Progress in molecular biology and translational science, volume 141

Of the various molecular mechanisms that impart selectivity, sensitivity and strength of trans- membrane signaling, ubiquitination of the receptor protein plays an important role because

P ROGRESS IN

MOLECULAR BIOLOGY AND TRANSLATIONAL

SCIENCE

Ubiquitination and

Transmembrane Signaling

P ROGRESS IN

MOLECULAR BIOLOGY AND TRANSLATIONAL

SCIENCE

Ubiquitination and

Transmembrane SignalingEdited by

Sudha K Shenoy

Department of Medicine, Duke University

Medical Center, Durham, NC, United States

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Typeset by Thomson Digital

T Fukushima

Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan; Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan

Transmission of diverse extracellular signals to the intracellular biochemicalmachinery is mediated by cell-surface receptors, and the intracellular chem-ical reactions triggered by these receptors elicit specific physical or physio-logical cellular responses Three classes of cell-surface receptors constitutethe primary conduits of transmembrane signaling: (1) chemically gated,multitransmembrane ion channels, (2) seven-transmembrane receptors,

or G protein–coupled receptors, and (3) single-transmembrane, containing enzymic receptors Accumulating evidence indicates that theposttranslational modification called ubiquitination has a significant impact

kinase-on the strength and duratikinase-on of transmembrane signaling For most surface receptors, ubiquitination of the receptor protein dictates its expres-sion level and longevity Additionally, for almost all cell-surface receptorsubiquitination regulates intracellular trafficking, signaling activity, andprotein–protein association that can create intracellular signaling complexes

cell-of receptors and other intracellular proteins

This volume summarizes the current state of knowledge on ubiquitination

of cell-surface receptors, associated kinases, effectors, and adaptors Chapter 1(P.-Y Jean-Charles et al.) describes the roles of ubiquitination in the regulation

of the largest family of cell-surface receptors—namely, seven-transmembranereceptors (7TMRs, also known as G protein–coupled receptors or GPCRs).Chapter 2 (M Torres) discusses the relationship between ubiquitination ofheterotrimeric G proteins and their signaling Chapter 3 (P Penela) presents anoverview of ubiquitin-dependent regulation of GPCR kinases and the impact

of their ubiquitination on signal transduction Chapter 4 (F.A Wright andR.J.H Wojcikiewicz) summarizes how ubiquitination regulates inositol 1,4,5-trisphosphate receptor–mediated Ca2+ responses in the cell Chapter 5(S.M Lamothe and S Zhang) is a comprehensive review of ubiquitin-dependent downregulation of ion channels and ion transporters Chapter 6,(A Conte and S Sigismund), Chapter 7 (L Girnita et al.), and Chapter 8(G.A Smith et al.) shed light on the ubiquitin-dependent regulation of growthfactor receptors and their signal transduction pathways Chapter 9 (P.-Y Jean-Charles et al.) highlights the functional roles of ubiquitination and deubiqui-tination of the versatile adaptor proteins called beta-arrestins, which also act

xiii

as critical scaffolds that connect a number of cell-surface receptors with theubiquitination machinery.

I hope the information included in this volume will provide the readerswith a broad perspective on the importance of ubiquitination in the reg-ulation of cell-surface receptors and the control of transmembrane signal-ing This volume was made possible, of course, only by the outstandingefforts of its contributors, to whom I am very grateful I thank P MichaelConn, the Chief Editor of this series (Progress in Molecular Biology andTranslational Science), for providing me the opportunity to synthesize avolume on the roles of ubiquitination in cell signaling I also thank MaryAnn Zimmerman, the Senior Acquisitions Editor and Helene Kabes,Senior Editorial Project Manager of Elsevier, for their help Finally, I thank

my postdoctoral mentor Robert J Lefkowitz for introducing me to thefascinating field of 7TMRs and signal transduction

SUDHAK SHENOYDurham, NC

Progress in Molecular Biology andTranslational Science, Volume 141

1

Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs Of the various molecular mechanisms that impart selectivity, sensitivity and strength of trans- membrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR Ubiquitination of proteins was originally discovered in the context of lysosome-indepen- dent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling To date about 40 GPCRs are known to be ubiquitinated For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes This chapter will focus

on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

1 INTRODUCTION

1.1 G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs), also known as seven-transmembranereceptors (7TMRs), constitute the largest family of cell-surface receptors, andare encoded by roughly 800 genes in the human genome.1GPCRs transducespecific intracellular signals in response to a wide variety of extracellular stimulithat range from photons, ions, organic odorants, amino acids, lipids, nucleo-tides, peptides, and proteins (Fig 1) Signal transduction by GPCRs is funda-mental for most physiological processes and include vision, smell, and taste aswell as neurological, cardiovascular, endocrine, and reproductive functions.2–4Consequently, the GPCR superfamily is a major target for therapeutic inter-vention and about 40% of prescription drugs target GPCR activity.5–7GPCRs have a conserved structural architecture of seven transmembranehelices that traverse the membrane bilayer such that the amino terminus isexposed to extracellular milieu and the carboxyl terminus is intracellular incontact with the cytoplasm (Fig 1) Upon ligand binding and activation,specific conformational changes are triggered in the GPCR molecule Thisconformational switch facilitates GTP/GDP exchange on the Gαsubunit ofbound heterotrimeric G proteins and results in the dissociation of active Gαand Gβγ This stimulates catalytic activation of various downstream effectors(eg, adenylyl cyclase) and activation of kinase cascades and subsequent phys-iological responses.3Agonist-activated GPCRs are rapidly phosphorylated

by cognate GPCR kinases or GRKs on specific intracellular seryl/threonyl

residues Phosphorylated GPCRs then recruit cytosolic adaptor proteinscalled β-arrestins (β-arrestin1 and β-arrestin2; also called arrestin2 andarrestin3).3,8–11 GRK phosphorylation and β-arrestin binding togetherblock G protein coupling and lead to signal desensitization GPCRs alsoelicit G protein independent signaling viaβ-arrestins, which bind to a host of

Stimulus (Light, ion, odorant, pheromone, peptide, proteins, and other molecules)

β-Arrestin-dependent trafficking

G protein binding and cause rapid desensitization of GPCR signaling GRK and β-arrestin binding not only block signal transduction by G proteins, but they also promote GPCR endocytosis and trigger alternate pathways of signal propagation leading to a G protein- independent cell response(s).

