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ava i l ab l e a t www.sc i enced i r ec t . com

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Review

Trafficking of receptor tyrosine kinases to the nucleus

Graham Carpenter⁎, Hong-Jun Liao

Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0146, USA

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author.E-mail address: graham.carpenter@vanderbi

0014-4827/$ – see front matter © 2008 Elseviedoi:10.1016/j.yexcr.2008.09.027

A B S T R A C T

Article Chronology:

Received 20 August 2008Accepted 19 September 2008Available online 11 October 2008

It has been known for at least 20 years that growth factors induce the internalization of cognatereceptor tyrosine kinases (RTKs). The internalized receptors are then sorted to lysosomes orrecycled to the cell surface. More recently, data have been published to indicate other intracellulardestinations for the internalized RTKs. These include the nucleus, mitochondria, and cytoplasm.Also, it is recognized that trafficking to these novel destinations involves new biochemical

mechanisms, such as proteolytic processing or interaction with translocons, and that thesetrafficking events have a function in signal transduction, implicating the receptor itself as asignaling element between the cell surface and the nucleus.

© 2008 Elsevier Inc. All rights reserved.

Keywords:

ErbB receptors

EGF receptorReceptor tyrosine kinaseNucleusTrafficking

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1557γ-Secretase-dependent trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1557

ErbB-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1557Ephrin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560CSF-1 receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560VEGF receptor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560Tie 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560IGF-1 and insulin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560

Non-secretase formation of RTK ICD fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560Trafficking of intact receptors to the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1561

ErbB-1 and translocon-mediated trafficking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1561Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1562References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1562

lt.edu (G. Carpenter).

r Inc. All rights reserved.

Table 1 – Receptor tyrosine kinases subject tointramembrane proteolys a

Receptortyrosine kinase

Stimulatingligand

ICD

Location Functionalevidence

ErbB-4 Neuregulin,TPA

Cytoplasm, nucleus,mitochondria

Yes

Ephrin Ephrin,ionomycin

Cytoplasm, nucleus No

CSF-1R CSF-1, LPS, TPA Cytoplasm, nucleus NoTie 1 VEGF, TPA Cytoplasm YesVEGFR1 PEDGF Cytoplasm YesInsulin, IGF-1 TPA Cytoplasm No

a References are in the text.

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Introduction

Growth factor binding to a cognate receptor tyrosine kinase (RTK)initiates receptor activation of several well-described signal trans-duction pathways that relay biochemical signals to points of signalreception, such as promoter elements in the nucleus, to effectcellular responses [1]. While receptor activation of these pathwaysoccurs predominantly at the cell surface, there are data indicatingthat signal transduction also occurs from intracellular RTKs [2,3].Coincident with the initiation of cell surface signaling, growthfactor:receptor complexes translocate to clathrin-coated pits andare rapidly internalized as endosomal complexes. Subsequently,the intracellular receptors, which remain active for severalminutes, are trafficked to the lysosome where both ligand andreceptor are degraded. While the lysosome is the predominantdestination and the trafficking pathway to it is reasonably wellunderstood, it is also clear, depending on cell content, that inter-nalized receptors can be recycled to the cell surface [3].

More recently, evidence has accumulated to support thetrafficking of the RTKs from the cell surface to other intracellulardestinations: cytoplasm, nucleus, and mitochondria. There is, insome instances, mechanistic information regarding the traffickingroute, as well as data pertaining to biologic significance. It is thefocus of this review to summarize these results. Mechanisms thatinvolve secretase-mediated RTK cleavage are addressed firstfollowed other less extensively understood mechanisms. AsErbB-1 and ErbB-4 are the best understood examples, they willbe described in more detail.

γ-Secretase-dependent trafficking

The role of secretase-dependent processing of cell surfacemolecules is most clear in the case of Notch [4]. In this case,ligand-binding initiates sequential proteolytic processing byα-secretase, which removes the ectodomain, and by γ-secretase,which cleaves within the transmembrane domain of the cell-associated receptor fragment to release an intracellular domain(ICD) fragment into the cytosol. The ICD subsequently escorts atranscription activation factor into the nucleus to initiate a cellularresponse to the ligand.

