Rab GTPases implicated in inherited and acquired disorders

Abstract

Endocytotic pathways ensure that internalized receptor complexes are routed in a highly orchestrated manner to the correct subcellular destinations. This, in turn, determines the consequences of receptor activation through targeting receptors and ligand for recycling or degradation as well as by the formation of signaling complexes within the cell that alter the kinetics and magnitude of activation of specific downstream signal transduction cascades. Thus the control of the fate of activated receptor complexes has profound physiologic and pathophysiologic implications. Rab GTPases, the largest subfamily of the RAS small GTPase superfamily, are the key regulators of endocytosis through decorating and targeting intracellular vesicles and cargoes to appropriate subcellular compartments. The six-fold increase in Rab family members from yeast to man correlates closely with the evolutionary increase in endomembrane complexity compatible with a rapid diversification of function of Rab GTPase decorated vesicles. As the vesicular cargo includes growth factors, nutrients, cytokines, integrins and even pathogens, aberrations in the pathway are likely to exhibit dire consequences. Several genetic diseases driven by mutations in Rab GTPases or their interacting proteins have been identified [1], [2], [3]. Aberrant Rab GTPase function has been implicated in diverse pathophysiologies including loss of hair, skin and eye pigmentation, loss of vision, loss of renal function, mental retardation, muscle skeletal degeneration, immune deficiency, infection, obesity, diabetes and cancer.

1. Introduction

Vesicular trafficking

We are only beginning to comprehend the far-reaching consequences of vesicular trafficking in health and disease. The endocytotic pathway regulates overall cell signaling and protein stability, cell polarity, motility, differentiation and survival of cells, as well as the integration of cellular processes into normal physiological function of tissues and organs [4], [5], [2], [6]. A dynamic endomembrane system compartmentalizes the eukaryotic cell into chemically distinct subcellular domains. Each domain has a signature lipid bilayer composition decorated by a unique set of Rab GTPases, optimized for seamless import, trafficking and export of biomolecular cargoes. The vesicular trafficking machinery ensures correct localization and routing of diverse protein complexes from their sites of synthesis or uptake to specific destinations, without any promiscuous interactions or deleterious mixing of organelle constituents. Dysregulation of vesicle trafficking leads to diverse pathological outcomes, some of which are life threatening while others significantly impair quality of life [2], [7]. A number of comprehensive reviews summarizing the molecular, physiological and pathological aspects of vesicular trafficking have been published [2, 5],[8],[9]. In this review, we update the role of the Rab GTPases as the “master organizers” of vesicular trafficking. We also use an interactive process based on experiments of nature such as monogenic inherited disorders, to identify functions of Rab GTPases and elucidate how disruption of vesicular trafficking can contribute to such monogeneic inherited disorders as well as multigenic conditions such as diabetes and cancer [10]. The concept that unraveling the role of derailed endocytosis in disease could increase the rate of development of useful therapies and biomarkers is driving current research [11], [12],[13].

Rab GTPases

The small GTPases (20–25 kDa) comprise the Ras superfamily members which includes the Ras, Rho, Rab, Ran and Arf families [14]. The processes controlling their dynamic interaction and hydrolysis of guanosine diphosphate (GDP; inactive) or guanosine triphosphate (GTP; active) makes the small GTPases effective molecular switches controlling diverse cellular processes. This review specifically focuses on the Rab GTPases, which constitute the largest subgroup of small GTPases [15].

Originally described as Ras-like proteins in brain (Rab), about 70 putative Rab family members have been identified in humans [1, 16]. Their conserved GTP binding sites and variable C-terminals impart the necessary specificity to act as molecular switches controlling downstream effector interactions. As byproducts of recent gene duplication, however, many Rab proteins have likely retained a degree of functional redundancy [17]. Overall, the Rab GTPases regulate various steps of dynamic assembly and disassembly of multiprotein scaffolds involved in vesicular traffic in both endocytic and secretory pathways [18] [19], [20]. Rab proteins have evolved to regulate endocytosis, recycling and degradation as well as exocytosis of various cargos within the cell, providing appropriate raw material as well as signals for cellular and overall tissue homeostasis. Importantly, their physical and functional links to signal transduction pathways provides the key to spatiotemporal regulation of signaling [6, 21]. Membranous vesicles with characteristic Rab GTPase/effector domains comprise the basic infrastructure of the cellular trafficking machinery. Rab proteins and their effectors couple endomembranes to motor proteins and the cytoskeleton [22] facilitating transport of vesicles and their cargoes to subsequent compartments. Thus, in a multistep process, Rab proteins facilitate vesicle formation via budding from the donor compartment, transport to the acceptor compartment, vesicle fusion and release of the vesicle content into the appropriate acceptor compartment [23–27].

Biochemistry and Regulation of Rab GTPases

A. GTP/GDP Cycle

Rab proteins have two highly conserved motifs housing the effector-interacting switch I and II regions. Binding of GDP or GTP in the switch region, results in marked conformational changes in Rab structure. The intrinsic GTP hydrolysis capacity of Rab GTPase proteins as well as GDP/GTP exchange rates is low. GDP/GTP cycling is controlled by two interrelated processes. The guanine-nucleotide-exchange factors (GEFs) promote formation of the active, GTP-bound form [28], whereas GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity to promote formation of the inactive GDP-bound form (Fig 1). Amongst the two nucleotide-bound states, only the GTP-bound conformation has high affinity for effector molecules.

Figure 1. RabGTPases Mediated Vesicular Trafficking.

Rab GTpases are involved in trafficking of cell membrane receptors including RTKs, GLUT4 and integrins.

