotide-bound transition state in vivo (6), stabilized by guanine nucleotide-free chaperones (GNFC) such as Mss4-like TCTP proteins. GTP-bound rab proteins interact with effector (E) molecules on ECV (8) and at their acceptor membranes, facilitating transport, tethering, and fusion of vesicles at their appropriate destinations. Hydrolysis of GTP is accelerated by GAPs, leading to dissociation of associated effector molecules (9,10). Removal of GDP-bound rab proteins from target membranes is mediated by GDI, which solubilizes rab proteins and targets rab proteins back to donor membranes (11). Interaction of GDI with GDF releases the rab protein allowing reinsertion into the donor membrane (12), and GDI recycles into the cytosol for additional rounds of extraction and transport (13,14). Abbreviations: REP, rab escort protein; rabGGTase, rab geranylgeranyl transferase; GEF, guanine exchange factor; TCTP, translationally controlled tumor-associated proteins; E1, E2, E3, effector molecules; ECV, endocytic carrier vesicle; GAP, GTPase activating protein; GDI, guanine dissociation inhibitor; GDF, GDI dissociation factor. donor membrane is facilitated by membrane-bound factors (GEFs). GEFs catalyze the exchange of GTP GDI-displacement factor (GDF) [31]. Upon release of for GDP; however, the molecular mechanisms govern- rabGDI, the geranylgeranyl moieties of GDP-rab rein- ing nucleotide exchange are not yet fully appreciated. sert into the donor membrane, readying the rab for A class of proteins sharing sequence similarity with another cycle of activation. Membrane association of the guanine nucleotide-free chaperones Mss4/Dss4 GDP-rab promotes nucleotide exchange from GDP to [32] has recently been identified. Translationally con- GTP; however, the intrinsic rate of nucleotide exchange trolled tumor-associated proteins (TCTP) are highly is very slow. The rate of nucleotide exchange is greatly expressed in a wide range of mammalian cells and may enhanced by specific guanine nucleotide exchange function as guanine nucleotide-free chaperones [33]. M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1427

Evidence for a functional role of nonnucleotide bound and as interfaces to intracellular signaling cascades. In rab proteins in endocytosis derives from recent evi- this context, rab proteins may control the fate of dence demonstrating that rab15 function requires Mss4 individual cargo molecules by interacting with cargo binding and that Mss4 is a rab15 effector [34]. Func- directly or via specialized effectors that are induced in tional roles for other rab proteins bound to nucleotide- response to signaling. Examples of rab proteins and free chaperones will undoubtedly be uncovered. Fur- their effectors serving in each of these capacities are thermore, understanding how cycling from GDP- to detailed below. nonnucleotide- to GTP-bound states is regulated will Principally, the interaction of rab proteins with be critical for determining how and when conforma- effector molecules facilitates membrane transport tional changes in the rab proteins allow for functional through a coordinate assembly process. The rab pro- interactions with downstream effector molecules. teins serve as molecular scaffolds for the sequential GTP hydrolysis leads to the cycling of rab proteins recruitment of proteins and lipids that are required for from their active GTP-bound to inactive GDP-bound transport vesicle budding, cytoskeletal motility, and state. Although all rab proteins contain some intrinsic finally vesicle tethering, docking, and fusion with the GTPase activity, the rate of phosphate hydrolysis is target membrane. Effector molecules for a number of greatly enhanced by the presence of GTPase activating rab proteins have been identified and characterized proteins (GAPs). In mammalian cells, cytosolic GAP (Table 2). The best-characterized set of rab effector activity has been demonstrated to exist for a number of proteins are those that interact with rab5. Analysis of rab proteins. However, only two mammalian rab the rab5 effectors suggest a general model by which GAPs have been definitively identified: GAPCenA, interactions between rab proteins and effector mole- which has sequence similarity to a yeast Ypt/rab GAP cules may lead to the formation of macromolecular [35]; and Rab3-GAP, which has no sequence similar- scaffolds for efficient and accurate membrane trans- ity to any known GAP proteins [36]. Acceleration of port [5]. GTP