signaling kinases.12–14The adaptor function ofβ-arrestins also links activatedGPCRs with the endocytic machinery and initiates intracellular trafficking

of the receptor, mostly through clathrin-coated vesicles.8,14–18 However,caveolin- or lipid-raft-mediated endocytosis as well asβ-arrestin-indepen-dent internalization have also been reported.15,19–21 After internalization,GPCRs localize in compartments called early endosomes where they can bedephosphorylated and routed back to the plasma membrane via recyclingvesicles or alternatively GPCRs can be targeted to lysosomal compartmentsfor degradation This degradation pathway serves as an “off switch” todesensitize GPCR signaling for a prolonged duration (Fig 2) GPCR sig-naling at the plasma membrane is revived by recycling of internalizedGPCRs back to the plasma membrane or by their de novo synthesis

Early endosome

Late endosome Multivesicular body

(MVB) Lysosome

Recycling

Sorting

Degradation

Recycling endosome

P

GRK

Figure 2 Agonist-stimulated intracellular trafficking itinerary of GPCRs Agonist stimulation

of GPCRs induces signaling via activated Gα and Gβγ units This is followed by rapid phosphorylation of the receptor ’s intracellular domains by GRKs and triggers recruitment of β-arrestins leading to uncoupling of G proteins (not shown) β-arrestins serve as endocytic adaptors and facilitate receptor internalization into clathrin-coated vesicles Internalized GPCRs are then mobilized into early endosomes followed by their journey to the late endosomes The late endosomes fuse into multivesicular bodies and eventually fuse with the lysosomes, which serve as degradation chambers for GPCRs During postendocytic sorting, GPCRs can be mobilized from the early endosomes into recycling endosomes that will redirect and recycle GPCRs to the plasma membrane for resensitization of receptor signaling.

GPCRs undergo many posttranslational modifications that can affecttheir expression, localization, and functional properties Among these, pal-mitoylation of conserved cysteines, phosphorylation of serines and threo-nines, and ubiquitination of lysines have been characterized for a number ofGPCRs.20–25Ubiquitination is a pervasive modification that affects numer-ous pathways and proteins in a cell Not surprisingly, a number of proteinswithin the GPCR signal transduction cascades are also regulated by ubiqui-tination This chapter will describe the functional consequences of GPCRubiquitination across a diverse sampling of GPCRs and the physiology thatthey regulate An overview of the mechanisms and the enzymatic machinerythat has been linked with GPCR ubiquitination will also be reviewed Thechapters that follow will highlight the significance of ubiquitination of signaltransducers (G protein, Chapter 2;β-arrestin, Chapter 9), a kinase (GRK2,Chapter 3), and a downstream effector (IP3R, Chapter 4).

1.2 Ubiquitination and Deubiquitination

Protein ubiquitination has been extensively studied for over three decadesand the reader is directed to excellent chapters or reviews for more infor-mation on this subject.26–33 Ubiquitination is an evolutionarily conservedand reversible posttranslational modification in which the carboxyl terminus

of the 76 amino-acid long polypeptide ubiquitin (Ub) is covalently attached

to amino group (lysyl side chain or the amino terminus) of substrate proteins.Often, successive rounds of ubiquitination occur so that Ub moieties areattached to the previously appended Ub, thus allowing the formation ofpolyubiquitin chains Additionally, each Ub has 8 such attachment points(amino terminus, and 7 lysines at positions 6, 11, 27, 29, 33, 48, and 63),which facilitate the structural diversity of these chains

Ubiquitination is achieved by the concerted activities of three types

of enzymes: Ub activating enzyme or E1, Ub conjugating enzyme or E2and E3 Ub ligases The E3 ligases define substrate specificity and timing ofubiquitination and are categorized as either HECT (Homologous to E6APCarboxyl Terminus) family ligases or RING (Really Interesting New Gene)family ligases based on the structure of their catalytic domain Human cellsexpress ∼30 HECT and ∼600 RING ligases that can selectively modifysubstrates by direct binding or indirect interaction facilitated by adaptorproteins Ubiquitination is also reversed by specific enzymes called deubi-quitinases (DUBs) that hydrolyze ubiquitin linkage from protein substrates.About 100 DUBs have been identified in human cells most of them belong-ing to ubiquitin specific proteases (USP) family

Ub was originally branded as a tag for protein degradation However, thisreversible modification is now considered as a pleiotropic tag that can reg-ulate a wide array of cellular processes This now includes protein activity,protein trafficking, and protein–protein interactions.27In general, poly-Ubchains with lysine-48 linkage marks the modified substrate for 26S protea-somal degradation On the other hand, Ub chains with lysine-63 linkagesignal vesicular trafficking or kinase activation via the modified protein.34Aside from its role as a covalently linked tag, ubiquitin can also serve as abinding platform for noncovalent association with a variety of ubiquitinbinding domains.30,35Thus ubiquitin tags and their binding partners provide

a network of protein complexes with “ubiquitin codes” that are tailored foreach substrate to effect protein conformation, function, localization, andperhaps signal transduction

2 UBIQUITINATION OF GPCRs

A substantial number of GPCRs are known to be ubiquitinated Therole of GPCR ubiquitination on internalization, signaling, vesicular traf-ficking, and/or degradation may vary based on the receptor type, ligandidentity and physiological factors involved In general, ubiquitination ofGPCRs functions as an obligatory tag for postendocytic sorting of inter-nalized GPCRs to the lysosomes.14,20,36–39For some GPCRs, ubiquitina-tion of the receptor is dispensable for lysosomal trafficking whereas forother GPCRs, ubiquitination of the receptor induces their proteasomaldegradation While ubiquitin does not generally function as a signal forinternalization of mammalian GPCRs, monoubiquitination is required forthe internalization of yeast GPCR STE2 and under certain conditionspolyubiquitination of mammalian GPCRs can serve as a trigger forinternalization.40–42A list of GPCRs that have been shown to be regulated

by ubiquitination is presented inTable 1 Specific GPCR examples alongwith the mechanisms involved are described in the following sections

Theβ2adrenergic receptor (β2AR) is a prototypical member of the GPCRfamily cloned in the 1980’s.102 β2AR is widely expressed, and has beenextensively characterized over the years for its biological and physiologicalroles.103,104Recently, crystallographic studies have enabled visualization of

and IUPHAR

and Other Adaptors

[41]

ligase complex

and IUPHAR

β-Arrestins and Other Adaptors

Role(s) of

recruitment, internalization, cell sorting and intracellular signaling

cell surface and recycling

FSH receptor Constitutive N.D N.D N.D Cell surface

[42,68–70]

UBPY

processing in MVBs

and IUPHAR

β-Arrestins and Other Adaptors

internalization Activation of p38

hormone receptor

during biosynthesis

and IUPHAR

β-Arrestins and Other Adaptors

[91,92]

internalization and lysosomal degradation

Art7

Internalization and degradation

β-Arrestin 2 Receptor

signaling Frizzled Family

RNF43

UBPY (USP8)

trafficking and degradation

[96–98]

lysosomal and proteasomal degradation

[99,100]