The Notch scenario is recapitulated to different extents byseveral RTKs, as indicated in Table 1. In the case of ErbB-4, allessential steps are repeated and the ErbB-4 data are reviewedbelow and illustrated in Fig. 1. Secretase processing is reported forseveral other RTKs (ephrin, CSF-1R, VEGFR1, Tie1, plus prelimina-rily for the insulin and IGF-1 receptors) and these data are alsodiscussed. It should be mentioned that the list can be expected tolengthen as additional RTKs are known to be subject to ectodomaincleavage and this is a necessary precursor step for intramembra-nous cleavage by γ-secretase. Since biochemical detection of ICDfragments is known to be problematic, as these fragments areproduced in substoichiometric amounts and are metabolicallylabile, more effective antibodies or protocols may be required.

In addressing these examples of RTK intramembranouscleavage two points are emphasized. First, is the cleavage processstimulatable by a ligand? Second, what is the evidence that thereleased ICD fragment produces a relevant biologic activity? Theseissues are important as it has been hypothesized that secretase

processing of transmembrane proteins may be a cellular house-keepingmechanism to degrade thesemolecules, as the presence ofa transmembrane domain(s) would seem to present a barrier toother proteolytic systems [5,6]. These are not, however, necessarymutually exclusive interpretations. For example, α- or β-secretaserelease of an ectodomain fragment may be biologically important,while the γ-secretase degradation of the remaining cell-associatedfragment may proceed as a housekeeping function. However,when the cleavage is stimulated by a ligand, especially the cognateligand, and there is a biologic function to the ICD fragment, then itseems very likely that these trafficking events also represent asignal transduction mechanism.

Also, it is instructive to note that an increasing number of non-RTK cell surface molecules are subject to secretase cleavage andthese are tabulated in Table 2. Within the RTK field of research, theprocessing of receptor phosphotyrosine phosphatases and growthfactor precursors are especially relevant. Also, within the RTK andligand categories are two ligand:receptor pairs: nueregulin1 TypeIII and ErbB-4 plus ephrin and the ephrin receptor. Availableevidence indicates that these are similar to the Notch system inthat formation of the ligand:receptor complex in a juxtacrinemanner initiates forward and backward signaling between twoadjacent cells in a secretase-dependent manner.

ErbB-4

Ectodomain proteolytic processing of ErbB-4 includes a basal level,which can be increased by TPA in all cells or by the addition ofneuregulin (heregulin) to certain cells [7,8]. As depicted in Fig. 1,this cleavage results in the formation of two receptor fragments: a120 kDa ectodomain fragment that is released into the media andan 80 kDa membrane-bound fragment, termed m80. Cleavagerequires ADAM 17 (TACE) and it is likely this is the enzyme thatexecutes cleavage of ErbB-4 between His651 and Ser652within theextracellular stalk or ecto-juxtamembrane region [9,10]. Hence, them80 fragment includes eight ectodomain residues, the transmem-brane domain and entire ICD.

Sensitivity to ectodomain shedding is likely determined, at leastin part, by the length of the stalk region in various transmembraneproteins, as demonstrated for the selectins [11]. There are twoErbB-4 isoforms termed Jm-a, in which the ectodomain is sensitiveto cleavage, and Jm-b, which is not cleavable [12]. Since ADAM-

Fig. 1 – Secretase-mediated trafficking of ErbB-4 from the cell surface to intracellular compartments. Following ligand bindingand receptor activation and dimerization, ErbB-4 is subject to ectodomain cleavage by ADAM17(TACE) with release of theectodomain fragment into the media. The cell-associated fragment m80 is then cleaved by γ-secretase to produce an ICDfragment also termed s80. The ICD fragment is translocated to intracellular organelles. In the case of nuclear translocation,transcription factors are reported to be chaperoned into the nucleus with the ICD.

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mediated cleavage events do not involve a defined sequence orcleavage site in the substrate, it seems that longer stalk regions insubstrates may simply permit accessibility of the protease.Interestingly, the stalk region in Jm-b is much shorter (6 residues)than the corresponding region of Jm-a (16 residues) and ErbB-1, -2and -3 also have relatively short stalk regions (6–9 residues) andare not subject to a significant level of metalloprotease mediatedectodomain cleavage [7]. Hence the unique sensitivity of the Jm-aErbB-4 isoform to secretase-dependent processing and signalingseem likely due to the length of its stalk region.