hydrolysis by GAPs results from the insertion of Rab5 effector interactions are tightly coupled to rab a common arginine finger motif found in yeast Ypt/ activation. GTP-rab5 initially recruits regulatory com- rabs, ras, and rho, into their substrate [37]. Rates of plexes of rabaptin-5 and rabex-5 to early endosomes. hydrolysis up to 1000-fold over baseline have been Rabex-5 acts as a rab5 nucleotide exchange factor and observed. rabaptin serves as an accessory factor for coated The factors regulating nucleotide exchange and vesicle formation [38], as well as vesicle tethering hydrolysis appear to be highly specific for individual [39]. The GTP-rab5 bound to early endosomes also rab proteins. The exchange of GTP for GDP by GEFs recruits the p150/hVPS34 complex to early endo- provide the molecular ‘‘on-switch’’ for rab proteins somal membranes by binding the p150 adapter pro- while the hydrolysis stimulated by GAPs provides the tein [40]. Once membrane associated, hVPS34, a ‘‘off-switch’’. These opposing reactions therefore play phosphatidylinositol (PI) 3V-kinase, generates PI 3V- a crucial role in regulating the temporal dynamics of phosphate (PI3P) in the plane of the early endosomal vesicular membrane trafficking. Thus, these proteins membrane. The newly formed PI3P microdomains may be attractive therapeutic targets to enhance or regulate endosome motility on microtubules [41] and diminish rab protein activity and modulate select endo- provide platforms for assembly of the fusion machin- cytic trafficking pathways. ery. Molecules containing the FYVE domain specif- ically and preferentially bind to PI3P [42,43].Two 2.4. Regulated function through specific effectors such FYVE-domain containing proteins specifically recognize PI3P on early endosomes and promote ve- Rab proteins serve to promote trafficking by coor- sicle docking and fusion [44–46]. EEA1, early endo- dinating the individual steps in vesicular transport. In somal autoantigen 1, is a tethering molecule that this context, the rab proteins can be viewed as molec- interacts with syntaxins 6 and 13 to establish vesicle ular scaffolds that promote temporal and spatial control contact with the target membrane [47]. Rabenosyn-5 through their compartment-specific localization and is required for SNARE complex formation and thus activation. Rab proteins also serve in cargo selection promotes fusion [48]. The assemblies of macromo- 1428 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437

Table 2 Table 2 (continued) Endocytic rab effector proteins Rab Rab effector Function References Rab Rab effector Function References Rab11-GTP Eferin EF-hand domain [119] Rab4-GTP Rabip4 Implicated in [106] containing protein, retrograde transport implicated in rab11 from REa to SE localization Rabaptin-5 Coordinates [107] Rab11BP Implicated in vesicle [120,121] endocytosis and (Rabphilin 11) recycling recycling from EE Rab11-GDP, Rab11-FIP Implicated in [122] Rabaptin4 Implicated in [108] Rab11-GTP recycling; homo- and transport from EE heterodimerization to to RE create protein RCP Implicated in [109] platforms recycling Rab34-GTP RILP Regulates [104] Rab5-GTP EEA1 EE homotypic [110] lysosomal tethering and fusion positioning Rabaptin-5 Forms a stable [38,111] a ARE, apical recycling endosome; CCV, clathrin-coated vesicle; complex with EE, early endosome; Eferin, EF-hands-containing Rab11-interacting Rabex-5; implicated protein; Rab11-FIP, rab11 family-interacting protein; LE, late in membrane endosome; M6PR, mannose 6-phosphate receptors; PI3K, phosphoi- docking and fusion; nositol-3V-kinase; RCP, rab coupling protein; RE, recycling endo- recently shown to some; SE, sorting endosome; TGN, trans-Golgi network. participate in clathrin adapter binding Rabaptin-5h Implicated in fusion [112] Rabenosyn-5 Required for CCV– [48] lecular complexes on clathrin-coated vesicles and EE and EE–EE early endosomes are triggered by rab5 activation. fusion Together, rab5, phospholipids, and a variety of rab p110h Class I PI3K [113] effector molecules contribute to the coordinate control catalytic subunit of early endocytic trafficking and culminate in regu- PI3K Class III PI3K [113] (hVPS34) lated endosomal fusion. These events provide a par- p150 Class III PI3K [40,114] adigm for other intracellular membrane trafficking adapter protein for and fusion events. The directionality and specificity hVPS34; serine/ of transport relies on the specific interactions between threonine