Orphan Family

intraluminal vesicles of multivesicular endosomes

its activation at a subatomic level.105 In humans, β2ARs are expressed incardiomyocytes, skeletal muscles, neurons, and in the smooth muscle cells ofblood vessels, lungs, intestine, and uterus.β2ARs promote smooth musclerelaxation and regulate cardiac contractility.106,107When stimulated by ago-nists,β2ARs activate the heterotrimeric Gsproteins and increase intracellularcAMP levels through the activation of adenylyl cyclase Agonist activationalso promotesβ-arrestin-dependent endocytosis of the β2AR108as well asβ-arrestin-dependent MAP kinase signaling.109,110

Individual GRKs createspecific phosphorylation codes on the carboxyl terminus of activatedβ2AR

to engage distinct functional conformations ofβ-arrestin GRK2 ylation of the β2AR promotes β-arrestin’s conformation required for itsendocytic functions and GRK6 phosphorylation promotesβ-arrestin’s alter-nate conformation that favors kinase scaffolding.111The dynamic contribu-tion of ubiquitination in the intracellular trafficking of agonist-activated

phosphor-β2AR is illustrated inFig 3

β2AR ubiquitination occurs within 15 min of isoproterenol stimulationand the signal decreases after hours of agonist exposure correlating withreceptor degradation.112A lysinelessβ2AR is not ubiquitinated upon agonisttreatment nor degraded in the lysosomes; nonetheless, it still internalizes intoearly endosomes as efficiently as the wild type receptor.112,113 The lysinestargeted for ubiquitination have been mapped by mass spectrometryapproaches to residues in the third intracellular loop (lys-263, lys-270) and

in the carboxyl tail or C-tail (lys-348, lys-372, and lys-375) of human

β2AR.113 G protein andβ-arrestins have been predicted to interact withthese two domains, namely third intracellular loop and C-tail in GPCRs.Therefore, the dynamics of ubiquitination might play a role in differentialcoupling or activation of these transducers in addition to ubiquitin-tagging

of activated receptors for lysosomal degradation Additionally, the nation site Lys-263 in the third intracellular loop is close to Glu-268 thatforms a salt-bridge with Arg-131 located in transmembrane helix 3 Thissalt-bridge constitutes an ionic lock, which maintains receptors quiescent inthe absence of an agonist As visualized in the crystal structure, the ionic lock

ubiquiti-is dubiquiti-isrupted upon receptor activation.114,115Therefore, ubiquitination of the

β2AR at these sites may alter or stabilize a new receptor conformation andmay be a direct effect that follows disruption of ionic lock and β2ARactivation

Ubiquitination of agonist-activated β2AR requires both receptor phorylation and β-arrestin2 binding.112

phos-A mutant β2AR in which all thephosphorylation sites are altered such that there is no agonist-induced

Recycling endosome

Early endosome

Clathrin-coated

Late endosome/

autophagosome Lysosome

cAMP

GRK Agonist

11 12

9

Figure 3 Roles of ubiquitination/deubiquitination in the life cycle of agonist-stimulated

β 2 AR (1) Within seconds of agonist exposure, β 2 ARs stimulate Gs, and adenylyl cyclase, increasing cellular cAMP (2) Agonist-occupied receptors are phosphorylated by GRKs on cytoplasmic domain seryl and/or threonyl residues, within seconds to minutes of agonist exposure (3) Cytosolic β-arrestin2 (β-arr2) translocates to phosphorylated receptors within 1 –5 min after agonist treatment Agonist-dependent β-arrestin ubiquitination (U) occurs immediately upon β-arrestin recruitment and is mediated by Mdm2 that is bound to β-arrestin β-arrestin recruitment prevents further G protein coupling and β-arrestin ubiquitination allows it to form signaling and endocytic complexes, facilitating both receptor endocytosis and MAPK signaling (4) β-Arrestin conformational changes that occur upon receptor binding allow its interaction with Nedd4, which displaces Mdm2 from β-arrestin (5–15 min after agonist treatment) (5) By interacting simultaneously with β 2 AR, clathrin and AP-2, β-arrestin2 facilitates β 2 AR endocytosis (6) β-Arrestin2 is deubiquitinated by USP33 starting at step 4 Nedd4 mediates ubiquitination of the internalizing β 2 AR (10 –15 min after agonist treatment) (7) Ubiquitinated β 2 ARs move on into early endosomes (at >15 min after activation) (8)

β 2 AR ubiquitination persists until about 6 h after agonist stimulation, when β 2 ARs move into late endosomal/lysosomal compartments (9) The level of ubiquitinated β 2 ARs decreases, as ubiquitinated receptors are degraded in lysosomes (6 –24 h or more after agonist stimulation) (10 –12) From the early and/or late endosomes, receptors may take up an alternate path and enter recycling endosomes (<15–30 min or 6 h after activation), in which β 2 ARs become dephosphorylated by a phosphatase and deubiquitinated by USP20 (or USP33), and return to the plasma membrane as “nạve receptors ” This figure has been modified from an originally published panel in the Journal

of Biological Chemistry by Shenoy, SK, et al 2008; 283(32):22166 –22176.

phosphorylation shows impaired ubiquitination as well as significantly reducedβ-arrestin interaction.8,112

In cells where theβ-arrestin2 gene is deleted or itsexpression is downregulated by RNAi,β2AR ubiquitination is not induced

by agonist-stimulation.112,116β-arrestin2 functions as an adaptor for the nate E3 ubiquitin ligase designated as “Neural Precursor Cell Expressed,Developmentally Downregulated 4” (NEDD4), which ubiquitinates activated

cog-β2ARs.45,116 NEDD4’s catalytic activity is attributed to its intact HECTdomain In addition, it has a C2 domain that facilitates NEDD4’s associationwith membranes and 3–4 WW domains that are characteristic binding surfacesfor polyproline (PPXY) motifs in various substrates or adaptor proteins.117,118β-arrestin2 and NEDD4 interaction, however, does not involve WW domainbinding and follows alternate mode of interaction.45 Althoughβ-arrestins 1and 2 lack the PPXY motif,β-arrestins still associate with NEDD4 Moreover,upon β2AR stimulation, NEDD4 binding with β-arrestin2 occurs withgreater affinity and faster kinetics than withβ-arrestin1.45

