www.elsevier.com/locate/addr

Advanced Drug Delivery Reviews 55 (2003) 1421–1437

Rab proteins 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 protein 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 (Griscelli syndrome), mental retardation, neuropathy (Charcot–Marie–Tooth), kidney

disease (tuberous sclerosis), and blindness (choroideremia) 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

0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2003.07.009

* 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: wness@unm.edu (A. Wandinger-Ness).

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371422

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

Table 1

Endocytic rab proteins

Rab Rab function References

Rab4 Localized on EEa and RE; regulates

sorting/recycling

[84,85]

Rab5 Formation of CCV from PM; CCV–EE

and EE–EE homotypic fusion;

endosome motility

[41,42,86–88]

Rab7 Required for EE–LE transport and

LE–Lys fusion

[10,89,90]

Rab9 Endosome to TGN transport [12,91]

Rab11 Recycling through perinuclear RE;

exocytosis from TGN to PM; implicated

in polarization of the Drosophila oocyte

[92–95]

Rab14 Implicated in endocytosis, lysosome

fusion, and phagocytosis

[96,97]

Rab15 Implicated in EE sorting and perinuclear

recycling

[6]

Rab17 Required for transcytosis in epithelial

cells

[98,99]

Rab20 Implicated in endocytosis and recycling

in epithelial cells

[100]

Rab22 Localized on EE and LE; implicated in

endocytosis

[101,102]

Rab25 Implicated in ARE recycling [93]

Rab34 Implicated in macropinosome formation

and lysosome distribution

[103,104]

Rab39 Implicated in endocytosis [105]

a ARE, apical recycling endosome; CCV, clathrin-coated vesicle;

EE, early endosome; LE, late endosome; RE, recycling endosome;

TGN, trans-Golgi network.

Numerous human diseases can be attributed to

alterations in endocytic trafficking. The rab GTPases

and associated proteins are critical regulators of endo-

cytic transport. Increasingly, rab proteins and their

effectors are found overexpressed or subject to loss of

function mutations in human disease. The alterations

impact cellular physiology by perturbing the homeo-

stasis of key cell surface receptors, lipid metabolism,

hormone processing, and specialized secretory path-

ways. Consequently, diverse diseases ranging from

mental retardation to cancer may be attributed to the

derangement of the regulatory machinery governing

endocytic membrane trafficking. This review begins

with an overview of rab protein function and regulation

and highlights how these processes are disrupted in

disease with the aim of identifying potential avenues

for therapeutic intervention.

1.1. Constitutive endocytosis and rab proteins

Endocytosis is a fundamental cellular process re-

quired for the regulated uptake and intracellular trans-

port of macromolecules. There are numerous routes

whereby molecules may be internalized including re-

ceptor-mediated endocytosis via clathrin-coated vesi-

cles, caveolar uptake, macropinocytosis, and phagocy-

tosis (reviewed in Ref. [1]). The discussions in this

review will center on receptor-mediated endocytosis.

Beginning with the initial steps of receptor seques-

tration and culminating with the delivery of cargo to

specific intracellular destinations, endocytosis is a

highly regulated process. Following internalization,

molecules in vesicular carriers derived from the plasma

membrane are transported to specific intracellular des-

tinations allowing for coordinate regulation of hor-

mone and growth factor receptor signaling, sampling

and presentation of antigens for immune recognition,

and general cellular homeostasis. The small ras-related

rab GTPases have emerged as important regulators of

control required for endocytic transport fidelity. Based

on our current understanding, the rab proteins regulate

individual transport steps by controlling cargo selec-

tion [2] and modulating vesicle budding, directed

targeting, and fusion [3]. As detailed further below,

rab protein activity depends on their selective activa-

tion and ability to act as scaffolds for the recruitment of

specific effectors in a spatially and temporally con-

trolled manner. Approximately 60 rab proteins are

encoded by the human genome, with additional rab

proteins possibly generated by alternative splicing [4].

A subset of 13 rab proteins is utilized on the endocytic

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–1437 1423

pathway (Table 1) and serves to determine the fate of

endocytosed cargo.

Althoughmany rab proteins have been implicated in

endocytic transport (Table 1), only a small subset has

been extensively characterized (Fig. 1). Early endo-

cytic events are primarily regulated by rab5 and rab15.

Rab5 facilitates segregation of cargo into clathrin-

coated pits and together with a number of identified

effector molecules, promotes cytoskeletal motility and

homotypic early endosome fusion (reviewed in Ref.

[5]). In contrast, rab15 inhibits transport to early endo-

somes, directly opposing the activity of rab5 [6,7].

Molecules that reach early endosomes are subsequently

sorted for recycling back to the plasma membrane or

Fig. 1. Schematic diagram of the rab-regulated endocytic pathway. Mole

endosomal compartments that are characterized by the presence of rab5

within early endosomes results in immediate transport back to the plasma m

transport either to perinuclear localized recycling compartments or tow

endosomes is subsequently followed by rab11-mediated recycling to the pl

is facilitated by rab7, and cycling of molecules from late endosomes to the T

mediated by rab7 and RILP. Finally, secretory lysosomes, such as those fou

calcium-regulated process. Abbreviations: CCP, clathrin-coated pits; CCV

LYS, lysosome; RE, recycling endosome; TGN, trans-Golgi network.

transport to lysosomes for degradation. Recycled mol-

ecules can either be sorted into rab4 containing micro-

domains within early endosomes that permit fast

recycling to the plasma membrane [8] or be transported

to perinuclear recycling endosomes where rab11 regu-

lates transport back to the plasma membrane [9].