It seems probable, though not formally demonstrated, that theshed ErbB-4 ectodomain may function to block receptor activationby binding neuregulin. The function of the m80 fragment,however, is known. The capacity of γ-secretase to cleave substratesrequires that the substrate have a short ectodomain region of 50 orfewer residues [13]. Hence, the ADAM-mediated removal of a largeportion of the ErbB-4 ectodomain is a prerequisite step forsubsequent γ-secretase cleavage of the m80 fragment [14].

γ-Secretase is a complex of at least four distinct transmem-brane proteins of which presenilin is the catalytic protease [15]. Ithas been shown that the nicastrin subunit of the γ-secretasecomplex recognizes transmembrane proteins with shortened ornub-like ectodomains and thereby acts as a targeting subunit forintramembrane cleavage by presenilin [16]. This would predictthat nicastrin recognition of the ErbB-4 m80 fragment initiates

intramembranous cleavage. As shown in Fig. 1, presenilin activityconverts the ErbB-4 m80 fragment to a soluble s80 or ICD frag-ment that is found in the cytoplasm, nucleus and, in one report,mitochondria [17,14].

The C-terminus of ErbB-4 encodes a PDZ domain recognitionmotif, which is required for presenilin cleavage of the m80fragment [18]. Deletion of this motif (TVV) does not influenceectodomain cleavage, but does abrogate presenilin associationwith the m80 fragment and production of the ICD fragment.Presenilin also contains a PDZ domain recognition motif, and, it ispossible that a scaffold of PDZ domain containing proteins may berequired for γ-secretase cleavage. Proteins that recognize the PDZdomain recognition motifs in ErbB-4 [149,150] and presenilin[151,152] have been reported.

Presenilin cleavage of substrates occurs within the transmem-brane domain and, based on APP and Notch processing, this mayoccur at multiple sites, producing several species of ICD fragmentsthat may have differing levels of metabolic stability based on theN-end rule [19]. Hence, mutation within the transmembranedomain can diminish cleavage and/or alter the metabolic stabilityof the ICD fragments. This is shown in the case of Notch where atransmembranemutation appears to prevent cleavage, but actuallyresults in a new ICD fragment that is very rapidly degraded due tothe presence of a metabolically destabilizing N-terminal residue[20].

Table 2 – Cell surface transmembrane protein subject to intramembrane proteolysis

Category Protein Stimulating ligand ICD References

Localization Functional evidence

Ligands TNFα None Cytoplasm Yes [64,65]NRG1 Type III ErbB-4 Cytoplasm, nucleus Yes [66,67]Delta, Jagged Notch Cytoplasm, nucleus Yes [68–70]Ephrin Eph, TPA Cytoplasm, nucleus Yes [71,72]

PTPases RPTPκ,μ High cell density, antibody Cytoplasm, nucleus Yes [73]LAR TPA Cytoplasm, nucleus Yes [74]

Ligand receptor Notch Delta, Serrate Cytoplasm, nucleus Yes [4] (ref therein)p75 BDNF, TPA Cytoplasm, nucleus No [75–81]IL-1R2 TPA Cytoplasm No [82]Growth hormone receptor TPA Cytoplasm, nucleus No [83]GluR3 None ICD not produced Yes [84]LDLRPs TPA Cytoplasm, nucleus Yes [85–90]CXCL1 and 16 None Cytoplasm None [91]IFNaR2 IFN-α, TPA Cytoplasm, nucleus Yes [92–94]

Channels Na channel-β2 None Cytoplasm No [95–97]Adhesion CD44 Ionomycin, TPA Cytoplasm, nucleus Yes [98–101]

Cadherins Apoptosis, Ca influx, toxin Cytoplasm Yes [102–108]DCC None Cytoplasm Yes [109]Syndecan 3 TPA, bFGF Cytoplasm Yes [110]L1 None Cytoplasm No [111]

Miscellaneous APP TPA Cytoplasm, nucleus Yes [112] (ref therein)TMEFF2 TPA Cytoplasm No [113]CD43 None Cytoplasm, nucleus Yes [114,115]CD74 None Cytoplasm, nucleus Yes [116]SorLA None Cytoplasm, nucleus Yes [117]Fibrocystin Ca+2 mobilization Cytoplasm, nucleus No [118]RAGE receptor Ionomycin Cytoplasm, nucleus No [119]HLA-A2 TPA Cytoplasm No [120]Bri 2 None Cytoplasm No [121]Nectin-1α TPA Cytoplasm No [122]Neogenin None Cytoplasm, nucleus Yes [123]NRADD None Cytoplasm No [124]Tyrosinase None Cytoplasm No [125]GnT-V None Cytoplasm No [126]