Whileβ-arrestin2 functions as the preferred adaptor for the tion of activated β2ARs and for NEDD4-mediated ubiquitination ofthe receptor, other vesicle-associated proteins that share a predicted struc-tural homology with β-arrestins may regulate postendocytic sorting ofGPCRs.14,116,119,120These proteins include the arrestin-domain containingproteins (ARRDC 2, 3, and 4) and VPS26, which associate with subpopula-tions of endocytic vesicles to whereβ2ARs and other GPCRs colocalize afterinternalization.116,121,122 Since ARRDC3 possesses polyproline (PPXY)motifs that bind the WW domains in HECT family E3 ligases (such asNEDD4), ARRDCs rather thanβ-arrestins have been implicated as ubiqui-tin ligase adaptors for theβ2AR.120However, downregulation of ARRDC3has no effect onβ2AR internalization, ubiquitination or lysosomal traffick-ing.116Despite the absence of polyproline motifs, bothβ-arrestins can func-tion as efficient adaptors for WW domain containing E3 ligases as demon-strated for the β2AR, CXCR4, and other membrane proteins.14,53,116,123The PPXY motifs in ARRDCs are critical for their interaction withNEDD4 and components of the endosomal sorting complexes requiredfor transport (ESCRTs) such as HRS (hepatocyte growth factor-regulatedtyrosine kinase substrate) that regulate cargo trafficking116,121It is likely thatARRDCs play an ancillary role inβ2AR trafficking to promote sorting ofinternalized receptors Additionally, they may recruit NEDD4 to ubiquiti-nate HRS, which can facilitate the sorting of internalized receptors to lateendosomes or multivesicular bodies.54 On the other hand, HRS has beenshown to regulate recycling of theβ2AR124even though other studies have

internaliza-concluded that HRS inhibits recycling of early endosomes.125More detailedcharacterizations of ARRDCs for their roles in the trafficking of additionalGPCRs will be required to understand the relationship ofβ-arrestins and therelated arrestin-like proteins in GPCR trafficking.

The beta-blocker carvedilol stimulatesβ-arrestin2-dependent signaling and

β2AR internalization.126Carvedilol also induces ubiquitination of theβ2AR;however, this does not involveβ-arrestin2 or NEDD4, but requires a trans-membrane RING-finger E3 ligase, Membrane-associated RING-CH2(MARCH2).41 MARCH2 is a ubiquitously expressed protein that regulatestrafficking between the trans-Golgi network and endosomes.127Interestingly,while specific lysines are ubiquitinated by agonist-stimulation of theβ2AR,carvedilol may trigger MARCH2-mediated ubiquitination of nonlysineresidues in theβ2AR Carvedilol-induced ubiquitination provokes internali-zation, lysosomal trafficking, and degradation of the β2AR Thus, whileagonist-induced ubiquitination is dispensable forβ2AR internalization, carve-dilol-induced ubiquitination is required for receptor endocytosis.41

In quiescent cellsβ2AR is also hydroxylated on C-tail proline residues byoxygen-dependent prolyl hydroxylase, Egl-9 family hypoxia-inducible fac-tor 3 (EGLN3).48The E3 ligase pVHL (von Hippel-Lindau) which is part of

a complex that includes elongin B, elongin C, and cullin-2, ubiquitinates thehydroxylatedβ2AR and promotes the degradation of the receptor via 26Sproteasomes.48Hypoxia inhibits prolyl hydroxylase and prevents ubiquitin-dependent proteasomal degradation of theβ2AR In addition to hypoxia,other mechanisms can regulate β2AR degradation For example,SUMOylation of caveolin 3 by the SUMO E3 ligase PIASγ appears toregulate β2AR expression.128While coexpression of wild type caveolin 3increases β2AR expression, a mutant caveolin 3 (Cav3 K7R) dramaticallyreducesβ2AR expression.128

Agonist-stimulation of the β2AR also triggers ubiquitination ofβ-arrestin2 that is mediated by the E3 ubiquitin ligase Mdm2 β-arrestin2ubiquitination serves as a critical tag required for high affinity interactionswith the β2AR, endocytic proteins and signaling kinases (see Chapter9).112,129 β-arrestins possess conserved SUMOylation motifs that contain alysine residue that is SUMOylated.14,130Bovineβ-arrestin associates weaklywith AP2 when the SUMOylation site is mutated and this inhibits β2ARinternalization SUMOylation ofβ-arrestin is not critical for its associationwith activated β2AR.130 β2AR activation also triggers ubiquitination ofvarious proteins: GRK2 (see Chapter 3), PDE4D5, Rab GTPases, which

in turn modulateβ2AR signaling and trafficking mechanisms.131–133

Multiple lines of evidence suggest that ubiquitination plays an importantrole in targeting activatedβ2ARs to lysosomal compartments, which are theprimary destruction sites for internalized β2ARs.41,45,46,112,113,116,134,135Additionally, reversible ubiquitination may regulate the partitioning of acti-vated receptors between the route of degradation and that of recycling to theplasma membrane.46 It is also tempting to speculate that the ubiquitinmoieties on theβ2AR can serve as a signal transduction platform at the lateendosomes as the ubiquitinatedβ2ARs are retained for prolonged periods inthese subcellular compartments before permanent destruction.45

2.2 Chemokine Receptors

The chemokine receptor family consists of 20 GPCRs that mainly couple tothe inhibitory heterotrimeric G proteins, Giand elicit signaling via phos-pholipase C-β and phosphatidylinositol 3-kinase (PI3K).136–139

Receptoractivation also triggers the recruitment ofβ-arrestin with distinctive effects

on receptor signaling, endocytosis and trafficking As much as 50 kines are promiscuously recognized by these receptors which engage either

chemo-in G protechemo-in- or β-arrestin-dependent signaling based on ligand, cell, ortissue characteristics.140–142Conventional chemokine receptors that couple

to G proteins are subdivided into 4 subgroups (CCR, CXCR, XCR, andCX3CR), based on the number and arrangement of conserved cysteineresidues in their cognate ligands In addition, a group of four chemokinereceptors have been designated as atypical chemokine receptors (ACKRs)because of their alternate signaling mechanisms that do not mirror theconventional chemokine signaling profiles.143 By governing migration,priming and homing of immune cells, the chemokine receptors not onlyregulate cellular and humoral immune responses, but are primary mediators

of cellular responses in inflammation and cancer.136A handful of chemokinereceptors have been studied for the effects of ubiquitination Surprisinglythere is hardly a consensus for the role(s) of ubiquitination Some chemo-kines induce ubiquitin-dependent lysosomal targeting whereas others pro-mote ubiquitin-dependent plasma membrane recycling of receptors (Fig 4).2.2.1 CXCR2

CXCR2 is expressed in neutrophils and mediates neutrophil migration tosites of inflammation Interleukin 8 (IL8) induces CXCR2 internalization to

a greater extent than other ligands CXCL6 and CXCL7.144The C-tail ofCXCR2 governs IL8-induced receptor phosphorylation and β-arrestinrecruitment and rapid internalization.145CXCR2 trafficking involves AP2

K327 poly-Ub is required for:

(1) β-arr2 recruitment.