Molecules destined for degradation are delivered in a

rab7-dependent transport step from early to late endo-

somes [10]. Rab7may also function downstream of late

endosomes facilitating transport to lysosomes in asso-

ciation with its one identified effector protein, RILP

[11]. An additional arm of the endocytic pathway

provides for rab9-mediated recycling of molecules

such as the mannose 6V-phosphate receptor (M6PR)

cules internalized by endocytosis are initially transported to early

and rab4. Sorting of molecules into rab4-containing microdomains

embrane while sorting into rab5-containing microdomains leads to

ard lysosomes for degradation. Transport to perinuclear recycling

asma membrane. Transport from early endosomes to late endosomes

GN is mediated by rab9. The transport of molecules to lysosomes is

nd in melanosomes, are delivered back to the plasma membrane in a

, clathrin-coated vesicle; EE, early endosome; LE, late endosome;

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371424

from late endosomes to the TGN [12]. These rab

proteins are the most highly understood of the consti-

tutively expressed endocytic rabs. Several additional

endocytic rab proteins, primarily localized to special-

ized cells, have also been characterized: rab27 (trans-

port of melanosomes), rab25 (apical recycling

endosome to TGN), and rab17 (endosomal recycling

in epithelia) [3]. Each endocytic rab protein character-

ized to date functions in concert with a relatively

unique set of effector molecules. Therefore, continued

characterization of the less well-characterized rab pro-

teins is imperative to achieve a more complete under-

standing of the molecular mechanisms governing

endocytosis.

1.2. Maintenance of normal cell physiology

Functional endocytic trafficking is central to normal

cellular physiology. For this reason, the manipulation

of constitutive or cell-type-specific rab proteins could

provide a means for treating diseases resulting from

aberrant intracellular transport. For example, overex-

pression and delayed degradation of epidermal growth

factor receptors, in particular ErbB2, has been associ-

ated with tumor formation and poor prognosis in breast

cancer patients [13]. Targeting ErbB2 receptors to

lysosomes might aid in reducing intracellular pools of

ErbB2 receptors resulting in decreased cell signaling

and reduced cell proliferation. In thyrocytes, increased

overall levels and increased membrane-bound fractions

of rab5a and rab7 have been linked to formation of

benign thyroid autonomous adenomas [14]. Altering

the amount and rate of thyroglobulin transport and

processing, and the subsequent basolateral secretion

of T3 and T4 by reducing rab5a and rab7 expression,

might lead to a reduction in tumor formation. In

Alzheimer’s patients, aberrant processing and transport

of amyloid precursor protein (APP) in axons leads to

the generation of Ah plaques and the progression of

neurodegenerative disease (reviewed in Ref. [15]).

Manipulating the transport of Ah-containing vesicles

may facilitate clearance of accumulated APP. Finally,

roles for rab proteins in G-protein-coupled receptor

endocytosis, desensitization, and downregulation

(reviewed in Ref. [16]) and in cholesterol and lipid

metabolism and trafficking [17,18] demonstrate that

rab proteins are critically important for a wide variety

of normal cellular functions.

2. Regulation of rab protein function

Rab proteins direct vesicular transport by means of

their localization to select intracellular compartments

and through specific interactions with multiple effec-

tor proteins. Downstream regulation of rab effector

proteins is in turn dependent on the tight regulation of

the rab proteins themselves. Regulation occurs at

multiple levels and in conjunction with numerous

regulatory proteins. Transcriptional and translational

mechanisms control rab protein expression. Mem-

brane localization is controlled through specific post-

translational modification and membrane recruitment.

Selective rab protein activation depends on nucleotide

exchange and hydrolysis, and localized function

occurs through interaction with unique effector pro-

teins. Thus, the rab proteins govern intracellular

vesicular trafficking by acting as localized scaffolding

platforms to exert temporal and spatial control of

transport. Below, the well-characterized regulatory

events associated with rab protein expression and

functional control are presented. As the identification

of factors that modulate rab function in vivo contin-

ues, additional regulatory mechanisms will surely be

revealed.

2.1. Regulated expression levels

Most of the endocytic rab proteins are constitutively

expressed in all mammalian cells. Nevertheless, indi-

vidual rab protein levels vary quantitatively between

cell types (reviewed in Ref. [19]). In addition, tissue-

specific expression of rab proteins in cells such as

epithelia and neurons fulfill the need to regulate

specialized transport processes in these polarized cells.

The data imply that individual cells express a partic-

ular repertoire of rab proteins to fulfill their cellular

vesicular transport needs.

Altered rab gene expression in response to various

inflammatory stimuli and diseases has been reported

and suggests additional plasticity is exerted through

the control of rab gene expression. Modulation of

expression offers another level of control that might

be manipulated for therapeutic purposes. Increased

expression of rab5a and rab7 occurs in response to

cAMP [14], while rab5a can also be increased by

interferon gamma in macrophages [20]. Conversely,

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].

The possibility of regulating membrane trafficking

through the control of rab protein expression is clearly

illustrated through these studies on inflammatory

responses and may serve as a paradigm for other

disease situations with altered rab expression. Indeed,

altered expression of rab proteins in diseases such as

low-grade dysplasia associated with Barret’s esopha-

gus (rab11) [22], cardiomyopathy (rab1, rab4, and

rab6) [23], lung tumor progression (rab2) [24], pros-

tate and liver cancer (rab25) [25,26], and possibly

atherogenesis (rab7) [27], point to the importance of

understanding RAB gene control and the identification

of factors affecting RAB expression. Such pathways

may then be targeted to selectively increase or decrease

rab protein expression and achieve the desired modu-

lation of endocytic transport.