protein rab proteins and their effector molecules while the kinase RIN2 Guanine nucleotide [115] temporal specificity of these interactions relies on the exchange nucleotide cycling of the rabGTPases. Rab5-GDP RIN1 Guanine nucleotide [52] New data document rab protein function in cargo exchange selection, offering unprecedented opportunities for Rab7-GTP RILP Regulates LE to [11] modulating the fate of specific molecules. Rab pro- lysosome transport through interactions teins may directly interact with some cargo molecules, with dynein as is the case for the interaction between rab3b and Rab9-GTP p40 Stimulates transport [116] polymeric IgA receptor [2,49]. Alternatively, rab of M6PR from LE to proteins may interact with specific cargo via a unique TGN intermediary, as illustrated by the requirement for TIP47 Bind to both M6PR [117] and Rab9-GTP; TIP47, which mediates the interaction between rab9 cytosolic recognition and the mannose 6-phosphate receptor [50,51]. Cargo factor for M6PR selection may also depend on interfaces with intracel- Rab11-GTP Rip11 Required for ARE to [118] lular signaling cascades that can serve to increase or apical PM transport decrease rab protein activity. The regulation of EGFR RCP Homologue of Rip11 [109] endocytosis illustrates the complexity of such stimuli. Ras activation induced by EGFR may on the one hand M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1429 increase endocytosis and promote receptor downregu- infections due to defects in T cell cytotoxicity and lation. Endocytosis, in this case, is stimulated by cytolytic granule release. Partial albinism results from Rin1, a protein with rab5 GEF activity that converts the accumulation of melanosomes in melanocytes. rab5 to its active form [52]. On the other hand, ras Hemophagocytic syndrome is caused by hyperstimu- activation may also attenuate endocytosis. In this case, lated T cells and macrophages [55]. The genetic rab5 is inactivated by the action of a specific rab5 defects responsible for GS2 include three distinct GAP, RN-Tre, which is recruited by the activated ras missense mutations in highly conserved residues and effector Eps8 [53]. Such opposing activities are most numerous microdeletions or larger deletions in probably temporally balanced during normal signal- RAB27A on 15q21[55]. Rab27a has ing, but may be aberrant in some disease states where been shown to be critically important for the transport EGFR is constitutively active. and release of melanosomes, a form of secretory In summary, selective manipulation of rab proteins lysosome, from melanocytes [56]. Similar defects in or their effectors offers the potential to disrupt specific secretory lysosome release from immune cells and transport steps and preferentially shunt cargo for recy- platelets account for the bleeding disorders and im- cling or degradation. In addition, as we learn more mune dysfunctions associated with the disease [57,58]. about specialized cargo selection pathways, it is con- A related syndrome, Griscelli syndrome type 1 ceivable that the trafficking of individual receptors may (GS1), is also characterized by partial albinism but be precisely controlled and modulated for therapeutic exhibits a primary severe neurological impairment benefit. without deficits in immune function [59]. The primary defect in GS1 was localized to MYO5A. MYO5A en- codes an unconventional myosinVa motor protein [60]. 3. Altered rab proteins in disease Mouse coat-color variants equated with GS1 (dilute [61]) and GS2 (ashen [58]) originally helped to define Increasingly, rab proteins and associated regulatory the intracellular transport pathway of melanosomes. An molecules or effectors are shown to be altered in additional mouse model, the leaden mouse, identified human disease. The alterations may be associated melanophilin [62] as a rab27a-interacting partner that with a loss of function, in which case, an underlying mediates the recruitment and binding of myosinVa to germline mutation is frequently responsible. Loss of [63,64]. Thus, human and mouse studies have function mutations in the rab protein or associated elucidated the components and fundamental mecha- regulatory molecules and effectors frequently have nisms requisite for secretory lysosome transport and similar phenotypic outcomes. Rab protein overexpres- release, demonstrating that phenotypically similar dis- sion or aberrant activation, triggered by somatic eases may arise due to disruptions