(2) CXCR2 internalization.

(3) signaling.

IL-8 induced poly-Ub

K327

U U

U U

U U

Mono-Ub is required for MVB sorting &

degradation.

MVB targeting requires β-arr1 ESCRTs &

VPS4.

CXCL12-induces de-ubiquitination &

recycling.

Constitutive poly-ub

is required for CCL19-induced trafficking and recycling.

CCL19 CXCL12

CXCL12

AIP4

IL-8

U U

Figure 4 Functional diversity of ubiquitination of chemokine receptors IL-8 stimulation induces polyubiquitination of CXCR2 on lysine 327,

the receptor CXCL12 triggers AIP4 recruitment and monoubiquitination of CXCR4, which is required for postendocytic sorting and lysosomal

CXCR7 to the cell surface The constitutive K63-linked polyubiquitination of CCR7 is not enhanced upon agonist stimulation but is required for CCL19-induced trafficking and recycling.

association and requires a type I PDZ ligand motif, which regulates lysosomaltrafficking and chemotactic responses, but does not influence receptor inter-nalization and receptor recycling.51A single lysine (K327) in the CXCR2C-tail serves as a major site for polyubiquitination CXCR2K327Rmutant isnot only impaired in ubiquitination but also significantly defective inβ-arrestin2 recruitment and CXCR2 internalization.52 CXCR2K327Rmutant is expressed at the plasma membrane at levels similar to the wild typeCXCR2 However, the CXCR2K327Rmutant is impaired in IL8-inducedERK activation as well as AP1 and NFκB-dependent transcription.52

Thussite-specific ubiquitination regulates both CXCR2 signaling and trafficking.The molecular mechanisms of CXCR2 ubiquitination remain to beidentified

of CXCR4.148 Although both nonvisual arrestins associate with activatedCXCR4,β-arrestin2 is recruited more readily than β-arrestin1 β-arrestin-binding requires GRK2-mediated phosphorylation of a distinct group ofserines in the C-tail of CXCR4.148 β-arrestin-dependent ERK activationproceeds only when β-arrestin binds CXCR4 that is phosphorylated onspecific serine residues by GRK3 and GRK6.148 CXCR4 downregulationrequires lysosomal trafficking and a tight regulation of CXCR4 expression isrequired for normal physiology An increase in CXCR4 expression (perhapscaused by a faulty degradation pathway) is seen in many aggressive cancers,which require CXCR4 signaling for growth and metastasis

CXCL12 stimulation induces monoubiquitination of CXCR4, which isdependent upon the phosphorylation of serines within a degradation motif

“SSLKILSKGK” in the C-tail of the receptor.55Mutation of either serines

or lysines in this motif affects both ubiquitination and ligand-induceddegradation of CXCR4 The HECT-domain E3 ligase called Atrophin-1-interacting protein 4 (AIP4) ubiquitinates CXCR4 and binds the receptorC-terminus via a direct interaction that involves seryl-phosphorylation of

CXCR4 and WW domains in AIP4.149CXCR4 lysosomal degradation alsorequires AIP4-mediated ubiquitination of the ESCRT-0 protein, HRS.54β-arrestins are not required for AIP4-mediated ubiquitination of CXCR4.However,β-arrestin1 is required for the lysosomal trafficking of CXCR4.53

The N-terminus of β-arrestin1 interacts with the WW domains of AIP4.Furthermore,β-arrestin1 interaction with the endosomal protein STAM-1(signal transducing adaptor molecule 1) is required for HRS ubiquitination,but not for CXCR4 or STAM-1 ubiquitination.56 Although β-arrestin2binds CXCR4 and HRS, it does not interact with STAM-1, hence theESCRT-dependent sorting of CXCR4 appears to be predominantly affected

byβ-arrestin1.56,116AIP4 and STAM-1 also associate directly through a proline region in AIP4 and an SH3 domain in STAM-1.150Ubiquitination ofSTAM-1 by AIP4 in caveolae is required for CXCR4-induced ERK activa-tion.150 The activity of AIP4, which is critical for the ubiquitination ofCXCR4 and ESCRT proteins is suppressed when the expression of aRING domain E3 ubiquitin ligase Deltex3L is elevated.151 In addition toDeltex3L andβ-arrestin1, whether AIP4 and its activity in CXCR4 endocytictrafficking involves the ARRDC proteins remains to be defined

poly-2.2.3 CXCR7

The chemokine receptor CXCR7 (also known as ACKR3) binds the mokine ligands CXCL11 (which also activates CXCR3) and CXCL12(which also activates CXCR4); however, it fails to activate G protein sig-naling.142,152CXCR7 associates withβ-arrestin and promotes MAP kinaseactivity as well as chemotaxis of rat aortic smooth muscle cells in aβ-arrestin-dependent manner.142CXCR7 expression has been correlated with increase

che-in tumor progression and cancer metastases che-in various tissues.153,154Since itbinds CXCL12 with considerable affinity, CXCR7 has also been regarded as

a means by which CXCR4 signaling can be titrated CXCR7 is also basallyubiquitinated and stimulation with CXCL12 reduces CXCR7 ubiquitina-tion mainly by deubiquitination because a drastic decrease in CXCR7protein levels does not ensue.59Additionally, CXCR7 recycles by default:after few hours of agonist binding the internalized receptor recycles to theplasma membrane Interestingly, a CXCR7 mutant receptor, in which theC-tail phosphorylation sites are eliminated,β-arrestin binding and receptorinternalization are dramatically reduced whereas CXCR7 ubiquitination isincreased.59The unique trafficking and deubiquitination profiles of CXCR7are attributed to the C-tail sequence of this receptor The E3 ligase and thedeubiquitinase that regulate CXCR7 are not known, but the data available

suggest thatβ-arrestins may have a role in mediating deubiquitination of thisreceptor after CXCL12 stimulation.