2.2. Regulated membrane association and localization

Following protein synthesis, rab proteins interact

with several common rab regulatory molecules, which

result in posttranslational modification and membrane

association of rab proteins. One or two cysteine

residues in the C-termini of all rab proteins are

modified with isoprenoid moieties to allow for mem-

brane localization. Modification occurs when newly

synthesized rab proteins first interact with rab escort

proteins (REP) (Fig. 2, steps 1–4). Next, the rab:REP

complex is specifically recognized by a rab geranyl-

geranyl transferase (RabGGTase), which catalyzes the

addition of geranylgeranyl groups to C-terminal

cycteine residues in the motifs CC, CXC, CXXX,

CCXX, or CCXXX (where X is any amino acid). The

geranylgeranylated rab proteins are subsequently de-

livered to intracellular membranes in a complex with

REP, which maintains the modified rab protein in a

soluble form. Targeting of REP proteins to specific

intracellular membranes may involve membrane

receptors, which have not yet been identified. Once

the geranylgeranylated rab protein is presented to the

specified target membrane, REP is released and

recycled to the cytosol for additional rounds of escort.

REP proteins share sequence homology with rab GDP

dissociation inhibitors (rabGDI), another rab regula-

tory protein.

Similar to REP, rabGDI binds GDP-bound rab

proteins, producing soluble rabGDI:GDP-bound rab

(GDP-rab) complexes in the cytosol. Importantly,

rabGDI extracts GDP-rab from intracellular mem-

branes and recycles GDP-rab back to donor mem-

branes (Fig. 2, steps 11–13). Major conformational

changes in rabGDI occur upon binding to membrane-

associated GDP-rab, resulting in the dissociation of the

rabGDI:GDP-rab complex from acceptor membranes

[28]. Recycling of GDP-rab proteins to specific mem-

brane compartments is mediated by compartment-

specific receptors that recognize the soluble rabG-

DI:GDP-rab complex. The membrane-bound Hsp90

chaperone complex has been identified as a putative

rabGDI:rab receptor [29]. Alternatively, the rab GDI

displacement factor (GDF, discussed below) may also

serve as a rabGDI:GDP-rab receptor. Upon binding to

donor membrane receptors, rabGDI is released from

GDP-rab, allowing recycling of rabGDI for additional

rounds GDP-rab extraction and transport.

Highly conserved sequences within REP and

rabGDI form a general binding platform that facilitates

rab protein recognition. Complementary conserved

residues within all mammalian rab proteins enable

rab protein binding to these and other common regu-

latory molecules [30]. Since the conserved binding

motifs within the regulatory molecules bind to a variety

of rab proteins, the disruption of multiple cellular

functions by mutation would be predicted to have dire

consequences. However, due to the expression of

multiple isoforms of rab regulatory molecules with

redundant function, mutation of REP1 results specifi-

cally in retinal degeneration whereas loss of GDI

function results in severe mental retardation. Modulat-

ing endocytosis through the manipulation of common

regulatory molecules is therefore unlikely to be a

tractable therapeutic strategy. Deficits in these regula-

tory molecules should be considered candidates for

gene therapy.

2.3. Regulated activation through nucleotide binding

and hydrolysis

In addition to cycling between membrane compart-

ments, all rab proteins also cycle between GDP-bound

(‘‘inactive’’) and GTP-bound (‘‘active’’) states (Fig. 2,

steps 5–10). After extraction from membranes by

rabGDI, rab proteins either remain in a cytosolic

complex associated with rabGDI or are delivered back

to donor membranes. Dissociation of rabGDI at the

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.

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371426

donor membrane is facilitated by membrane-bound

GDI-displacement factor (GDF) [31]. Upon release of

rabGDI, the geranylgeranyl moieties of GDP-rab rein-

sert into the donor membrane, readying the rab for

another cycle of activation. Membrane association of

GDP-rab promotes nucleotide exchange from GDP to

GTP; however, the intrinsic rate of nucleotide exchange

is very slow. The rate of nucleotide exchange is greatly

enhanced by specific guanine nucleotide exchange

factors (GEFs). GEFs catalyze the exchange of GTP

for GDP; however, the molecular mechanisms govern-

ing nucleotide exchange are not yet fully appreciated.

A class of proteins sharing sequence similarity with

the guanine nucleotide-free chaperones Mss4/Dss4

[32] has recently been identified. Translationally con-

trolled tumor-associated proteins (TCTP) are highly

expressed in a wide range of mammalian cells and may

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

rab proteins in endocytosis derives from recent evi-

dence demonstrating that rab15 function requires Mss4

binding and that Mss4 is a rab15 effector [34]. Func-

tional roles for other rab proteins bound to nucleotide-

free chaperones will undoubtedly be uncovered. Fur-

thermore, understanding how cycling from GDP- to

nonnucleotide- to GTP-bound states is regulated will

be critical for determining how and when conforma-

tional changes in the rab proteins allow for functional

interactions with downstream effector molecules.