Rab5 facilitates fusion of clathrin-coated vesicles containing activated receptor to EE. The cargo is then sorted either to Rab4 decorated vesicles for fast-track recycling or to Rab11 decorated long loop slow recyling endosomes. Alternatively, for attenuation of signal transduction, the cargo laden vesicles are targetted to the lysosome via Rab7 and Rab9 coated late endosomes. The Endosomal Sorting Complex (ESCRT) recognizes ubiquitinated proteins and routes them for degradation. Rab GTPases such as Rab3, Rab10 and Rab27 play critical role in the exocytosis/secretory pathway, while Rab1 and 2 are necessary for ER-Golgi transport. Receptor ligand complexes are differentially sensitive to pH, a fact that is utilized by the vesicular trafficking machinery to uncouple the signalosome.

B. Membrane Insertion Cycle

Rab proteins undergo a membrane insertion and extraction cycle, partially in tandem with the GDP/GTP cycle, to allow exchange of cargo between specific membrane domains (Fig 1). Posttranslational lipidation (prenylation), dictated by divergent C-terminal sequences, is critical for membrane insertion into the appropriate compartments [29, 30]. De novo, Rab, as synthesized by cytosolic ribosomes, is GDP bound and inactive, with very low affinity for enzymes that mediate lipidation, such as geranylgeranyltransferase (RabGGTase or GGTaseII) [29, 31]. In contrast to other GTPases, the GDP-Rab requires an additional escort protein, referred to as the Rab escort protein (Rep) [32], to be presented to the prenylating enzyme. This posttranslational modification occurs at the C-terminal by addition of the 20-carbon isoprenoid geranylgeranyl, mediated by RabGGTase. Next, the REP sequesters the prenyl groups attached to the C-terminus of Rab to form a soluble complex until the prenylated protein is delivered to its appropriate membrane compartment. GDP dissociation inhibitor (GDI) binds to prenylated GDP-Rab, however, masking its isoprenyl anchor and thereby maintaining the Rab protein in the cytosol. Membrane attachment of Rab proteins therefore requires the function of a GDI displacement factor [33]. REP is also required to release the GDI from the Rab molecule so it can be delivered to its specific membrane. Once dissociated from GDI and associated with the appropriate membrane compartment, the Rab is available for GEF-stimulated GTP binding. Thus the importance of Rep is underscored by the fact that lethal phenotypes or pathological conditions occur where Rep activity is aberrant [34]. The active, membrane-bound Rab participates in various functions in membrane traffic by binding to one or more effector molecules. After inactivation by its specific GAP, the GDP-bound Rab is extracted from the membrane by GDI and recycled back to the cytosol [29], [35]. This allows for a dynamic change in the Rabs decorating particular vesicles, allowing maturation of the vesicle and sorting to different compartments.

Rab Effectors

Using a variety of approaches, such as the yeast two-hybrid system, genetic screens and affinity purification, a plethora of potential Rab effectors have been implicated in diverse aspects of membrane transport. Rab effectors are a highly heterogeneous group of proteins with broad Rab-binding specificity. Some are coiled-coil proteins involved in membrane tethering or docking, while others are enzymes or cytoskeleton-associated proteins regulating molecular events at restricted membrane locations and microtubule-dependent motility of endocytic structures [36] [37]. Thus in addition to the GTP switch and delivery of Rab proteins to specific compartments by lipidation, the coordinate interactions of Rab proteins with specific effector molecules adds an additional level of complexity allowing Rab proteins to regulate an even more diverse set of cellular processes and functional outcomes.

Regulation of Cargo Delivery by Rab GTPAses

Rab GTPases are not mere transporters of cargoes; they also facilitate the arrival of cargoes at the appropriate vesicle (Fig 2). As an example, mannose 6-phosphate receptors recycle between trans-Golgi networks via late endosomes, carrying lysosomal hydrolases laden with mannose 6-phosphate. The late endosomal GTP-Rab9 effector protein, TIP 47 by binding to the cytoplasmic tail of the mannose 6-phosphate receptor captures and enriches these receptors within Rab9-containing vesicles, thus ensuring their transport back to the Golgi [38], [39]. Thus the Rab proteins ensure the delivery of the mannose 6-phosphate receptors and lysosomal hydrolases to the correct compartments. Rab GTPases also influence vesicle tethering and fusion through their effects on members of the family of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE), which are upstream of various Rab effectors [40], [41], [23],[42]. An example of a more direct mechanism, the coiled-coiled EEA1, a Rab5 effector, bridges Rab5-bearing donor and acceptor membranes [43], [44]. Thus the Rab proteins and their effectors act in a coordinate manner to ensure delivery of vesicles and cargoes to the correct compartment as well as to ensure that inappropriate mixing of vesicle and cargoes does not occur.

Figure 2. GTP Exchange and Membrane Association Cycles.

The newly translated GDP bound Rab in the ER is recognized by REP, and presented to GTT enzyme for prenylation. In many cases, following prenylation, a GDP dissociation inhibitor (GDI) associates with the prenylation group, allowing the Rab to remain in the cytosol, prior to being translocated to the correct endosomal compartment. At this compartment, GDI is removed by GDF allowing a guanine exchange factor (GEF) to facilitate GDP-GTP exchange.

The GTP bound Rab can now interact with its effectors and perform various trafficking functions. The membrane associated active GTP Rab is inactivated by GTPase activating proteins (GAPs), thus returning back to the inactive GDP bound state. In some cases, the GDI extracts the GDP bound Rab back into the cytosol, allowing the process to reinitiate.

Vesicles are often actively transported through the cytoplasm toward their target membrane by either actin-dependent motors (myosins) or microtubule-dependent motors (kinesins or dyneins). During evolution, Rabs became functionally connected to motors of the actin cytoskeleton. This collaboration facilitates tethering of vesicle membranes to molecular motors, which can then efficiently transport the cargo to specific cellular addresses. For example, Rab27A uses its effector melanophilin as a linker to Myosin 5a [45] and active Rab6 binds the microtubule motor Rabkinesin-6 as a means to deliver vesicles from the Golgi to the endoplasmic reticulum [46],[47].