2.2.4 CCR7

CCR7 is mainly expressed by naı¨ve lymphocytes and mature dendritic cells.CCR7 signaling is important for immune responses needed for the homingand interaction of various cells types within lymphoid tissues.155,156CCR7 isactivated by two endogenous chemokine ligands CCL19 and CCL21, whichhave similar binding affinities and equal efficacy for G protein signaling andchemotaxis However, only CCL19-stimulation triggers robust CCR7phosphorylation, β-arrestin binding, and CCR7 internalization, whereasCCL21 shows only weak effects.157 Both CCL19 and CCL21 engageGRK6 activity, however, only CCL19 induces GRK3-mediated CCR7phosphorylation, thus displaying a kinase bias in GPCR signaling.158CCR7 is constitutively appended with lysine-63 linked polyubiquitina-tion and this is not further increased by stimulation with either CCL19 orCCL21.50CCR7 ubiquitination is not dependent upon AIP4 or NEDD4ligases, which have been shown to ubiquitinate other GPCRs.14,45,54However, CCR7 ubiquitination is dramatically decreased when the lysines

in the cytoplasmic loops are replaced with arginine residues The defective CCR7-7K7R mutant is expressed at the plasma membrane tosimilar levels as the wild type CCR7 While the constitutive trafficking ofCCR7-7K7R is impaired, the agonist-induced internalization is notaffected.50Unlike other GPCRs that are sorted into lysosomes, internalizedCCR7 does not colocalize with LAMP1 and even after 4–5 h of agonist-stimulation, CCR7 does not degrade Interestingly, when the agonistCCL19 is removed to induce CCR7 recycling, the wild type CCR7 recyclesback to the plasma membrane but the mutant CCR7-7K7R does notrecycle.50 This suggests that ubiquitin chains on CCR7 carry a specificsignal that facilitates recycling of CCR7 In addition the agonist-inducedchemotactic responses are not elicited by the CCR7-7K7R mutant While

ubiquitin-WT CCR7 bindsβ-arrestin with high affinity after CCL19 stimulation, it isunknown whether CCR7-7K7R can recruitβ-arrestin Some of the defectsexhibited by CCR7-7K7R can be attributed to either loss of β-arrestinbinding and/orβ-arrestin ubiquitination, which remain to be defined

2.3 Proteinase Activated Receptors

A subfamily of four homologous GPCRs called Proteinase ActivatedReceptors (PAR1, PAR2, PAR3, and PAR4) is activated by endogenous

proteases that cleave the N-terminal domain of the receptor to expose a newN-terminus that acts as a tethered ligand.159–162 PARs 1, 3, and 4 areactivated by thrombin and PAR2 by trypsin and trypsin-like proteases.PARs are expressed predominantly in vascular, immune, and epithelial cells,astrocytes and neurons, and transmit cellular responses to coagulant proteases

as well as other proteases expressed in tissues PAR expression and signaltransduction orchestrate hemostasis, thrombosis, inflammation, and perhapstissue repair PARs may also participate in the progression of specific cancers.PARs 1 and 2 have been extensively studied whereas very little characteri-zation has been performed for PARs 3 and 4 Activated PAR1and PAR2couple to multiple heterotrimeric G-protein subtypes including Gi, Gq, and

PAR2internalizes and signals in aβ-arrestin-dependent manner.163–166

Thephosphorylation sites specifically targeted by individual GRKs have not beendefined for the PARs.159–161,167 Both PAR1 and PAR2 are regulated byubiquitination; however, the mechanisms involved and functional conse-quences are completely different between these two PARs (Fig 5)

2.3.1 PAR1

PAR1 ubiquitination occurs basally and is also modulated by agonists.168PAR1ubiquitination inhibits its constitutive internalization and PAR1lyso-somal trafficking occurs independent of ubiquitination.78,168The postendo-cytic trafficking of PAR1is also not dependent on the activity of ESCRT-0components, HRS and TSG101 that are required for the ubiquitin-dependent lysosomal trafficking of other GPCRs.168Agonist-independentconstitutive internalization of PAR1is completely dependent on the expres-sion of the clathrin adaptor protein complex-2 (AP-2) and requires AP-2binding to a tyrosine-based motif (YSIL) in PAR1 C-tail.79 In contrast,agonist-dependent internalization of PAR1 requires agonist-induced ubi-quitination of PAR1as well as Epsin1 and involves a ubiquitin-interactionmotif in Epsin1.79 Despite the requirement of PAR1 ubiquitination foragonist-induced PAR1 internalization, ubiquitin-defective PAR1 mutantreceptors internalize and traffic to lysosomes upon agonist-stimulation at arate comparable to the wild type PAR1

Another tyrosine containing motif YPXXXL in the second intracellularloop of PAR1 recruits an ESCRT-III associated protein called apoptosis-linked gene 2-interacting protein X (ALIX) which mediates the lysosomaldegradation of both ubiquitinated and un-ubiquitinated PAR1, but does notbind or regulate PAR2, which lacks the YPXXXL interacting motif.80ALIX

independent trafficking &

Ubiquitin-lysosomal degradation.

Requires ALIX ubiquitination

by WWP2 bound to the adaptor ARRDC3.

induced K-63 poly-Ub that is not required for lysosomal trafficking but required to activate p38 kinase via a non-canonical pathway.

induced multi mono-Ub is required for MVB sorting &

Agonist-degradation.

NEDD4-2

c-CBL

U U U U

Agonist peptide or thrombin

Agonist peptide or trypsin

Post-endocytic de-ubiquitination

by AMSH and USP8 promotes degradation.

U U

linked polyubiquitination by NEDD4-2, with no effect on receptor trafficking and degradation, but inducing p38 activation via a noncanonical

mediates PAR1’s interaction with the ESCRT-III protein, charged MVBprotein4-ESCRT-III (CHMP4).80ALIX-PAR1association also requires theexpression of adaptor protein complex-3 (AP-3) because AP-3 RNAi

or mutagenesis of yet another proximal tyrosine based motif (YSIL) inPAR1 C-tail eliminates the association of PAR1 and ALIX.169 AlthoughPAR1 ubiquitination has no role in this receptor’s lysosomal degradation,ubiquitination of ALIX by a HECT-domain ligase called WWP2 is criticalfor PAR1’s sorting to the lysosomes.80 ALIX ubiquitination requiresARRDC3 as an E3 ligase adaptor for WWP2.81

Although initial studies suggested that PAR1 is deubiquitinated uponagonist activation,78 subsequent studies revealed that thrombin as well asthe peptide agonist SFLLRN trigger robust PAR1 ubiquitination.77 TheHECT domain E3 ubiquitin ligase NEDD4-2 mediates PAR1 ubiquitina-tion that consists of either monoubiquitin (detected by the ubiquitin IgGcalled P4D1) or lysine-63 linked polyubiquitin.77Ubiquitinated PAR1initi-ates the assembly of signalosomes by recruiting TAB1-TAB2 and leads to theactivation of p38 MAP kinase independent of its canonical upstream kinasesMKK3 and MKK6.77The TAB2-, TAB1-, and NEDD4-2–dependent p38signaling is critical for activated PAR1-stimulated endothelial barrier per-meability in vitro and PAR1-induced p38 signaling is essential for vascularleakage in vivo.77 However, the mechanisms by which TAB1-dependentp38 signaling specifically regulates endothelial barrier disruption remain to