GTP hydrolysis leads to the cycling of rab proteins

from their active GTP-bound to inactive GDP-bound

state. Although all rab proteins contain some intrinsic

GTPase activity, the rate of phosphate hydrolysis is

greatly enhanced by the presence of GTPase activating

proteins (GAPs). In mammalian cells, cytosolic GAP

activity has been demonstrated to exist for a number of

rab proteins. However, only two mammalian rab

GAPs have been definitively identified: GAPCenA,

which has sequence similarity to a yeast Ypt/rab GAP

[35]; and Rab3-GAP, which has no sequence similar-

ity to any known GAP proteins [36]. Acceleration of

GTP hydrolysis by GAPs results from the insertion of

a common arginine finger motif found in yeast Ypt/

rabs, ras, and rho, into their substrate [37]. Rates of

hydrolysis up to 1000-fold over baseline have been

observed.

The factors regulating nucleotide exchange and

hydrolysis appear to be highly specific for individual

rab proteins. The exchange of GTP for GDP by GEFs

provide the molecular ‘‘on-switch’’ for rab proteins

while the hydrolysis stimulated by GAPs provides the

‘‘off-switch’’. These opposing reactions therefore play

a crucial role in regulating the temporal dynamics of

vesicular membrane trafficking. Thus, these proteins

may be attractive therapeutic targets to enhance or

diminish rab protein activity and modulate select endo-

cytic trafficking pathways.

2.4. Regulated function through specific effectors

Rab proteins serve to promote trafficking by coor-

dinating the individual steps in vesicular transport. In

this context, the rab proteins can be viewed as molec-

ular scaffolds that promote temporal and spatial control

through their compartment-specific localization and

activation. Rab proteins also serve in cargo selection

and as interfaces to intracellular signaling cascades. In

this context, rab proteins may control the fate of

individual cargo molecules by interacting with cargo

directly or via specialized effectors that are induced in

response to signaling. Examples of rab proteins and

their effectors serving in each of these capacities are

detailed below.

Principally, the interaction of rab proteins with

effector molecules facilitates membrane transport

through a coordinate assembly process. The rab pro-

teins serve as molecular scaffolds for the sequential

recruitment of proteins and lipids that are required for

transport vesicle budding, cytoskeletal motility, and

finally vesicle tethering, docking, and fusion with the

target membrane. Effector molecules for a number of

rab proteins have been identified and characterized

(Table 2). The best-characterized set of rab effector

proteins are those that interact with rab5. Analysis of

the rab5 effectors suggest a general model by which

interactions between rab proteins and effector mole-

cules may lead to the formation of macromolecular

scaffolds for efficient and accurate membrane trans-

port [5].

Rab5 effector interactions are tightly coupled to rab

activation. GTP-rab5 initially recruits regulatory com-

plexes of rabaptin-5 and rabex-5 to early endosomes.

Rabex-5 acts as a rab5 nucleotide exchange factor and

rabaptin serves as an accessory factor for coated

vesicle formation [38], as well as vesicle tethering

[39]. The GTP-rab5 bound to early endosomes also

recruits the p150/hVPS34 complex to early endo-

somal membranes by binding the p150 adapter pro-

tein [40]. Once membrane associated, hVPS34, a

phosphatidylinositol (PI) 3V-kinase, generates PI 3V-phosphate (PI3P) in the plane of the early endosomal

membrane. The newly formed PI3P microdomains

regulate endosome motility on microtubules [41] and

provide platforms for assembly of the fusion machin-

ery. Molecules containing the FYVE domain specif-

ically and preferentially bind to PI3P [42,43]. Two

such FYVE-domain containing proteins specifically

recognize PI3P on early endosomes and promote ve-

sicle docking and fusion [44–46]. EEA1, early endo-

somal autoantigen 1, is a tethering molecule that

interacts with syntaxins 6 and 13 to establish vesicle

contact with the target membrane [47]. Rabenosyn-5

is required for SNARE complex formation and thus

promotes fusion [48]. The assemblies of macromo-

Table 2

Endocytic rab effector proteins

Rab Rab effector Function References

Rab4-GTP Rabip4 Implicated in

retrograde transport

from REa to SE

[106]

Rabaptin-5 Coordinates

endocytosis and

recycling from EE

[107]

Rabaptin4 Implicated in

transport from EE

to RE

[108]

RCP Implicated in

recycling

[109]

Rab5-GTP EEA1 EE homotypic

tethering and fusion

[110]

Rabaptin-5 Forms a stable

complex with

Rabex-5; implicated

in membrane

docking and fusion;

recently shown to

participate in clathrin

adapter binding

[38,111]

Rabaptin-5h Implicated in fusion [112]

Rabenosyn-5 Required for CCV–

EE and EE–EE

fusion

[48]

p110h Class I PI3K

catalytic subunit

[113]

PI3K

(hVPS34)

Class III PI3K [113]

p150 Class III PI3K

adapter protein for

hVPS34; serine/

threonine protein

kinase

[40,114]

RIN2 Guanine nucleotide

exchange

[115]

Rab5-GDP RIN1 Guanine nucleotide

exchange

[52]

Rab7-GTP RILP Regulates LE to

lysosome transport

through interactions

with dynein

[11]

Rab9-GTP p40 Stimulates transport

of M6PR from LE to

TGN

[116]

TIP47 Bind to both M6PR

and Rab9-GTP;

cytosolic recognition

factor for M6PR

[117]

Rab11-GTP Rip11 Required for ARE to

apical PM transport

[118]

RCP Homologue of Rip11 [109]

Table 2 (continued )

Rab Rab effector Function References

Rab11-GTP Eferin EF-hand domain

containing protein,

implicated in rab11

localization

[119]

Rab11BP

(Rabphilin 11)

Implicated in vesicle

recycling

[120,121]

Rab11-GDP,

Rab11-GTP

Rab11-FIP Implicated in

recycling; homo- and

heterodimerization to

create protein

platforms

[122]

Rab34-GTP RILP Regulates

lysosomal

positioning

[104]

a ARE, apical recycling endosome; CCV, clathrin-coated vesicle;

EE, early endosome; Eferin, EF-hands-containing Rab11-interacting

protein; Rab11-FIP, rab11 family-interacting protein; LE, late

endosome; M6PR, mannose 6-phosphate receptors; PI3K, phosphoi-

nositol-3V-kinase; RCP, rab coupling protein; RE, recycling endo-

some; SE, sorting endosome; TGN, trans-Golgi network.