of multiple compo- mutation or altered signaling, also underlie a number nent on the same intracellular pathway. of disease states. Recently, a second genetic disorder was pinpointed to defects in a rab gene. Mutations in the RAB7 gene 3.1. Loss of function mutations in rab proteins, rab cause Charcot–Marie–Tooth type 2B neuropathy regulatory molecules, or rab effectors [65]. The disease is marked by sensory and motor neuron impairment, distal muscle weakness and atro- Genetic mutations affecting rab proteins and asso- phy, and ulcerations often requiring amputation. Mis- ciated regulatory molecules have recently come to sense mutations in RAB7 were localized to exons 3 light. To date, two diseases have been characterized and 4, where either a C to T transition led to substi- with causal mutations in rab , while others tution of Leu129 for Phe or a G to A transition resulted impact accessory factors as detailed below. in mutation of Val162 to Met [65]. The Val162 residue The first human disease identified to result from a is highly conserved in all species and Leu129 is mutation of a rab gene was Griscelli syndrome type 2 localized adjacent to the GTP-binding domain of (GS2). GS2 is a rare autosomal recessive disorder rab7. As such, both mutations are predicted to disrupt originally described in 1978 [54]. Patients exhibit rab7 function. A second form of Marie–Charcot– immune impairment and increased susceptibility to Tooth disease (MCT4B1) results from a genetic defect 1430 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 in myotubularin-related protein 2 [66], a dual speci- membrane association of rab proteins. Altered regula- ficity phosphatase required for PI3P and PI3,5P2 tion of rab5 nucleotide hydrolysis promoted by dis- metabolism [67]. A third form of Marie–Charcot– crete cofactors has been implicated in both prostate Tooth disease (MCT2A) was previously characterized cancer and tuberous sclerosis. In the case of tuberous based on missense mutations in KIF1B [68].The sclerosis, this is precipitated by germline mutation of KIF1B gene encodes a kinesin motor protein thought the cofactor’s coding sequences, while in the case of to play a crucial role in synaptic vesicle transport. By prostate cancer, the cofactor protein is highly overex- analogy with Griscelli syndrome, it seems likely that pressed. The nature of a causal mutation in prostate rab7, myotubularin, and KIF1B are also part of a cancer has not been defined, but may arise from common molecular pathway such that disruption of somatic rearrangements. any one of the genes causes a similar disease pheno- Choroideremia, a form of retinal degeneration due type that is manifested as peripheral neuropathy. In this to loss of retinal epithelium, choroids, and retinal context, it is of interest to consider our observations photoreceptor cells, ultimately causes blindness in demonstrating that rab7 and a PI 3V-kinase, hVPS34, affected individuals [30]. A number of point mutations coordinately control late endocytic transport*. The in rab escort protein1 (REP1), as well as translocations rab7-regulated formation of PI3P on late endosomes that disrupt REP1 gene expression, have been de- may facilitate kinesin-regulated membrane recycling, scribed [72]. Identification of two REP genes, REP1 particularly in motor neurons. Turnover of PI3P may and REP2, suggest that functional redundancies in rab subsequently be temporally regulated by myotubu- regulatory molecules may exist. However, tissue-spe- larin-related protein phosphatase, providing a molec- cific expression of REP1 or the specificity of particular ular ‘‘off-switch’’ for the kinesin-mediated vesicular rab proteins for REP1 could result in an absolute transport. Further characterization of the intracellular requirement for REP1 in a given tissue. This appears pathways governed by rab proteins and identification to be the case in retinal epithelia, where loss of REP1 of rab effector molecules will undoubtedly uncover activity results in retinal degeneration and loss of additional molecules that are mutated to cause a eyesight in affected individuals. Functional redundan- variety of human diseases. Consequently, effective cy through the expression of REP2 may partially gene therapy strategies for reconstitution of these compensate for loss of REP1 activity, except where molecules will be increasingly important. REP1 activity is absolutely required. In the case of rab27, the disease etiology