Multiple Rab cascades feed into each other to relay their cargo to the correct subcellular compartment. Rab domains and effectors can be structurally and functionally linked allowing coordination of transfer of cargoes between specific compartments. As an example, Rab4 and Rab5 share a common set of effectors, namely, Rabaptin4, Rabaptin5, Rabenosyn5 and Rabip4 [48], [49], [50] coordinating endocytic traffic mediated via Rab5 with recycling traffic mediated via Rab4. First Rab5 is recruited to the membrane where it is activated by the GEF Rabex5, facilitating interaction with its effector, Rabaptin5. Subsequently, Rabaptin5 binds to Rabex5, which increases the guanine nucleotide exchange activity of Rabex5 for Rab5, generating a positive feedback loop which outcompetes the GAP inactivation and GDI-mediated membrane extraction effects on Rab5 [51]. As the enlarging Rab5-bearing early endosomes migrate toward the center of the cell, they progressively lose the Rab5/EEA1 complex. Rab7 begins to replace Rab5 in these vesicles along with late endosome-specific cargo [51]. Such Rab exchange also alters the endosome qualitatively as degradative properties typical of late endosomes develop. These interrelated mechanisms where several aspects of a process are regulated by a single molecule such as the GEF of a downstream Rab GTPase (Rabex5) also serving as an effector of an upstream Rab protein (Rab4) suggests that it will be necessary to implement systems biology and modeling approaches to determine the consequences of mutation or change in levels of specific Rab GTPases and their regulators and effectors to capture and understand the complexity of the process.

Distribution and Localization of Rabs

Some Rabs are expressed ubiquitously in human tissues, whereas others demonstrate tissue type specificity [52]. Despite the variability among different tissues, Rabs appear to cluster in unique groups based on similarities in their expression patterns across different tissues [53], [18], [54] [55],[15],[56] suggesting a coordinated action within the groups. In addition to coordinate expression across tissues, the function of a specific Rab is governed by their subcellular distribution combined with the unique tissue/subcellular distribution of requisite GDIs, Reps, GEFs, GAPs, and effector proteins. It is thus likely that Rab proteins demonstrate markedly different functions based on the cellular context combined with the formation of Rab-regulated protein hubs.

A. Housekeeping Rabs

Gurkan et al., used a hierarchical clustering approach to propose a functional organization for Rab GTPases [53]. They proposed that a class of Rabs delegated to maintenance of constitutive exocytic and endocytic pathways includes Rab1 isoforms (involved in endoplasmic reticulum-to-Golgi transport in the exocytic pathway), and isoforms of Rab 4, 5, 7 and 11 involved in early and late endosome function [55] be designated housekeeping Rabs. However as several of these Rab proteins are aberrant in human disease resulting in discrete phenotypes and the nature of cargoes and functions are altered by levels of these Rabs and their effectors, they likely subserve important cell specific functions independent from the proposed “housekeeping” role [53].

B. Specialized Rabs

The abundance of Rabs in higher eukaryotes suggests that they have evolved to contribute to specific subcellular trafficking pathways, forming a class of specialized Rabs. For example, Rab3A, which is expressed predominantly in brain and synaptic vesicles, specifically tethers and docks synaptic vesicles prior to fusion, a critical step in regulated release of neurotransmitters [57]. In contrast, Rab27, a phylogenetically distinct protein, functions in the secretory lysosome pathway in a variety of specialized subcellular organelles such as cytotoxic T lymphocyte granules, antigen-processing compartments involved in major histocompatibility complex class II presentation, platelet granules and melanosomes in melanocytes [58], [59]. These Rabs however, do not appear to exhibit functions in other tissue types.

2. Rab GTPase Associated Diseases

A summary of the various monogenic and multigenic diseases associated with Rab GTPases are listed in Table 1 and broadly discussed in the following paragraphs.

Table 1.

Rab GTPases implicated in monogenic and multigenic diseases

RAB Isoforms	Cellular Locations	Functions	Diseases
Rab1 A, B	ER, Golgi	ER-Golgi trafficking	Elevated in tongue squamous cell carcinoma Oculocerebrorenal Syndrome Viral infections
Rab2 A,B	ER, Golgi Plasma membrane Melanosomes	ER-Golgi trafficking	Elevated in various cancers
Rab3 A, B, C, D	Plasma membrane	Neurotransmitter release Fusion of synaptic vesicle Exocytosis (insulin) Protein transport	Cancers of nervous system, Neuro-endocrine related cancers Pituitary adenomas Warburg Micro Syndrome
Rab4 A,B	Plasma membrane	Protein transport	Obesity, Diabetes
Rab5 A, B, C	Plasma membrane, Early endosome, Melanosomes	Fusion of PM to EE Protein transport	Rab5A elevated in lung adenocarcinoma, hepatocellular carcinoma, thyroid carcinoma 5B decreased in metastatic melanoma Tuberous Sclerosis Complex Oculocerebrorenal Syndrome Neurodegenerative Diseases Huntington’s Disease Bacterial infection Diabetes
Rab6 A, A′, B	Golgi membrane	Golgi-ER trafficking	Oculocerebrorenal Syndrome
Rab7 A, B	Late endosome, Lysosome Phagosome Melanosomes	Late endocytic transport, Maturation of phagosome	Hereditary Sensory and Autonomic Neuropathies Charcot-Marie-Tooth Types 2B Niemann-Picktype CPC Alzheimer’s Disease Chagas Disease Thyroid carcinoma
Rab8 A, B	Plasma membrane	Vesicular trafficking Neurotransmitter release Dendrite Extension Ciliogenesis	Melanoma Retinal degeneration in mice/Glaucoma Microvillous Inclusion Disease Bardet-Biedel Syndrome
Rab9 A, B	Late endosome	Endosome-TGN trafficking	Niemann-Picktype C Disease
Rab10	Plasma membrane	Neurotransmitter release Vesicular trafficking Phagosome Maturation Glut4 translocation	Type 2 Diabetes
Rab11 family A,B,C Rab11C synonymous with Rab25	Recycling endosome Rab25 in Apical Recycling compartments	Log loop recycling Rab25 implicated in integrin recycling Cell motility Ciliogenesis	Breast, lung, colon, ovarian endometrial, prostrate, bladder carcinomas. Rab11 implicated in Chagas Disease Rab11A in Diabetes Rab25 in various Cancers
Rab13	Tight junction	Polarized transport, Tight junction activity	Crohn’s disease Upregulated in Tuberculosis
Rab23	Plasma membrane	Negative regulator of sonic hedgehog signaling Vesicular Trafficking	Carpenters Syndrome Craniosynostosis Hepatocellular carcinoma Gastric cancer
Rab24	ER-Golgi, Late Endosome	Autophagy	Tuberculosis
Rab27 A, B	Plasma membrane, Melanosomes	Exocytosis	Choroidermia Griscelli Syndrome 2 Type 2 Diabetes Breast cancer
Rab31	Plasma membrane Trans-Golgi network	Anterograde transport	Metastatic breast cancer
Rab33 A, B	Golgi	Autophagosome formation	Rab33A downregulated in Tuberculosis
Rab38	Melanosomes	Melanosomal transport, docking, sorting TYRP1	Hermansky Pudlak Syndrome