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371428

lecular complexes on clathrin-coated vesicles and

early endosomes are triggered by rab5 activation.

Together, rab5, phospholipids, and a variety of rab

effector molecules contribute to the coordinate control

of early endocytic trafficking and culminate in regu-

lated endosomal fusion. These events provide a par-

adigm for other intracellular membrane trafficking

and fusion events. The directionality and specificity

of transport relies on the specific interactions between

rab proteins and their effector molecules while the

temporal specificity of these interactions relies on the

nucleotide cycling of the rabGTPases.

New data document rab protein function in cargo

selection, offering unprecedented opportunities for

modulating the fate of specific molecules. Rab pro-

teins may directly interact with some cargo molecules,

as is the case for the interaction between rab3b and

polymeric IgA receptor [2,49]. Alternatively, rab

proteins may interact with specific cargo via a unique

intermediary, as illustrated by the requirement for

TIP47, which mediates the interaction between rab9

and the mannose 6-phosphate receptor [50,51]. Cargo

selection may also depend on interfaces with intracel-

lular signaling cascades that can serve to increase or

decrease rab protein activity. The regulation of EGFR

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-

lation. Endocytosis, in this case, is stimulated by

Rin1, a protein with rab5 GEF activity that converts

rab5 to its active form [52]. On the other hand, ras

activation may also attenuate endocytosis. In this case,

rab5 is inactivated by the action of a specific rab5

GAP, RN-Tre, which is recruited by the activated ras

effector Eps8 [53]. Such opposing activities are most

probably temporally balanced during normal signal-

ing, but may be aberrant in some disease states where

EGFR is constitutively active.

In summary, selective manipulation of rab proteins

or their effectors offers the potential to disrupt specific

transport steps and preferentially shunt cargo for recy-

cling or degradation. In addition, as we learn more

about specialized cargo selection pathways, it is con-

ceivable that the trafficking of individual receptors may

be precisely controlled and modulated for therapeutic

benefit.

3. Altered rab proteins in disease

Increasingly, rab proteins and associated regulatory

molecules or effectors are shown to be altered in

human disease. The alterations may be associated

with a loss of function, in which case, an underlying

germline mutation is frequently responsible. Loss of

function mutations in the rab protein or associated

regulatory molecules and effectors frequently have

similar phenotypic outcomes. Rab protein overexpres-

sion or aberrant activation, triggered by somatic

mutation or altered signaling, also underlie a number

of disease states.

3.1. Loss of function mutations in rab proteins, rab

regulatory molecules, or rab effectors

Genetic mutations affecting rab proteins and asso-

ciated regulatory molecules have recently come to

light. To date, two diseases have been characterized

with causal mutations in rab genes, while others

impact accessory factors as detailed below.

The first human disease identified to result from a

mutation of a rab gene was Griscelli syndrome type 2

(GS2). GS2 is a rare autosomal recessive disorder

originally described in 1978 [54]. Patients exhibit

immune impairment and increased susceptibility to

infections due to defects in T cell cytotoxicity and

cytolytic granule release. Partial albinism results from

the accumulation of melanosomes in melanocytes.

Hemophagocytic syndrome is caused by hyperstimu-

lated T cells and macrophages [55]. The genetic

defects responsible for GS2 include three distinct

missense mutations in highly conserved residues and

numerous microdeletions or larger deletions in

RAB27A on chromosome 15q21[55]. Rab27a has

been shown to be critically important for the transport

and release of melanosomes, a form of secretory

lysosome, from melanocytes [56]. Similar defects in

secretory lysosome release from immune cells and

platelets account for the bleeding disorders and im-

mune dysfunctions associated with the disease [57,58].

A related syndrome, Griscelli syndrome type 1

(GS1), is also characterized by partial albinism but

exhibits a primary severe neurological impairment

without deficits in immune function [59]. The primary

defect in GS1 was localized to MYO5A. MYO5A en-

codes an unconventional myosinVa motor protein [60].

Mouse coat-color variants equated with GS1 (dilute

[61]) and GS2 (ashen [58]) originally helped to define

the intracellular transport pathway ofmelanosomes. An

additional mouse model, the leaden mouse, identified

melanophilin [62] as a rab27a-interacting partner that

mediates the recruitment and binding of myosinVa to

actin [63,64]. Thus, human and mouse studies have

elucidated the components and fundamental mecha-

nisms requisite for secretory lysosome transport and

release, demonstrating that phenotypically similar dis-

eases may arise due to disruptions of multiple compo-

nent on the same intracellular pathway.