matches X-linked nonspecific mental retardation (MRX) has the expression profile of the protein. Rab27 is highly also been associated with genetic defects in a rab expressed in keratinocytes, platelets, and lymphocytes, regulatory molecule, rabGDIa. Both a truncation mu- where it promotes secretory lysosome fusion. In the tation in rabGDI (MRX48) and a T to C transition that absence of rab 27, secretory lysosome fusion is dis- resulted in a nonconservative amino acid change rupted, leading to bleeding and pigmentation disor- (Leu92 to Pro) in GDIa were described in two distinct ders, as well as immune dysfunction. In contrast, rab7 families with MRX [73].RabGDIa is critical for is a ubiquitous rab that controls transport between neurotransmitter release and recycling in the early and late endosomes. Determining how mutations brain, and the introduction of proline at position 92 in in rab7 result in sensory and motor neuropathy and rabGDIa decreased the affinity of rabGDIa for rab3a. identifying how the loss of rab7 function is compen- Consequently, this mutation might cause inefficient sated for in other cells, tissues, and organs remain recycling of rab3a from synaptosomes, possibly lead- critical unresolved issues for understanding the com- ing to mental defects. Furthermore, rabGDIa has been plexities of rab function in vivo. implicated in neuronal development based on expres- Genetic defects in rab regulatory molecules are sion at embryonic day 9 and its requirement for axonal associated with retinal degeneration in choroideremia, extension [73]. Expression of additional rabGDI pro- X-linked mental retardation, and kidney disease in teins and promiscuous binding of rab proteins to tuberous sclerosis [69–71]. Choroideremia and X- rabGDIs must allow for sufficient recycling of rab linked mental retardation result from germline muta- proteins in tissues and organs outside the brain. Similar tions in general regulatory factors that impact the to the case of REP2, expression of additional rabGDI M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1431 proteins does not compensate for loss of rabGDIa These results suggest that rab7, cholesterol, kinesin expression in brain. motor proteins, and the NPC1 and NPC2 proteins may Genetic defects in less well-characterized, rab-re- interact on late endosomes, resulting in the proper lated proteins may also result in severe disease. One transport and maintenance of cellular lipids. A greater example, tuberous sclerosis (TSC), is an autosomal understanding of these lipid transport processes will be dominant disease with a variety of manifestations invaluable, providing information both about the NPC including the formation of benign tumors called disease process as well as identifying molecules re- hamartomas, mental deficits that include behavioral quired for lipid homeostasis. With increased under- and learning difficulties, and renal complications in- standing, manipulation of cholesterol and lipid cluding but not limited to renal lesions. Genetic defects transport processes may provide therapeutic benefit in two distinct loci, TSC1 and TSC2, account for the to those suffering from lipid storage diseases. majority of cases. TSC1 encodes hamartin, a 130-kDa protein of unknown function while TSC2 encodes 3.2. Altered rab expression or activation in disease tuberin, a protein that stimulates GTP hydrolysis on rab5 and rap1 and thus behaves like a GAP [74]. As discussed above, many proteins participate in Hamartin and tuberin interact with one another in vivo the regulation of rab protein expression and localiza- and act as tumor suppressor genes [75,76]. Defects in tion and genetic defects in rabs or their regulatory either TSC1 or TSC2 are predicted to disrupt the molecules can result in disease. In addition, control intracellular complex formed by these proteins, there- over the levels of rab protein expression must exist. by inhibiting the unknown activity of hamartin. A role Overexpression of requisite endocytic rab proteins or for the tuberin/hamartin complex in intracellular trans- regulatory proteins has been associated with human port was suggested by recent findings that polycystin- thyroid, vascular, and lung diseases, as well as some 1, an integral membrane component of basolateral cancers [4,14,24,26,27]. Overexpression may be pre- adherens junctions, was mislocalized to the Golgi in cipitated by somatic rearrangements as in the case of cells deficient