Monogenic Diseases Resulting from Rab GTPase Aberrations

Many of the monogenic disease driven by aberrations in Rab GTPase and their effectors are rare orphan diseases, affecting only a limited number of families. However, these monogenic diseases have the potential to provide important information on the function of specific Rab GTPases as well as on the integration of vesicle trafficking into cellular and organ functions. Thus understanding these diseases is not only important for members of the affected families and their caregivers but also of great importance based on the application of lessons from these “experiments of nature” to more common multigenic diseases.

A. Choroidermia

Mutations in the Rab escort protein 1 (Rep1) are the cause of choroidermia, a rare X-linked disorder that manifests as degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye, resulting in night blindness and loss of peripheral vision [60]. Although Rep1 is phylogenetically similar to GDI, the latter cannot compensate for the loss of Rep function [34]. Interestingly, despite Rep2 being able to substitute for Rep1 for several Rabs, REP2 is unable to interact with Rab27A, the predominant Rab in retinal pigment epithelium cells. Loss of Rep1 thus results in accumulation of unprenylated Rab27A in the eye, leading to specific deficits due to an essential role for Rab27A. Although there is a diagnostic test for this disorder there are no current interventions.

B. X-Linked Mental Retardation

Mutations in neuronal Rab GDIa, one of the three members of a mammalian GDI family bearing close resemblance to Rep, are the cause of one form of X-linked mental retardation. Rab GDIa, a potential negative regulator of synaptic function, is essential for neurotransmission (along with Rab3A) as evinced by onset of neuronal hypersensitivity and epileptic seizures in Rab GDI-deficient mice [61]. More recently, Rab39B has also been linked to X-linked mental retardation that is associated with autism, epilepsy, and macrocephaly [62] suggesting a major role for Rab proteins in maintenance of normal neuronal function.

C. Griscelli Syndrome

Griscelli syndrome is a rare autosomal recessive disorder characterized by pigment dilution in hair and skin and variable immune deficiency that can lead to a life-threatening hemophagocytic condition. Griscelli syndrome is classified into three subtypes based on genetic and molecular features. GS3 is restricted to hypopigmentation, while the two other subtypes are additionally characterized by immunological defects (GS2) or neurological dysfunction (GS1). All three types demonstrate impaired vesicular trafficking manifest as perinuclear accumulation of melanosomes [63, 64]. GS2 is attributed to mutations in Rab27A gene [65].

Skin and hair melanocytes store melanin pigment in specialized lysosome-related melanosomes. Mature melanosomes traverse along microtubule tracks from the cell center to the cell periphery. Their bidirectional transport engages dynein motors to reach the minus end of the microtubule [66] and kinesin motors to arrive at the plus end before they reach their final peripheral destination [67]. Finally, from the microtubule, they are transported and trapped into the distal actin-rich regions of dendritic filaments [68, 69]. Rab27A and the actin motor Myo5A [70], co-localize with melanosomes [71] with the help of linker melanophilin (Rab27 effector), forming a tripartite complex that leads to attachment to the actin network [28],[72], [45], [33], [32]. Thus aberration in any component of this process could lead to a common phenotypic outcome, Griscelli syndrome.

The recurring mutations in Rab27A are homozygous nonsense or frame shift mutations, with only a few reported cases of missense mutations. Typically a missense mutation results in either a nonfunctional protein or complete loss of the protein through altered stability of the mRNA or protein, thus diminishing the available cellular pool of Rab27A [73]. If the mutation results in a premature truncation of the C-terminal geranylgeranylation motif (an early stop codon insertion), the Rab27A proteins cannot be targeted to the correct vesicle, resulting in loss of functionality.

D. Hermansky-Pudlak Syndrome

Hermansky-Pudlak syndrome similar to Griscelli syndrome is characterized by deficiency of the melanin pigment in the eyes, skin and hair; other features include bleeding diathesis and lung disease. This disease is associated with alterations in Rab38, which is highly expressed in melanocytes of the skin and alveolar type II cells in the lungs. In rodents, two different mutations causing inactivation of Rab38 reduced membrane binding or enhanced intracellular degradation, which resulted in oculocutaneous albinism and reduced clotting. Alteration in Rab38 also affected surfactant homeostasis and alveolar function in rodent lungs. Thus a genetic abnormality of Rab38 can affect multiple lysosome-related organelles, resulting in lung disease in addition to oculocutaneous albinism [74, 75].