Recently, a second genetic disorder was pinpointed

to defects in a rab gene. Mutations in the RAB7 gene

cause Charcot–Marie–Tooth type 2B neuropathy

[65]. The disease is marked by sensory and motor

neuron impairment, distal muscle weakness and atro-

phy, and ulcerations often requiring amputation. Mis-

sense mutations in RAB7 were localized to exons 3

and 4, where either a C to T transition led to substi-

tution of Leu129 for Phe or a G to A transition resulted

in mutation of Val162 to Met [65]. The Val162 residue

is highly conserved in all species and Leu129 is

localized adjacent to the GTP-binding domain of

rab7. As such, both mutations are predicted to disrupt

rab7 function. A second form of Marie–Charcot–

Tooth disease (MCT4B1) results from a genetic defect

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371430

in myotubularin-related protein 2 [66], a dual speci-

ficity phosphatase required for PI3P and PI3,5P2metabolism [67]. A third form of Marie–Charcot–

Tooth disease (MCT2A) was previously characterized

based on missense mutations in KIF1B [68]. The

KIF1B gene encodes a kinesin motor protein thought

to play a crucial role in synaptic vesicle transport. By

analogy with Griscelli syndrome, it seems likely that

rab7, myotubularin, and KIF1B are also part of a

common molecular pathway such that disruption of

any one of the genes causes a similar disease pheno-

type that is manifested as peripheral neuropathy. In this

context, it is of interest to consider our observations

demonstrating that rab7 and a PI 3V-kinase, hVPS34,coordinately control late endocytic transport*. The

rab7-regulated formation of PI3P on late endosomes

may facilitate kinesin-regulated membrane recycling,

particularly in motor neurons. Turnover of PI3P may

subsequently be temporally regulated by myotubu-

larin-related protein phosphatase, providing a molec-

ular ‘‘off-switch’’ for the kinesin-mediated vesicular

transport. Further characterization of the intracellular

pathways governed by rab proteins and identification

of rab effector molecules will undoubtedly uncover

additional molecules that are mutated to cause a

variety of human diseases. Consequently, effective

gene therapy strategies for reconstitution of these

molecules will be increasingly important.

In the case of rab27, the disease etiology matches

the expression profile of the protein. Rab27 is highly

expressed in keratinocytes, platelets, and lymphocytes,

where it promotes secretory lysosome fusion. In the

absence of rab 27, secretory lysosome fusion is dis-

rupted, leading to bleeding and pigmentation disor-

ders, as well as immune dysfunction. In contrast, rab7

is a ubiquitous rab that controls transport between

early and late endosomes. Determining how mutations

in rab7 result in sensory and motor neuropathy and

identifying how the loss of rab7 function is compen-

sated for in other cells, tissues, and organs remain

critical unresolved issues for understanding the com-

plexities of rab function in vivo.

Genetic defects in rab regulatory molecules are

associated with retinal degeneration in choroideremia,

X-linked mental retardation, and kidney disease in

tuberous sclerosis [69–71]. Choroideremia and X-

linked mental retardation result from germline muta-

tions in general regulatory factors that impact the

membrane association of rab proteins. Altered regula-

tion of rab5 nucleotide hydrolysis promoted by dis-

crete cofactors has been implicated in both prostate

cancer and tuberous sclerosis. In the case of tuberous

sclerosis, this is precipitated by germline mutation of

the cofactor’s coding sequences, while in the case of

prostate cancer, the cofactor protein is highly overex-

pressed. The nature of a causal mutation in prostate

cancer has not been defined, but may arise from

somatic rearrangements.

Choroideremia, a form of retinal degeneration due

to loss of retinal epithelium, choroids, and retinal

photoreceptor cells, ultimately causes blindness in

affected individuals [30]. A number of point mutations

in rab escort protein1 (REP1), as well as translocations

that disrupt REP1 gene expression, have been de-

scribed [72]. Identification of two REP genes, REP1

and REP2, suggest that functional redundancies in rab

regulatory molecules may exist. However, tissue-spe-

cific expression of REP1 or the specificity of particular

rab proteins for REP1 could result in an absolute

requirement for REP1 in a given tissue. This appears

to be the case in retinal epithelia, where loss of REP1

activity results in retinal degeneration and loss of

eyesight in affected individuals. Functional redundan-

cy through the expression of REP2 may partially

compensate for loss of REP1 activity, except where

REP1 activity is absolutely required.

X-linked nonspecific mental retardation (MRX) has

also been associated with genetic defects in a rab

regulatory molecule, rabGDIa. Both a truncation mu-

tation in rabGDI (MRX48) and a T to C transition that

resulted in a nonconservative amino acid change

(Leu92 to Pro) in GDIa were described in two distinct

families with MRX [73]. RabGDIa is critical for

neurotransmitter release and rab3a recycling in the

brain, and the introduction of proline at position 92 in

rabGDIa decreased the affinity of rabGDIa for rab3a.

Consequently, this mutation might cause inefficient

recycling of rab3a from synaptosomes, possibly lead-

ing to mental defects. Furthermore, rabGDIa has been

implicated in neuronal development based on expres-

sion at embryonic day 9 and its requirement for axonal

extension [73]. Expression of additional rabGDI pro-

teins and promiscuous binding of rab proteins to

rabGDIs must allow for sufficient recycling of rab

proteins in tissues and organs outside the brain. Similar

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

expression in brain.