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It has been reported that the Val675Ala [21] or Val673Ile [22]mutations within the ErbB-4 transmembrane domain abrogateγ-secretase cleavage, as judged by the inability to detect the ICDfragment. In view of the Notch mutagenesis data, it is not clearwhether these mutations actually prevent cleavage or result in aless stable ICD fragment. Given the low level of ICD fragmentnormally detectable, a modest change in stability may render thefragment undetectable by the same methodology.

In terms of the physiological relevance of the ErbB-4 ICD, it isnow clear that endogenous generation of the ICD by γ-secretase isrequired for control of astrogenesis in the developing mouse [23].In this system the ICD fragment interacts with TAB2, an adaptorprotein, and thereby with N-CoR, a co-repressor, and chaperonesthis complex into the nucleus. A similar chaperone mechanismbetween the ErbB-4 ICD and STAT5 has been proposed to beoperative in mammary differentiation in vitro [24] and it is clearthat ErbB-4 is functionally involved in mammary development inthe animal [25]. While ErbB-4 nuclear localization has beenobserved in normal and tumor mammary tissue and exogenousICD expression provokes differentiation events, it has not yet beendemonstrated that ErbB-4 cleavage is physiologically relevant inthis tissue. Also, in line with a role of the ErbB-4 ICD fragment invarious cell differentiation systems, is the report that γ-secretase

inhibition prevents neuregulin generation of the nuclear ErbB-4ICD in oligodendrocytes and maturation of this cell type [26].

In addition to STAT 5 and the TAB 2:N-CoR complex mentionedabove, several other proteins (Eto-2 [27], YAP [28,29], WWOX [30],ER [31], Mdm 2 [32], AIP4/Itch [33]) have been reported toassociate with the ErbB-4 ICD. Eto-2, YAP and ER are transcriptionfactors/co-activators and the ICD may regulate their nuclearlocalization similar to STAT 5 and N-CoR. WWOX is a cytoplasmicprotein and its interaction with the ICD attenuates nucleartranslocation of the ICD, while AIP4/Itch is a cytoplasmic ubiquitinligase that modulates the levels of intact ErbB-4 and the ICD. TheICD is an active tyrosine kinase [34] that phosphorylates Mdm2, aregulator of p53, which is predominantly localized in the nucleus[32].

As mentioned above, the ErbB-4 ICD has also been localized inmitochondria and in that location may function as a proapoptoticprotein. This is based on the capacity of the ICD to induce celldeath, the presence of a BH3 domain in the ICD, the loss ofapoptotic capacity following mutagenesis of this domain, anddetection of an interaction with the antiapoptotic protein BCL-2,which, when over-expressed, abrogated ICD-induced cell death[17]. This and other aspects of the ErbB-4 ICD are reviewedelsewhere [25].

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Ephrin receptor

Addition of the ephrin receptor ligand ephrin provokes secretasecleavages of the receptor releasing the ectodomain fragment andan ICD fragment [35]. The cleavage events are also stimulatable byionomycin or activation of the NMDA receptor, agents that mediateCa2+ influx into cells. In this system, ephrin-mediated cleavageevents require endocytosis, while cleavagemediated by ionomycinin NMDA receptor activation occurs on the cell surface. A similarendocytosis relationship between neuregulin and TPA mediatedcleavage of ErbB-4 was noted [8]. To date it is unclear whether thereceptor ICD fragment is translocated from the cytoplasm toanother organelle and there is no data related to a physiologicalfunction in mediating ligand cell responsiveness.

CSF-1 receptor

Secretase cleavage of the CSF-1R can be stimulated by CSF-1, LPSand TPA [36–38]. LPS is a ligand for the Toll4 receptor and agonistsfor other Toll-like receptors also stimulate cleavage of CSF-1R. Thisheterologous stimulation of CSF-1R cleavage may be related to thefact that in macrophages both receptor systems are thought to beinvolved in producing innate immune responses. While the CSF-1ICD fragment does appear in both cytoplasm and nucleus, aphysiologic function has not been identified.