E. Charcot-Marie-Tooth Type 2B

Charcot-Marie-Tooth disease type 2B (CMT2B) disease is a genetically and clinically heterogeneous group of autosomal-dominant axonal disorders affecting specifically peripheral neurons with a median motor conduction velocity of >38 m/s [76]. Manifestations include severe sensory loss, distal muscle weakness and a high frequency of foot ulcers, infections and even amputations because of recurrent infections. CMT2B maps to chromosome 3q13-q2, in a 2.5-cM region with four missense mutations in the small GTPase late endosomal protein Rab7. Rab7 plays a role in vesicular transport to late endosomes and lysosomes in the endocytic pathway in most cell types [77], [78]. As with many other inherited disorders, it is unknown how mutations in a ubiquitously expressed Rab GTPase with such a generalized functions result in defects in a specific cell population. The most likely explanation is based on another Rab protein demonstrating redundancy with Rab7 either not being expressed in the target neurons or by analogy a particular effector of Rab7 being rate limiting for neuronal function but not for functions of other tissues.

F. Hereditary Sensory and Autonomic Neuropathy

Hereditary sensory and autonomic neuropathy (HSAN) is an autosomal recessive disorder, more common in Jewish individuals [79], affecting the autonomous nervous system. Some clinical symptoms include loss of pain and temperature sensation, alacrima, excessive sweating and absence of fungiform tongue papillae. Some patients exhibit piercing pain. The HSAN type 1 subtype is associated with mutations in Rab7 [79] and in serine palmitoyl transferase, a rate-limiting step in sphingolipid synthesis [80], [81]. Rab7 plays a role in the transport of synthesized lipid from early to late endosomes and late endosomes to lysosomes in many cell lineages and mutations in the broadly expressed serine palmitoyl transferase result in selective disorders of cells in the autonomous nervous system. Why Rab7 mutations cause Charcot-Marie-Tooth and HSAN in different families is unknown. Interestingly, HSAN type II, which shares common traits with Charcot-Marie-Tooth Type 2B suggesting that HSAN and Charcot-Marie-Tooth Type may represent different manifestations of the same syndrome potentially due to the presence of important modifier genes.

G. Warburg Micro Syndrome and Martsolf Syndrome

The Warburg Micro and Martsolf syndromes are autosomal recessive disorders with developmental abnormalities of the eye, central nervous system including severe mental retardation, and microgenitalia [82]. Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. Isoforms of Rab3A, the most abundant Rab in the brain, and its effectors are involved in Ca²⁺-dependent exocytosis during neurotransmitter release from nerve terminals [83], [84]. The Warburg Micro and Martsolf syndromes results from failure of exocytic release of ocular and neuro-developmental trophic factors, implicating Rab3. Indeed, mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GAP were recently found to cause Warburg Micro syndrome and Martsolf syndrome, respectively [84] [85]. As predicted above, aberrations in Rab proteins as well as their regulators and effectors alter vesicle function.

H. Tuberous Sclerosis Complex (TSC)

TSC is characterized by the presence of benign hamartomas in multiple organs, including the central nervous system, lung, kidney, heart and skin, as well as learning and behavioral difficulties and renal complications (hematuria, renal cysts). TSC2 functions as a GAP for Rab5 and also for Rheb. Both Rab5 and Rheb interact in the mTOR pathway [86, 87]. How aberrations in the ubiquitously expressed Rab5 “housekeeping” Rab or in Rheb lead to the development of benign hamartomas in particular tissues remains unknown. However, the observation that inhibition of mTOR, a downstream target of Rab5 and Rheb, has marked clinical activity in Tuberous Sclerosis supports the contention that an understanding of the physiology and pathophysiology of vesicle function could have marked clinical relevance [88, 89].

I. Carpenter Syndrome

Carpenter syndrome is pleiotropic disorder manifested by craniosynostosis, polysyndactyly, obesity and cardiac defects. This autosomal recessive disorder is caused by truncation or missense mutations in the Rab23 gene. How aberrations in Rab23 that regulates trafficking of a plethora of proteins, including members of the intraflagellar transport complex and associated motor proteins leads to Carpenter syndrome remains to be elucidated. However, as Rab23 plays a major role in sonic hedgehog (SHH) signaling [90], [91] and drugs targeting this pathway are under development, they may alleviate some of the problems associated with this disease.

J. Niemann-Pick Type C Disease

Niemann-Pick type C (NPC) disease, which is associated with aberrant cholesterol trafficking at endosomes, is characterized by progressive neurological degeneration associated with hepatosplenomegaly. Symptoms first appear at age 2 to 4 years, resulting from accumulation of abnormal amounts of cholesterol and other lipids (including glycosphingolipids, sphingomyelin, lysobisphosphatidic acid and phospholipids) in the late endosomal/lysosomal compartment. In neurons, abnormal distribution of cholesterol in the cytoplasm rather than the endosome appears as the key lesion.

NPC is caused by mutations in the NPC1 or NPC2 transmembrane proteins that localize in late endosomes, facilitating relocation of free cholesterol to other cell compartments. It is possible that activating the Rab-mediated late endosomal recycling machinery, such as that regulated by Rab7 or Rab9 [92], [93] could reverse the effects of NPC.

At the cellular level, NPC shares some traits with Alzheimer disease including endosome enlargement, elevated hydrolase content and accumulation of p-cleaved amyloid precursor protein and amyloid precursor peptides within endosomes [94]. Although mutations in Rab proteins, regulators or targets have not be identified in Alzheimer disease, upregulation of Rab5 and Rab7 mRNA have been observed, implicating early endosomal dysfunction in Alzheimer’s disease progression [95]. Thus an exploration of endosomal trafficking could provide potential insight leading to effective treatments for this common and devastating disease.