be identified.77

2.3.2 PAR2

PAR2 is multi-monoubiquitinated by the RING domain E3 ligase c-Cblupon agonist-stimulation.82 PAR2 activation induces c-Cbl tyrosyl phos-phorylation by c-Src and triggers localization of c-Cbl to the plasma mem-brane and early endosomes to associate with PAR2 c-Cbl-mediated ubiqui-tination of PAR2is not required for receptor endocytosis but is necessary fortranslocation of the receptor from early endosomes to late endosomes andlysosomes, where its degradation irrevocably terminates signaling.82A PAR2

mutant in which all the 14 intracellular lysines are replaced with argininescouples to G proteins and internalizes as efficiently as the wild type PAR2,but does not get ubiquitinated and is not sorted into lysosomal compartmentslike the wild type PAR2.82 Since PARs are irreversibly activated by thecleavage of their N-terminal domain by proteases, lysosomal degradation

is the main mechanism to permanently stop their signaling In fact, whileendocytosed PAR2does not recycle, it induces ERK signaling in aβ-arrestin

dependent matter.165,170 Thus, termination of pain and inflammationinduced by PAR2signaling in epithelial or inflammatory cells would requirethe degradation of these receptor molecules mostly by ubiquitin-dependentmechanisms Accordingly, defects in PAR2 ubiquitination might prolongPAR2signaling and lead to uncontrolled pain and inflammation.

2.4 Opioid Receptors

Opioid receptors (ORs) are part of the rhodopsin family of GPCRs andhave been reviewed previously.171 Briefly, the ORs include the μ-OR(MOR), ∂-OR (DOR), κ-OR (KOR), and the nociceptin receptor(NOR) ORs are stimulated by endogenous peptide agonists and ORsignaling controls a multitude of central nervous system mediated behaviorsthat include motivation/reward, pleasure, and nociception Interestingly,the ORs display a divergent utilization of ubiquitination for trafficking(Fig 6) Polyubiquitination of the KOR within the C-tail of the receptordoes not regulate receptor internalization but does regulate sorting fromendosomes to lysosomes73 In contrast, ubiquitination of DORs is notnecessary for their internalization or sorting to lysosomes, but is requiredfor proteasomal degradation of newly synthesized misfolded recep-tors.71,172,173 However, subsequent studies did demonstrate that theDOR is ubiquitinated by the E3 ubiquitin ligase AIP4 and deubiquitinated

by the DUB called associated molecule with the SH3 domain of STAM(AMSH) and USP8 (also known as UBPY).72 This ubiquitination cyclewas found to be important for receptor proteolysis and targeting of thereceptors to the lysosomal compartments.68,72The MORs are arguably themost well known for their clinically useful analgesic properties whenstimulated by morphine Unfortunately, opiate drugs of abuse, like mor-phine and heroin, target MORs, evoke a sense of euphoria and are highlyaddictive.171 Therefore, several years of research have been underway todetermine if the clinically important analgesic properties of MORs can beselectively targeted while bypassing the unwanted euphoric and addictiveside-effects of opiates

Internalization and desensitization of MORs are important for regulatingtheir signaling and in vivo models suggest that the unwanted side effects ofmorphine may be due to its inability to effectively initiate receptor mediatedendocytosis.174 Typically, ligand mediated activation of GPCRs leads torapid recruitment of arrestins and clathrin-mediated endocytosis Thisoccurs for MORs when stimulated by many compounds in the alkaloid

β-arrestin2 dependent ubiquitination.

Ubiquitinated receptors signal timely scission of CCVs.

Agonist-induced K-63 poly-Ub required for β- arrestin & GRK2 dependent κ-OR degradation.

CYLD

or U50, 488H DADLE

U U

multivesicular bodies While ubiquitination by AIP4 facilitates the proteolytic processing of the receptor for lysosomal degradation, the

enhanced by a dominant negative mutant of the deubiquitinating enzyme CYLD.

opiate family However, very early observations demonstrated that morphine

is unable to initiate appreciable MOR internalization orβ-arrestin ment, unless GRK2 was overexpressed.175Unexpectedly, in vivo analysis ofmorphine elicited behaviors inβ-arrestin2 knockout mice illustrated that acomponent of this response does requireβ-arrestin2.176–179These conflict-ing results were resolved with a more sensitive assay forβ-arrestin recruit-ment that demonstrated morphine activated MORs can weakly recruitβ-arrestin2 but not β-arrestin1.180

recruit-Subsequent studies have confirmed thisfinding and also demonstrated that morphine biases translocation ofβ-arrestin2 to MORs and reduces ubiquitination of MORs A differentscenario is observed with other MOR ligands (ie, DAMGO) that driveβ-arrestin1/β-arrestin2 translocation and enable ubiquitination of MOR.The functional consequences of MOR ubiquitination have been quite sur-prising When stimulated with a MOR-specific agonist DAMGO, thereceptor is ubiquitinated in aβ-arrestin1-dependent manner.69

On the otherhand, stimulation with another ligand DADLE (that stimulates bothμ and δopioid receptors) triggers MOR ubiquitination in aβ-arrestin2-dependentmanner In contrast to many other GPCRs, which are ubiquitinated inintracellular loop 3 (ICL3) or the C-tail, ubiquitination for the MOR occurs

at two lysine residues located in intracellular loop 1 (ICL1).181This tination is not required for sorting the receptor into recycling compartments

ubiqui-or endosomes nubiqui-or destines the MOR fubiqui-or lysosomal degradation.181BlockingMOR ubiquitination delays the scission of the clathrin-coated vesicles andslows the rate of endocytosis This mechanism is driven by β-arrestin2recruitment to the MOR and by β-arrestin2’s scaffolding of the E3-ubiquitin ligase SMURF2 The mobilization of clathrin-coated vesicles byubiquitinated MORs demonstrates yet another mechanism by which inter-nalization of GPCRs is tightly regulated.42

2.5 Noncanonical GPCRs and Ubiquitin Ligases

The prototypical 7-transmembrane architecture is a defining structural ture of GPCRs However, for a few GPCRs, their ability to directly couple

fea-to G protein is quite controversial These so-called noncanonical GPCRseither do not couple to G proteins or their full signaling mechanism remainsincompletely characterized Many of these noncanonical GPCRs signal bydirectly interacting with additional membrane proteins to transduce signals.For instance, there are 10 frizzled receptors (Fzd) that are part of the Frizzledclass of GPCRs (Class F) that transduce Wnt/β-catenin signaling.182 In

addition, also part of this class of GPCRs is the smoothened receptor (Smo)that together with its repressor Patched (Ptc) controls hedgehog signaling.183These signaling pathways are important for tissue patterning, embryonicdevelopment, stem cell mediated repair and regeneration during adulthood,and remain implicated in cancer Not surprisingly trafficking and ubiquiti-nation appear important for the regulation of signaling via Fzd (Fig 7) as wellSmo (Fig 8).