VEGF receptor 1

Pigment epithelium-derived factor (PEDF) binds to an unknownreceptor and promotes an anti-angiogenic response and canoppose the capacity of VEGF to promote endothelial cell prolifera-tion. The addition of PEDF to endothelial cells promotes theγ-secretase mediated cleavage of VEGFR1 with release of its ICDfragment [39]. This ICD fragment was only present when cells weretreated simultaneously with PEDF and VEGF and the fragment wasdetected in the cytoplasm, but not in the nucleus. In this system,the intact VEGFR1 molecule is found in the nucleus following theaddition of VEGF (see below) and PEDF reduces VEGF-inducedangiogenesis and the nuclear level of intact VEGFR in a mannerdependent on γ-secretase activity. This implies that the PEDFstimulated production of the VEGFR1 ICD fragment negativelyregulates intact VEGFR1 levels in the nucleus and VEGF-inducedangiogenesis, based on the action of γ-secretase inhibitors.

Table 3 – Intact receptors trafficked to nucleus or mitochondria

Receptor tyrosine kinase Stimulating ligand Organell

ErbB-1 EGF NucleusErbB-1 Src MitochondErbB-1 vIII None NucleusErbB-2 Over-expression NucleusErbB-3 None NucleusFGF-R1, R2 FGF NucleusIFN-γR1 IFN-γ NucleusTrkA NGF, LPS NucleusTGF-β TGF-β NucleusGH Growth hormone NucleusIL-15R2 IL-15 NucleusVEGFR1/FLT1 VEGF NucleusMet HGF Nucleus

However, other signaling systems (Notch, etc.) will also be blockedby pharmacologic γ-secretase inhibitors.

Tie 1

Tie 1 is an orphan receptor that forms a hetero-oligomeric complexwith Tie 2, the receptor for angiopoietin 1 (Ang 1). Addition of Ang1 activates Tie 2 and provokes tyrosine phosphorylation of Tie 1. Inthis system ectodomain cleavage of Tie 1 is stimulated by a varietyof agents (TPA, VEGF, TNFα, sheer stress) and increases Ang 1activation of Tie 2, apparently by allowing greater access of theligand to its Tie 2 binding site. Following ectodomain release, theTie1 cell-associated cleavage fragment (45 kDa) is processed byγ-secretase to produce a 42 kDa cytoplasmic ICD fragment [40]. Inthis receptor system, the ectodomain secretase action is physio-logically important: however, the significance of γ-secretaseactivity may be simply to remove the highly tyrosine phosphory-lated 45 kDa fragment. While the addition of Ang 1 promotes rapidendocytosis and degradation of Tie 2, Tie1 is not cleared from thecell surface by this same route. Therefore, a secretase mechanismmay provide the means by which Ang1-phosphorylated Tie 1 isinactivated.

IGF-1 and insulin receptors

Inpreliminary reports, it has beendemonstrated that the insulin andIGF-1 receptors can be cleaved by secretase action to produce ICDfragments [41,42]. However, while TPA stimulates formation of theICD fragments neither cognate ligand was demonstrated to do so.

Non-secretase formation of RTK ICD fragments

In the case of several RTKs (ErbB-2 [43–45], Ret [46], ALK [47], TrkC[48], Met [49–51]) there is evidence that caspases cleave thecytoplasmic domain to produce an ICD fragment. Since thefragment is often produced by two cleavage events within thecytoplasmic domain, the fragment is often considerably smallerthan that produced by intramembrane proteolysis. In no reportedcase are these caspase cleavages stimulated by ligand binding or byTPA and in some studies the presence of the cognate ligandprevents cleavage. In the above RTKs the formation of caspase ICDfragments is functionally associated with the induction of apop-

e Functional evidence References

Yes [53,57,127,60,52,56,54,55,58]ria No [128]

Yes [129]Yes [130,131]No [132]Yes [133,134] (ref therein)Yes [135–137]No [138,139]No [140]Yes [141–143]No [144]No [145–147]No [148]

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tosis and in one instance [44] the fragment has been localized tothe mitochondria. Thus, this group of RTKs can be added to the listof dependence receptors in which the receptor mediates opposingcellular responses (apoptosis, cell proliferation) depending on theabsence or presence of ligand.