K. Oculocerebrorenal Syndrome of Lowe

The Oculocerebrorenal Syndrome of Lowe, first described in 1952, is an X-linked multi-organ disease caused by mutation of OCRL1 an inositol polyphosphate 5-phosphatase. OCRL1 catalyzes the conversion of phosphatidylinositol (4, 5)-biphosphate to phosphatidylinositol 4-phosphate. The disease is characterized by mental retardation, congenital cataracts and reduced ammonia production by the kidney. Upon growth factor stimulation, OCRL1 translocates from the Golgi apparatus and early endosomes to the lamellipodia, a journey intimately associated with several members of the Rab family of small GTPases [96], [97],[98]. Overexpression or depletion of OCRL1 inhibits trafficking of proteins from early endosomes to the trans-Golgi networks, suggesting a role in clathrin-mediated trafficking between these compartments [98]. Since clathrin adaptors mediate transport of different proteins, including lysosomal hydrolases from trans-Golgi networks to endosomes, defects in OCRL1 could disrupt normal endosomal trafficking. The Golgi-associated Rab1 and Rab6 and endosomal Rab5 have been proposed to play a dual role in targeting OCRL1 to both the Golgi apparatus and endosomes and in directly stimulating the 5-phosphatase activity of OCRL1 following membrane recruitment.

Multigenic Diseases Associated with Rab GTPases

A. Neurodegenerative Diseases

Neuronal Rab5 is essential for synaptic plasticity and neuroprotection [99], playing an important role in AMPA receptor internalization and overall neurotransmission. Not surprisingly, Rab5 defects are detected in various neurodegenerative syndromes such as autosomal recessive motor neuron disease caused by loss of function of Alsin (encoded by ALS2), which is a GEF for Rab5 [100]. Rab5-alsin interaction modulates insulin-like growth factor 1 and other neurotrophic signaling factors likely contributing to the progression of the ALS syndrome [101].

In one study, Huntingtin and Huntingtin-associated protein (HAP40) were identified as Rab5 effectors that regulate endosome motility [102]. Increased HAP40, as observed in Huntington’s disease, reduces mobility of Rab5 endosomes and derails endosomal transport, contributing to neurodegeneration [103]. Moreover, in a recent report, aberrant Rab11 mediated trafficking of a neuronal glutamate transporter was linked to oxidative stress and cell death leading to neurodegeneration in Huntington’s disease [104].

During juvenile onset of Parkinson disease, ER-Golgi transport is disrupted because of mutations in the α-synuclein gene. Overexpression of Rab1GTPase can reverse the toxic effects of α-synuclein and protect against neurodegeneration [105–107] suggesting a potential therapeutic approach.

B. Cystic Fibrosis

CTFR is a chlorine-selective ion channel belonging to the ATP-binding cassette transporter superfamily, which is often mutated in cystic fibrosis and other genetic diseases. The clinical symptoms include exocrine pancreatic insufficiency, increase in sweat concentration of sodium chloride, male infertility and airway disease. Ion channel transporters travel along the canonical endosomal recycling pathway regulated by Rab GTPases. Transport of the cystic fibrosis transmembrane conductance regulator (CTFR) from the plasma membrane to early endosomes is regulated by Rab5; Rab11 recycles it back to the plasma membrane, while Rab7 and Rab9 control its trafficking from early endosomes to late endosomes and finally to the trans-Golgi networks, respectively. As Rab GTPases act as molecular switches that regulate CTFR, they could demonstrate therapeutic utility [108, 109].

Rab Proteins in Immune Response and Infection

A. Crohn’s Disease

Crohn’s disease is associated with deregulated immunological responses and chronic inflammation in the intestinal mucosa. The underlying cause is increased permeability of the intestine due to dislocation of tight junction proteins. Rab13, along with vasodilator-stimulated phosphoprotein and zonula occludin-1, an apical tight junction protein, are often dislocated to the basolateral position in patients with inactive Crohn’s disease, with little alteration in F-actin. The immune response is altered when tight junctions are compromised, allowing invasion of gut antigens [110]. Whether mutations in Rab13, its effectors, or regulators contribute to Crohn’s disease is as yet unknown but clearly warrants investigation.

B. Infection

Rab proteins regulate the entry and exit of a number of bacteria and virus cargoes in the cell. Rab5 is essential for routing internalized biomolecules from the plasma membrane to the endosomal trafficking pathway, which eventually directs bacteria contained in internalized phagosomes to the lysosome for degradation of the pathogen. A number of bacterial pathogens have developed survival mechanisms allowing escape from the phagosome into the cytoplasm by disrupting Rab function, and thus eluding lysosomal degradation. Immune responses initiate with cytokine stimulation of natural killer cells and T-helper cells, which upregulates expression of Rab5a. Subsequently Rab5a facilitates intracellular degradation of the pathogen by recruiting Rac2 to phagolysosomes [111]. The effect of Rab5 is effectively countered by Listeria monocytogenes by preventing extraction of Rab5 from the membrane. In another example, Salmonella type III uses secretory proteins such as SopE with GEF activity to stabilize GTP-Rab5 on the phagosome, thus preventing extraction by RabGDI. These strategies ensure that the phagosome fails to reach lysosome, allowing the bacteria avoid degradation. In another study, gene expression arrays showed a signature of downregulated Rab33A, a T-cell regulator, and upregulated Rab33 and Rab14 in patients with tuberculosis (Mycobacterium tuberculosis) [112, 113]. In sexually transmitted Chlamydia infection, a yeast two-hybrid system linked a Rab4A effector, a Chlamydia inclusion membrane protein Cpn0585, to pathogenesis of the disease [114, 115]. In Chagas disease, the causative protozoan (Trypanosoma cruzi) impairs endocytosis in the host by altering the recycling Rab7 and Rab11 [116].

Viruses enter the host cells via receptor-mediated endocytosis. However, they must escape endosomal vesicles to avoid degradation. In the case of the hepatitis C virus, the host Rab1 GAP (TBC1D20), implicated in regulation of anterograde traffic between the endoplasmic reticulum and the Golgi complex, is hijacked by a viral nonstructural protein to promote viral membrane-associated RNA replication [117] and propagation.