Genetic defects in less well-characterized, rab-re-

lated proteins may also result in severe disease. One

example, tuberous sclerosis (TSC), is an autosomal

dominant disease with a variety of manifestations

including the formation of benign tumors called

hamartomas, mental deficits that include behavioral

and learning difficulties, and renal complications in-

cluding but not limited to renal lesions. Genetic defects

in two distinct loci, TSC1 and TSC2, account for the

majority of cases. TSC1 encodes hamartin, a 130-kDa

protein of unknown function while TSC2 encodes

tuberin, a protein that stimulates GTP hydrolysis on

rab5 and rap1 and thus behaves like a GAP [74].

Hamartin and tuberin interact with one another in vivo

and act as tumor suppressor genes [75,76]. Defects in

either TSC1 or TSC2 are predicted to disrupt the

intracellular complex formed by these proteins, there-

by inhibiting the unknown activity of hamartin. A role

for the tuberin/hamartin complex in intracellular trans-

port was suggested by recent findings that polycystin-

1, an integral membrane component of basolateral

adherens junctions, was mislocalized to the Golgi in

cells deficient for TSC2 expression [77]. Further detail

regarding the functions of tuberin and hamartin in

vivo, as well as their relationships to rab5 and other

components of the endocytic regulatory machinery, is

essential to gain clues about the molecules that are

most acutely affected by the defects in TSC.

A role for rab proteins in the transport and mainte-

nance of cellular cholesterol and lipids was recently

revealed. Niemann–Pick type C (NPC) disease is a

genetic disorder in which the accumulation of lipids in

late endosomes ultimately causes a severe neurode-

generative disorder resulting in premature death. The

vast majority of NPC cases result from genetic muta-

tions in NPC1, while a small percentage result from

mutations within a second gene, NPC2 [78]. These

genes encode proteins required for lipid trafficking,

although the precise molecular mechanisms have not

yet been elucidated. Overexpression of rab7 or rab9 in

NPC cells alleviated cholesterol accumulation and

restored transport of several glycospingolipids, lacto-

sylceramide, and GM1 to the Golgi [17]. Importantly,

the motility of rab7-containing late endosomes is

directly altered by cholesterol accumulation, possibly

through the inhibition of motor protein binding [18].

These results suggest that rab7, cholesterol, kinesin

motor proteins, and the NPC1 and NPC2 proteins may

interact on late endosomes, resulting in the proper

transport and maintenance of cellular lipids. A greater

understanding of these lipid transport processes will be

invaluable, providing information both about the NPC

disease process as well as identifying molecules re-

quired for lipid homeostasis. With increased under-

standing, manipulation of cholesterol and lipid

transport processes may provide therapeutic benefit

to those suffering from lipid storage diseases.

3.2. Altered rab expression or activation in disease

As discussed above, many proteins participate in

the regulation of rab protein expression and localiza-

tion and genetic defects in rabs or their regulatory

molecules can result in disease. In addition, control

over the levels of rab protein expression must exist.

Overexpression of requisite endocytic rab proteins or

regulatory proteins has been associated with human

thyroid, vascular, and lung diseases, as well as some

cancers [4,14,24,26,27]. Overexpression may be pre-

cipitated by somatic rearrangements as in the case of

some prostate cancers or may arise in response to

sustained stimuli from intracellular signaling.

Thyroid hormone production requires the uptake

and processing of thyroglobulin (Tg) from apical

extracellular colloidal stores, endocytosis and pro-

cessing of Tg in late endosomes and lysosomes,

and transport and release of mature thyroid hormone

at the basal surface. Thyroid autonomous adenomas

(AA) are benign tumors of the thyroid associated

with elevated levels of rab5a and rab7 and decreased

levels of follicular Tg [14]. Membrane association of

both rab5a and rab7 are also increased in AA,

suggesting that rab5 and rab7 are both activated in

AA. Increased expression of rab5a and rab7 increase

the rate of thyroglobulin endocytosis and processing

in response to elevated cAMP [14]; however, it

remains to be determined if the enhanced processing

of Tg results in tumor formation or if additional

factors are involved.

Alterations in endo- and exocytic rab protein

expression have also been revealed in several models

of human disease and in a variety of cancers. Upre-

gulation of rab1a, rab4, and rab6, and altered Golgi

morphology were observed in a h2-adrenergic recep-

M.-P. Stein et al. / Advanced Drug Delivery Reviews 55 (2003) 1421–14371432

tor model of cardiomyopathy [23], while increased

expression of rab7 was observed in a rabbit model of

atherogenesis [27]. Similarly, in a mouse model for

lung tumor progression, increased expression of rab2

was detected [24] whereas in prostate cancer cell

lines, alterations in rab25 expression were noted

[25]. Recent work identified six rab and three Arf/

Sar proteins that are upregulated in human liver

cancers, including hepatocellular carcinomas and

cholangiohepatomas [26]. Thus, alterations in expres-

sion of a variety of rab proteins, as determined by

gene expression profiling, suggest that rab proteins

play a multitude of roles in maintaining normal

cellular physiology.

The novel prostate cancer gene 17 (PRC17) pro-

vides an example where overexpression of a rab

regulatory factor decreases rab activation and results

in disease. PRC17 was recently identified from a panel

of prostate tumors to contain a GTPase-activating

domain that can interact with rab5 [4]. PRC17 was

shown to be highly upregulated in metastatic prostate

tumors to transform mouse fibroblast 3T3 cells and

when mutated in the GAP domain, to lose its trans-

forming capability [4]. These results demonstrate that

the GAP activity of PRC17 is responsible for its

oncogenic activity and suggest that upregulation of

GAP activity might alter rab5 and/or rap1 activity in

human prostate disease. Thus, defects or alterations in

rab proteins or their effectors may account for a number

of diseases in which the molecular mechanisms have

yet to be identified.