Trafficking of intact receptors to the nucleus

An accounting of recently published reports demonstrating theappearance of intact RTKs and other cell surface receptors in thenucleus is presented in Table 3. In a few instances the data relies onimmuno-histochemistry alone and it is not clear that intact recep-tor is distinguishable from an ICD fragment. In nearly all cases,however, it does appear that the receptor is present in the nucleo-plasm and not the nuclear envelope. The presence of a transmem-brane protein in a non-membranous environment requires amechanism to extract the intact receptor from the surroundinglipid bilayer. Such a mechanism has recently been reported and isdescribed below and illustrated in Fig. 2. Whether this traffickingroute can be applied to other receptors listed in Table 3 remains tobe seen.

ErbB-1 and translocon-mediated trafficking

The capacity of EGF to induce trafficking of the intact EGF receptor(ErbB-1) to the nucleus was first reported in 2001 and a nuclear

Fig. 2 – Translocon-mediated trafficking of ErbB-1 (EGF receptor) frreceptor activation and dimerization, the receptor is internalized awith the Sec 61 translocon mediates extraction from the lipid bilayinto the cytoplasm. Subsequent association with importin-β leads

function was also identified [52]. The nuclear receptor wasreported to recognize the promoter of cyclin D1 and to transacti-vate this promoter in a reporter system. Subsequently, the samegroup established the following points: 1) other promoters are alsorecognized by the ErbB-1 (though direct binding to any promoterremains to be shown [53–55]), 2) importin β is required for ErbB-1nuclear localization [56], 3) a nuclear localization sequence ispresent in the ErbB-1 sequence [57], and 4) the nuclear receptorassociates with PCNA in the nucleus and modifies its stability [58].The nuclear receptor was identified by both biochemical fractiona-tion and morphological methods and shown to be in a non-membranous environment. Furthermore, the ligand (EGF) wasreported to also be present in the nucleus [52].

While no RTK trafficking system was known to extract thetransmembrane receptor from its lipid bilayer, another groupsuggested that a protein translocon could provide such a step [59].Specifically, the Sec 61 translocon located in the endoplasmicreticulum was known to mediate the trafficking of certainextracellular toxins from the cell surface to the cytoplasm andalso, as part of the ERAD pathway, to retrotranslocate malfoldedtransmembrane proteins from the endoplasmic reticulum to thecytoplasm. Subsequent testing of this possibility showed that EGFinduced trafficking of ErbB-1 to the endoplasmic reticulum whereit interacted with the Sec61 translocon that then mediatedreceptor retrotranslocation to the cytoplasm and import into thenucleus [60]. The data showed that knock-down of a Sec61 subunitabrogates both EGF nuclear localization of ErbB-1 and EGF

om the cell surface to the nucleus. Following ligand binding andnd trafficked to the endoplasmic reticulum (ER). Interactioner, interaction with chaperones, such as Hsp 70, and extrusionto nuclear import.

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induction of cyclin D1. This trafficking pathway is depicted in Fig. 2.HSP 70 has been shown to be essential in the in vitro retro-translocation process [60] and likely functions by interacting withthe receptor transmembrane domain and thereby maintaining thereceptor in a soluble state following extraction from the ERmembrane. The route by which the receptor is trafficked from thecell surface to the endoplasmic reticulumwas not determined, butprecedent exists with both toxins [153,154] and the SV40 virus[155]. In the former case, the Golgi serves as an intermediate, whilein the latter caveosomes translocate virus to the endoplasmicreticulum.

The translocon pathway described above for ErbB-1 is distin-guished from the endocytic pathways leading to lysosomaldegradation or recycling to the cell surface on the basis of timeand quantity of receptors involved. The translocon pathway isrelatively slow and involves a smaller fraction of the receptorpopulation [60]. Therefore it is not clear whether the transloconpathway represents a third destination for receptors internalizedthrough clathrin-coated pits or whether receptors destined for thetranslocon and nucleus are internalized by a separate cell surfacemechanism.

There are several examples of ligand/receptor pairs thattranslocate to the nucleus, perhaps as a complex. However, thishas not been convincingly demonstrated. The interested reader isreferred to recent reviews that address the issue of ligandtrafficking to the nucleus [61–63].

Acknowledgments

The author appreciates the efforts of Sue Carpenter in manuscriptpreparation and acknowledges support of NIH grant CA125649.

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