for TSC2 expression [77]. Further detail some prostate cancers or may arise in response to regarding the functions of tuberin and hamartin in sustained stimuli from intracellular signaling. vivo, as well as their relationships to rab5 and other Thyroid hormone production requires the uptake components of the endocytic regulatory machinery, is and processing of thyroglobulin (Tg) from apical essential to gain clues about the molecules that are extracellular colloidal stores, endocytosis and pro- most acutely affected by the defects in TSC. cessing of Tg in late endosomes and lysosomes, A role for rab proteins in the transport and mainte- and transport and release of mature thyroid hormone nance of cellular cholesterol and lipids was recently at the basal surface. Thyroid autonomous adenomas revealed. Niemann–Pick type C (NPC) disease is a (AA) are benign tumors of the thyroid associated genetic disorder in which the accumulation of lipids in with elevated levels of rab5a and rab7 and decreased late endosomes ultimately causes a severe neurode- levels of follicular Tg [14]. Membrane association of generative disorder resulting in premature death. The both rab5a and rab7 are also increased in AA, vast majority of NPC cases result from genetic muta- suggesting that rab5 and rab7 are both activated in tions in NPC1, while a small percentage result from AA. Increased expression of rab5a and rab7 increase mutations within a second gene, NPC2 [78]. These the rate of thyroglobulin endocytosis and processing genes encode proteins required for lipid trafficking, in response to elevated cAMP [14];however,it although the precise molecular mechanisms have not remains to be determined if the enhanced processing yet been elucidated. Overexpression of rab7 or rab9 in of Tg results in tumor formation or if additional NPC cells alleviated cholesterol accumulation and factors are involved. restored transport of several glycospingolipids, lacto- Alterations in endo- and exocytic rab protein sylceramide, and GM1 to the Golgi [17]. Importantly, expression have also been revealed in several models the motility of rab7-containing late endosomes is of human disease and in a variety of cancers. Upre- directly altered by cholesterol accumulation, possibly gulation of , rab4, and rab6, and altered Golgi through the inhibition of motor protein binding [18]. morphology were observed in a h2-adrenergic recep- 1432 M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 tor model of cardiomyopathy [23], while increased nomas, it may be sufficient to transiently downregulate expression of rab7 was observed in a rabbit model of rab protein expression, while in the case of genetic loss atherogenesis [27]. Similarly, in a mouse model for of function diseases, it will be crucial to have sustained lung tumor progression, increased expression of rab2 and permanent reconstitution of protein function. Fi- was detected [24] whereas in prostate cancer cell nally, it is important to consider therapeutic interven- lines, alterations in rab25 expression were noted tions that can restore rab protein function, as well as [25]. Recent work identified six rab and three Arf/ those that can block function. Such situations arise Sar proteins that are upregulated in human liver when the trafficking of specific cargo may need to be cancers, including hepatocellular carcinomas and modulated. cholangiohepatomas [26]. Thus, alterations in expres- sion of a variety of rab proteins, as determined by 4.1. Modalities for stimulating rab protein expression gene expression profiling, suggest that rab proteins and/or function play a multitude of roles in maintaining normal cellular physiology. As discussed above, rab proteins may be regulat- The novel prostate cancer gene 17 (PRC17) pro- ed at the level of expression or activation. Changes vides an example where overexpression of a rab in expression or activation may be achieved either regulatory factor decreases rab activation and results by overexpressing specific genes or by modulating in disease. PRC17 was recently identified from a panel signaling. of prostate tumors to contain a GTPase-activating Rab proteins that function on constitutive endocytic domain that can interact with rab5 [4]. PRC17 was pathways are continuously cycling between active and shown to be highly upregulated in metastatic prostate inactive states. Nevertheless, interfaces with intracel- tumors to transform mouse fibroblast 3T3 cells and lular