4. Therapeutic targets

Based on the prevalence of altered rab protein

expression and/or regulation as an underlying cause

of human disease, it is of significant interest to consider

the therapeutic potential of modulating rab protein

function. There are several considerations of import

in this regard. The first issue pertains to the requirement

for cell- or tissue-specific targeting. This is an impor-

tant consideration for ubiquitous rab proteins, which

may exhibit altered function only in select tissues as

well as for rab proteins expressed in a tissue-specific

manner. The second issue pertains to transient versus a

more permanent modulation of expression. For exam-

ple, in the treatment of some cancers or thyroid ade-

nomas, it may be sufficient to transiently downregulate

rab protein expression, while in the case of genetic loss

of function diseases, it will be crucial to have sustained

and permanent reconstitution of protein function. Fi-

nally, it is important to consider therapeutic interven-

tions that can restore rab protein function, as well as

those that can block function. Such situations arise

when the trafficking of specific cargo may need to be

modulated.

4.1. Modalities for stimulating rab protein expression

and/or function

As discussed above, rab proteins may be regulat-

ed at the level of expression or activation. Changes

in expression or activation may be achieved either

by overexpressing specific genes or by modulating

signaling.

Rab proteins that function on constitutive endocytic

pathways are continuously cycling between active and

inactive states. Nevertheless, interfaces with intracel-

lular signaling cascades afford considerable plasticity

in membrane trafficking. Signaling may result in en-

hanced rab5 and rab7 protein expression and activation

with notable enhancements caused by cAMP [14],

interferon g [20], and ras activation [79]. Conversely,

signaling via the stress-induced MAP kinase p38

promotes rab5–GDI association and thus decreases

endocytosis [80]. Intracellular signaling may also af-

fect the fate of internalized cargo by altering transport

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].

Therefore, treatments that stimulate or interfere with

these signaling cascades present one avenue for ma-

nipulating endocytic transport and rab function.

Rab protein and regulatory protein overexpression

may be achieved using available gene therapy strate-

gies. However, caution must prevail since overexpres-

sion can in some cases have deleterious effects and

result in disease. Rab5 might be modulated to affect

immune cell function and increase phagocytosis and

intracellular killing, while enhancing rab7 function

may have utility in treating lipid storage diseases such

as Niemann–Pick type C disease, stimulating bone

resorption by osteoclasts [82] and treating Charcot–

Marie–Tooth neuropathy. Upregulation of both rab5

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-

lation of rab4 function might enhance plasma mem-

brane aVh3 integrin recycling and promote cell

adhesion and differentiation. Reconstitution of rab27

or associated cofactors could restore secretory lyso-

some function in melanosomes, platelets, and immune

cells. Similarly, reconstitution of the rab regulatory

proteins REP1 and GDIa may ameliorate choroider-

emia and X-linked mental retardation. Thus, a variety

of possibilities exist for therapies that increase rab

expression or activity with the caveat that overexpres-

sion must be carefully controlled and/or transient

where possible.

4.2. Modalities inhibiting rab protein expression and/

or function

Diseases, such as certain cancers, lung, and vascular

diseases, where rab protein expression is elevated, will

require careful analysis to pinpoint which pathway or

rab protein is central to disease pathogenesis. Once

clarified, small interfering RNA (siRNA) approaches

may be combined with adenoviral vectors to reduce the

expression of the ‘‘offending’’ rab protein. In the case

of thyroid adenomas, the elevated expression of rab5

and rab7 might be reduced with specific antagonists of

cAMP-mediated signaling. In prostate cancer, reduc-

tion of PRC17 expression via siRNA or inhibition of

function with specific inhibitors of its GAP activity

may block metastatic spread.

In some cases, diseases result from the improper

sorting and trafficking of key cargo molecules along

rab-regulated pathways. For example, processing of

amyloid precursor protein to its plaque-producing

products by g secretase is enhanced by treatments that

inhibit cholesterol transport. Thus, ensuring normal

cellular cholesterol levels and enhancing transport to

rab7-positive late endosomes may positively reduce

aberrant APP processing [83]. A number of cancers

result from the increased recycling of growth factor

receptors. Therefore, implementing therapies that re-

sult in receptor downregulation in place of recycling,

effectively shifting the balance between interconnected

pathways, would be expected to positively impact

outcomes. Such therapies could be achieved either by

selectively blocking rab protein function or by stimu-

lating cofactors that shunt cargo transport along the

desired pathway.

5. Concluding remarks

In summary, the rab GTPases and associated regu-

latory factors are frequent targets of mutation and/or

altered expression in a variety of human disease states.

Although our understanding of the molecular mecha-

nisms of rab protein function have made significant

progress in the recent past, research to identify and

characterize additional rab-interacting proteins that are

required for endocytosis will undoubtedly provide

crucial insights into disease processes. Treatments

may vary from gene therapy to small molecule inter-

ventions, geared toward restoring normal function or

modulating pathways central to normal physiology.

*Note: Data concerning rab7 and hVPS34 interaction

now published:M.P. Stein, Y. Feng, K.C. Cooper, A.M.

Welford, A.Wandinger-Ness, Human VPS34 and p150

are rab7 interacting partners, Traffic 4 (2003) 1–18.

Acknowledgements

This work was supported by the National Science

Foundation under grant number MCB9982161 to

A.W.N. Partial salary support was provided to MPS

through a grant from the University of New Mexico

Cancer Center.

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