Rab Trafficking and Metabolic Diseases

A. Obesity and Impaired Lipid Trafficking

Obesity is a major health concern in the modern world, as it is an underlying contributor to multiple diseases including diabetes, hypertension, atherosclerosis and cancer. Rab GTPases are not merely transporters of proteins and carbohydrates; one of their most critical functions is proper lipid trafficking. Cells regulate the levels and distribution of lipids such as cholesterol, a key structural component of membranes. In adipose tissue, cholesterol homeostasis is regulated via lipogenesis and lipolysis. Impairment of the lipolytic pathways in adipose tissue and skeletal muscle are important factors in increased triglyceride storage, contributing to obesity, insulin resistance and type-2 diabetes. Small GTPases, especially Rab7 and Rab5 GTPases, by virtue of their roles in trafficking to and from the endosome/lysosome organelles, are implicated in the pathology of these diseases.

Loss of the recycling Rab4b, possibly as a compensatory modification to counter faulty glucose transport [118] in adipocytes, increased obesity in rodents implicating Rab4 proteins in obesity. In neurons, the Rab7-mediated endocytic pathway is under the regulation of Tub1, and mutations in Tub1 were shown to lead to increased fat storage in mice [119]. Whether the regulation of Rab7 contributes to this function of Tub1 remains to be elucidated.

B. Type 2 Diabetes

Cellular uptake of glucose from the circulation occurs through the translocation of glucose transporters to the plasma membrane in response to insulin. In type 2 diabetes, insulin resistance results in insufficient amounts of GLUT4 being translocated from the endosomal recycling compartment, the Golgi complex and the trans-Golgi networks [120], [121] to the plasma membrane despite normal GLUT4 expression. The AKT substrate AS160, also a Rab GAP, was shown recently to connect AKT signaling downstream of insulin to GLUT4 trafficking [122]. Interestingly, in a cochlear hair cell model, downregulation of another Rab, the recycling Rab11a, and two of its effectors, Rip11 and Rab11FIP2, reduced the plasma membrane abundance of GLUT4, providing further evidence of a potential role of Rab GTPases in pathogenesis of metabolic diseases such as diabetes [123, 124].

Rab27 mutant (Ashen) mice demonstrate glucose intolerance of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. This appears to be due to a role for the Rab27a in F-actin mediated exocytosis of insulin containing granules in pancreatic beta cells [125],[121], [126]. Similar mutations in human diabetes have not yet been identified.

C. Cancer

Dynamic genomic changes drive tumorigenesis by deregulating cell proliferation and homeostasis. Signals emanating from membrane-bound growth factor receptors and integrins regulate these critical proliferation and survival pathways. Derailed endocytosis imparts growth factor and extracellular matrix autonomy to cancer cells by shunting degradation and promoting recycling of growth factors as well as integrins (see [4, 5] for recent reviews ). Thus aberrant endocytosis, vesicle targeting and receptor recycling, which are instrumental in altering cell adhesion, migration, proliferation, polarity, asymmetrical division and overall survival, represent emerging hallmarks of cancer.

I. Aberrant Rab GTPase expression in Cancer

Integrative genomic studies have implicated aberrations in Rab pathways as contributing to the initiation and progression of multiple cancer lineages. Elevated levels of Rab23 are associated with invasive gastric cancer [127], Rab38 with melanoma [128], Rab2B with colon carcinoma and Rab1A with tongue cancer [129, 130]. Upregulation of Rab5a and Rab7b occurs in thyroid-associated adenomas [131], while the Rab27A effector JFC1/Slp1 is an important regulator of exocytotic processes in prostate carcinoma cells [132, 133]. Hepatocellular carcinomas may be particularly sensitive to Rab deregulation, as Rab1B, Rab4B, Rab10, Rab22A, Rab24 and Rab25 were shown to be commonly upregulated in these cancers [134]. Rab27 isoforms, which are secretory GTPases that control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment, are associated with poor outcome in invasive breast cancers. Rab27A promotes invasion and metastasis of human breast cancer cells through regulation of insulin-like growth factor II secretion [135]. Rab27B, on the other hand, activates heat shock protein 90 (Hsp90) and matrix metalloproteinase 2 (MMP2), thus increasing G₁-S transition, proliferation, invasive tumor growth and lymph node metastasis in an estrogen receptor–positive mammary carcinoma xenograft model [26]. Rab31 is also associated with poor outcome in invasive breast cancer [136]. Systematic analysis of ovarian cancers demonstrated upregulation of almost half of known RAB or RAB-associated genes [2].

The phosphatidylinositol 3 kinase pathway has been implicated in a broad variety of cancers. Intriguingly, PIK3R1, which encodes p85alpha and is mutated in a number of cancers, exhibits GAP activity towards early endosomal Rabs Rab4 and Rab5 [137]. A GAP-defective mutant p85R274A induces sustained levels of activated platelet-derived growth factor receptors and enhanced downstream signaling [137]. Rab4 interacts with the N-myc–downregulated gene protein 1 (NDRG1) altering the kinetics of E-cadherin recycling and stability. In metastatic breast and prostate cancers, downregulation of NDRG1 is associated with poor prognosis [138] Rab5-mediates trafficking of Rac to the cell membrane during Rac-mediated actin assembly and cell migration as well as the internalization and recycling of β1 integrins increasing Rac-dependent cell migration [139, 140]. Thus the consequences of mutation of PIK3R1 in cancer may be mediated through aberrations in Rab function.