signaling cascades afford considerable plasticity when mutated in the GAP domain, to lose its trans- in membrane trafficking. Signaling may result in en- forming capability [4]. These results demonstrate that hanced rab5 and rab7 protein expression and activation the GAP activity of PRC17 is responsible for its with notable enhancements caused by cAMP [14], oncogenic activity and suggest that upregulation of interferon g [20], and ras activation [79]. Conversely, GAP activity might alter rab5 and/or rap1 activity in signaling via the stress-induced MAP kinase p38 human prostate disease. Thus, defects or alterations in promotes rab5–GDI association and thus decreases rab proteins or their effectors may account for a number endocytosis [80]. Intracellular signaling may also af- of diseases in which the molecular mechanisms have fect the fate of internalized cargo by altering transport yet to be identified. along specific rab5- or rab4-regulated pathways, as is the case for EGF-regulated trafficking of its receptor [52,53] and PDGF-regulated integrin recycling [81]. 4. Therapeutic targets Therefore, treatments that stimulate or interfere with these signaling cascades present one avenue for ma- Based on the prevalence of altered rab protein nipulating endocytic transport and rab function. expression and/or regulation as an underlying cause Rab protein and regulatory protein overexpression of human disease, it is of significant interest to consider may be achieved using available gene therapy strate- the therapeutic potential of modulating rab protein gies. However, caution must prevail since overexpres- function. There are several considerations of import sion can in some cases have deleterious effects and in this regard. The first issue pertains to the requirement result in disease. Rab5 might be modulated to affect for cell- or tissue-specific targeting. This is an impor- immune cell function and increase phagocytosis and tant consideration for ubiquitous rab proteins, which intracellular killing, while enhancing rab7 function may exhibit altered function only in select tissues as may have utility in treating lipid storage diseases such well as for rab proteins expressed in a tissue-specific as Niemann–Pick type C disease, stimulating bone manner. The second issue pertains to transient versus a resorption by osteoclasts [82] and treating Charcot– more permanent modulation of expression. For exam- Marie–Tooth neuropathy. Upregulation of both rab5 ple, in the treatment of some cancers or thyroid ade- and rab7 function might be used to enhance EGFR M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1433 downregulation, and slow tumor growth and upregu- 5. Concluding remarks lation of rab4 function might enhance plasma mem- brane aVh3 integrin recycling and promote cell In summary, the rab GTPases and associated regu- adhesion and differentiation. Reconstitution of rab27 latory factors are frequent targets of mutation and/or or associated cofactors could restore secretory lyso- altered expression in a variety of human disease states. some function in melanosomes, platelets, and immune Although our understanding of the molecular mecha- cells. Similarly, reconstitution of the rab regulatory nisms of rab protein function have made significant proteins REP1 and GDIa may ameliorate choroider- progress in the recent past, research to identify and emia and X-linked mental retardation. Thus, a variety characterize additional rab-interacting proteins that are of possibilities exist for therapies that increase rab required for endocytosis will undoubtedly provide expression or activity with the caveat that overexpres- crucial insights into disease processes. Treatments sion must be carefully controlled and/or transient may vary from gene therapy to small molecule inter- where possible. ventions, geared toward restoring normal function or modulating pathways central to normal physiology. 4.2. Modalities inhibiting rab protein expression and/ or function *Note: Data concerning rab7 and hVPS34 interaction Diseases, such as certain cancers, lung, and vascular now published: M.P. Stein, Y.Feng, K.C. Cooper, A.M. diseases, where rab protein expression is elevated, will Welford, A. Wandinger-Ness, Human VPS34 and p150 require careful analysis to pinpoint which pathway or are rab7 interacting partners, Traffic 4 (2003) 1–18. rab protein is central to disease pathogenesis. 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