2.5.1 Wnt/β-Catenin Signaling

The developmentally important Fzds lack many of the discrete GPCR motifsthat are critical for G protein coupling, including the “DRY” motif intransmembrane 3 and the “NPXXY” domain in transmembrane 7.182Even still, as previously reviewed, many reports suggest that coupling to Gproteins is important for their function.184,185Regardless of their ability tocouple to G proteins, this family of GPCRs is critical for initiating Wnt/β-catenin signaling Fzds, together with their coreceptor lipoprotein recep-tor-related protein (LRP) tyrosine kinase, bind to Wnt This activated Wnt-receptor complex exerts its effects by inhibiting the phosphorylation, ubi-quitination, and proteasomal degradation of β-catenin Free β-catenin isthen able to translocate into the nucleus and activate a Wnt/β-catenin geneexpression program.186,187Therefore, much like canonical GPCR signaling,controlling Wnt-receptor levels at the plasma membrane is a potent modethat controls the Wnt/β-catenin signaling pathway Wnt/β-catenin signaling

is a critical mediator of organogenesis, stem cell fate, and tissue tion.187In addition there are many well-known oncogenic mutations withinthe Wnt/β-catenin signaling pathway Therefore, antibodies, peptides, andsmall molecule therapies have been developed to regulate Wnt/β-cateninsignaling These myriad approaches target Wnt-ligand secretion, antagonizeWnt-receptor binding, or promoteβ-catenin destruction.188,189An attractiveand alternative approach would be to target the surface expression and traf-ficking of the Wnt-receptors

regenera-Important progress was made in 2010 when Fzd expression at the plasmamembrane and its recycling was shown to be regulated by ubiquitination.96Inthis mechanism, ubiquitination of endocytosed Fzd sorts the receptor to thelysosome while deubiquitination by USP8, enables Fzd recycling back to theplasma membrane.96 Recent work has identified the E3 ubiquitin ligasesresponsible for the clearance of Fzd from the membrane and its sorting towardlysosomal degradation In a screen for Wnt/β-catenin responsive genes, twogenes RNF43 (RING finger-43) and ZNRF3 (Zinc and RING finger-3)

Fzd-LRP complex ubiquitination and lysosomal degradation.

Fzd-LRP complex stabilized at the membrane.

ZNRF3 RNF43

Wnt Wnt

R-spo

Wnt Signal down-regulated.

Wnt Signal up-regulated.

Figure 7 Ubiquitination and Wnt-Signaling (A) Wnt-signaling is initiated upon Wnt binding to the Wnt-receptors Fzd and Lrp5/6 This facilitates

“A.” R-spondin causes scaffolding of Lgr4/5 together with ZNRF3/RNF43 This association sequesters away the ubiquitin ligases so that the receptor complex escapes ubiquitination Therefore, the Fzd/Lrp complex is more stably expressed at the plasma membrane and Wnt-signal transduction is upregulated.

USP8 de-ubiquitinates Smo and promotes recycling upon Hh stimulation.

USP8

P

P P

Hh promotes Ptc ubiquitination and degradation, which increases Smo signaling.

Increased Gli signaling

SMURF1 SMURF2 Hh

Figure 8 Ubiquitination and Smoothened-Signaling (A) Ubiquitination of Smo: In the absence of Hh, Smo is ubiquitinated within the Smo autoinhibitory domain (SAID) This facilitates endocytosis and degradation of Smo Ptc activation by Hh results in Smo phosphorylation within the SAID domain and prevents ubiquitination of Smo The net effect is to increase Smo at the plasma membrane and promote Smo-signaling A key enzyme in this process is the deubiquitinase (DUB) USP8 (B) Ubiquitination of Ptc: Ptc internalization and degradation is also regulated by ubiquitination Hh-binding to Ptc drives Smo-signaling, which in turn also fosters ubiquitination of inactive Ptc and clears this negative regulator

of Smo-signaling from the plasma membrane This positive feedback mechanism is driven through Smo-mediated upregulation of the E3 ubiquitin ligases SMURF1/2 Smo itself may directly scaffold SMURF1/2 to Ptc.

were found These genes encode proteins that contain both a brane domain and an E3-ubiquitin ligase domain Current reports suggestthat these E3-ubiquitin ligases are functional homologs, that they areexpressed at the plasma membrane, and that they attenuate Wnt/β-cateninsignaling through ubiquitination and clearance of Fzd proteins.190Activityappears to depend on the scaffolding of RNF43/ZNRF3 to Fzd/LRP bythe adaptor protein disheveled (DVL).97Therefore, increasing the levels ofRNF43/ZNRF3 at the plasma membrane attenuates Wnt/β-catenin sig-naling by clearing and degrading Wnt-receptors Conversely, loss ofRNF43/ZNRF3 at the cell surface results in decreased ubiquitination ofWnt-receptors and a concomitant increase in their expression at the cellsurface Together this results in potentiated Wnt/β-catenin signaling andcan lead to tumorigenesis.98

transmem-Recent work illustrates that membrane expression of RNF43/ZNRF3 istightly controlled by R-spondins and their cognate receptors Lgr4 andLgr5.191Lgr4 and Lgr5 are members of the rhodopsin class of GPCRs andare part of a family of GPCRs characterized by leucine-rich repeat domains

in the extracellular N-terminus (like the glycoprotein hormone receptors).Lgr4 and Lgr5 were originally cloned in 1998 but the inability to find theircognate ligands slowed their biochemical characterization.192–194 Morerecently, lineage tracing analysis from a multitude of tissues demonstratedthat adult intestinal stem cells can be identified solely on the basis of Lgr5expression.195 Lgr4 expression also appears to mark an expanded pool ofprogenitor cell types that includes both stem cells and their transientlyamplifying progeny.196 Lgr5-expressing cells also appear to be the cell oforigin in intestinal cancer.197,198 Therefore, the search for Lgr4 and Lgr5ligands has been reinvigorated

Several groups demonstrated that Lgr4 and Lgr5 bind R-spondins andthat this results in potentiation of Wnt/β-catenin signaling Similar to Fzds,this appears to be independent of G protein coupling or recruitment ofβ-arrestins.196,199–201

However, unlike Fzds, Lgr4, and Lgr5 both containmore discrete GPCR domains such as “DRY” and “NPXXY.” In addition,Lgr5 is able to recruit β-arrestin in vitro when GRKs are overexpressedsuggesting that a component of this signaling mechanism remains to beclarified.202 Several lines of biochemical and molecular evidence demon-strated that trafficking of Lgr5 and Lgr4 are important to their function Lgr4and Lgr5 constitutively internalize from the plasma membrane.Furthermore, upon its internalization, Lgr5 also appears to retrograde traffic

to the trans-Golgi network (TGN) However, the precise roles for trafficking