II. Special Focus on the Rab11 Family

Evidence suggests that long loop recycling members (Rab11A, Rab11B, Rab11C/Rab25 and its effectors) play a particularly important role in cancers of multiple lineages, including breast, colon, lung, ovarian, renal, endometrial, prostate, bladder and carcinoid types [5]. Tumor cells maintain high levels of incessant growth (RTK) and survival (integrins) signals by blocking steps of endocytosis that lead to degradation and favoring loops that promote recycling. Further, endosomes provide a compartment for the formation of active signaling complexes that does not occur at the cell surface [11, 141]. These complexes can signal within the cell or on return of the endosome to the cell surface. This can also be ligand specific with transforming growth factor alpha (TGFα) dissociating from the epidermal growth factor (EGF) receptor (EGFR) in the acidic environment of late endosomes allowing its prompt return to the plasma membrane by recycling endosomes, while the more stable EGF/EGFR complex is delivered to the lysosome compartment for degradation and attenuation of signaling [142]. Thus TGFα, by virtue of it recycling status, becomes a more potent mitogen that EGF. Detailed genomic and transcriptomic analysis of the frequent 1q22 amplicon in ovarian and breast cancer implicated the apical recycling Rab25 (aka Rab11c) as a likely driver of the amplicon [2, 4, 12]. Ectopic expression of Rab25 markedly increases tumorigenicity and metastatic potential in ovarian cancer cell models [12]. Rab25/Rab11 is also associated with aberrant growth factor and integrin recycling in 3D culture of ovarian cancer cells [141]. Subsequent to these studies, elevated levels of Rab25 have been observed in hepatocellular carcinoma, prostate cancer [143], transitional cell carcinoma of the bladder and testicular germ cell tumors [144].

Rab-coupling protein (RCP; also known as RAB11FIP1), an effector of Rab11 and Rab25, is a “driver” of the 8p11–12 amplicon in human breast cancer [145]. In mouse xenograft models of mammary carcinogenesis, RCP conferred aggressiveness to mammary tumors. Further overexpression of RCP in normal human mammary epithelial cells (MCF10A) conferred tumorigenic properties such as loss of contact inhibition, growth-factor independence and anchorage-independent growth [145], further supporting a role for Rab25 and its effectors in tumor initiation and progression.

Rab25 facilitates cell survival during stress including that induced by chemotherapy, hypoxia or anoikis at least in part by activation of the phosphoinositide-3 kinase pathway leading to AKT activation. Hypoxia can also stabilize microtubules by diminishing phosphorylation of Tau proteins facilitating Rab11–mediated trafficking of α5β6 via AKT activation and subsequent inhibition of glycogen syntheses kinase 3 beta [146]. Rab25 and Rab11 regulate α5β6 recycling and subsequent association with the EGFR leading to directed motility and invasion [11]. Overall, Rab25 through an interaction with AKT and integrins contributes prosurvival cues that coordinately allow cytoskeletal changes required for aggressive invasion through the extracellular matrix.

The effects of Rab25 may be conditional dependent on the cell lineage and the expression of effectors such as Rab11FIP1/RCP. For example, loss of Rab25 appears to confers aggressiveness to claudin low breast cancers [147] and Rab25 is present at low levels in Barrett esophagus [148] and in colorectal adenocarcinomas [149]. Further, deletion of Rab25 in mice promotes the development of intestinal neoplasia [150]. Together the data is most consistent with the function of Rab25 being context and cell type dependent and determined by the relative levels and activities of effectors molecules.

Why is Rab25, which is closely related to Rab11, more frequently implicated in tumorigenesis than Rab11? One possibility is that the sequence of GTP-binding site of Rab25 is DTAGLE instead of the DTAGQE found in Rab11 and most other Rab family members. This sequence is similar to the oncogenic Ras mutation (Q61L) that maintains a RAS in constitutively active form, which binds more efficiently to effectors.

III. Altered Developmental Pathways

The diverse roles that SHH, WNT and Notch signals play during the development and maintenance of normal tissues are recapitulated in different forms of cancer, and evidence supports an interesting role for Rab GTPases in their function and regulation. In Xenopus, the noncanonical WNT pathway requires Rab function, while in Drosophila a Rab6 homolog is essential for Notch signaling. In mammalian systems, Rab11-mediated recycling of the Notch effector Delta is critical for its activity, abnormalities in the function of Rab11 are implicated in both developmental defects and cancer [151].

In basal cell carcinoma and skin cancer [152] cancers of the prostate [153], stomach [154] and pancreas [155]; and juvenile medulloblastomas, SHH signaling is aberrant. Interestingly, Rab23 appears to be a key inhibitor of SHH signaling during development [156–158] affecting an as yet undefined step between Smoothened and Gli-transcription factors [159]. Analysis of Gli3 protein suggests that Rab23 also has a role in promoting production of the Gli3 repressor. Blockade of SHH signaling leads to tumor shrinkage and remission in preclinical tumor xenograft models. Thus Rab GTPases present a future therapeutic biomarker for various cancers with alterations in this pathway.

3. Concluding Remarks

The role of aberrations in the Rab family in monogenic as well as complex diseases is just beginning to be elucidated. We have provided examples of where aberrant Rab function is both a target for therapy and a biomarker predicting clinical outcomes. We expect that a concerted systems approach to the role of vesicle function including endosome recycling will extend the relevance of derailed endocytosis to the pathophysiology of many diseases including complex diseases such as diabetes, obesity and cancer. While it has proven difficult to selectively target RAS superfamily members with current therapeutic approaches, a number of new methods to “target the untargetable” such as siRNA and stapled peptides as well as the potential to selectively target Rab effectors raises the exciting potential that targeting the function of Rab proteins in regulating vesicle function will eventually enter the therapeutic lexicon.

Abbreviations

ER

Endoplasmic Reticulum

EEA

Early Endosome Antigen

GDP

Guanosine Diphosphate

GDI

GDP Dissociation Inhibitor

GEF

Guanine Exchange Factor

GAP

GTPase Activating Protein

Glut4

Glucose Transporter 4

GTP

Guanosine Triphosphate

MAPK

Mitogen Activated Protein Kinase

PI3K

Phosphoinositide-3 Kinase

RAB

Ras in the Brain

REP

Rab Escort Protein

RTK

Receptor Tyrosine Kinase

SNARE

Soluble NSF Attachment Receptors

SHH

Sonic Hedgehog

TGN

Trans-Golgi Network