plasma membrane - located trail - r1/r2 induce both cell death and non - apoptotic signaling pathways.tumor cells show elevated intracellular levels of trail - r1/r2.nuclear trail - r2 interacts with the microprocessor complex , inhibits let-7 maturation and enhances tumor cell proliferation . plasma membrane - located trail - r1/r2 induce both cell death and non - apoptotic signaling pathways . nuclear trail - r2 interacts with the microprocessor complex , inhibits let-7 maturation and enhances tumor cell proliferation . what is the mechanism of nuclear import / export of trail - r2 and how is this regulated?which mirnas , beside let-7 , are regulated by ntrail - r2?what are additional specific functions of ntrail - r2 ? is there any impact of other trail - rs ( trail - r1 , -r3 and -r4 ) on nuclear localization and function of trail - r2?do trail - r1 , trail - r3 and trail - r4 possess independent specific nuclear activities?is there a specific function of cytoplasmic trail death receptors ? what is the mechanism of nuclear import / export of trail - r2 and how is this regulated ? which mirnas , beside let-7 , is there any impact of other trail - rs ( trail - r1 , -r3 and -r4 ) on nuclear localization and function of trail - r2 ? do trail - r1 , trail - r3 and trail - r4 possess independent specific nuclear activities ? the death ligand tnf - related apoptosis - inducing ligand ( trail ) , a member of the tnf cytokine superfamily , interacts with five different receptors , plasma membrane - expressed trail - r1 - 4 and a soluble receptor , osteoprotegerin ( opg ) , all members of the tnf receptor superfamily . two trail receptors , trail - r1/dr4 and trail - r2/dr5/trick / killer , contain a cytoplasmic death domain ( dd ) and are hence able to induce apoptosis . trail has governed particular interest because of its apparent tumor cell - specific apoptosis - inducing capacity . consequently , trail and agonistic trail death receptor - specific antibodies are regarded as promising therapeutic agents for treatment of various malignancies and are currently under clinical investigation . however , many tumor cells are resistant to trail - mediated apoptosis and , furthermore , trail death receptors may induce pro - inflammatory , invasion- and metastasis - promoting non - apoptotic signaling pathways under certain conditions . these observations point to the necessity to improve trail - r1/r2-targeting therapies by combination with molecules sensitizing to cell death and at the same time inhibiting potentially harmful non - apoptotic signaling . overexpression of trail - r1 and/or trail - r2 has been demonstrated for many tumor entities ( table 1 ) . importantly , these studies detected mainly an intracellular localization of the receptors , suggesting that intracellular trail - r1 and trail - r2 may provide a growth advantage or other benefit for tumor cells . indeed , correlation of the expression status with clinical parameters disclosed prevalent prognostic significance of high death receptor expression , identifying predominantly trail - r2 and occasionally also trail - r1 as negative prognostic markers ( table 1 ) . for example , high trail - r2 expression correlated with higher tumor grade , positive nodal status and shortened survival of breast cancer patients . strong trail - r2 expression also negatively correlated with disease - free survival of patients suffering from metastatic hnscc . for stage i and ii nsclc , high trail - r2 expression correlated with poorly differentiated tumors and significantly reduced patient survival . similarly , poorly differentiated areas of stage iii nsclc showed high expression of trail - r1 and trail - r2 , but only trail - r2 positivity was associated with an increased risk of death . moreover , pancreatic cancer cells with stem cell characteristics also express trail - r2 at increased levels . correspondingly , high intracellular expression of trail - r2 has been demonstrated for stem cells at the base of the intestinal crypts in the small intestine . thus , trail - r1 was identified as an independent prognostic marker for a more favorable course of colorectal carcinoma . in bladder cancer , higher levels of either trail - r1 or trail - r2 correlated with longer recurrence - free survival . in glioblastoma multiforme , high expression of both trail - r1 and trail - r2 , of note mainly expressed at the cell surface , was found to be associated with longer overall survival of the respective patients . so far , mainly the overall tissue expression level of trail receptors and occasionally also their presence at the cell surface were taken into account when estimating their values as a prognostic marker for cancer . however , recent discoveries on trail - r2 functions revealed that even though an enormous body of information on death receptors has been gathered , our knowledge of the cellular functions of these receptors is still incomplete . the authors report that nuclear trail - r2 ( ntrail - r2 ) is engaged in microrna ( mirna ) maturation , thus performing functions entirely different from its classical role as a plasma membrane - located signal transducer . with this finding , the frequently observed high intracellular expression of trail - rs in tumors gains importance and demands careful re - evaluation . here we review the known functions of human trail death receptors with respect to their subcellular compartmentalization . the novel nuclear functions of trail - r2 will be discussed in comparison to nuclear functions of other conventionally plasma membrane - located cytokine and growth factor receptors . the best understood function of trail - r1 and trail - r2 is the induction of apoptosis ( reviewed in gonzalvez and ashkenazi and see figure 1 ) . upon trail ligation , trail death receptors cluster into higher multimeric complexes and gain the capability of assembling a disc ( death - inducing signaling complex ) at their intracellular dd . through this domain , trail - r1 and trail - r2 recruit the adaptor protein fadd ( fas - associated protein with death domain ) that in turn , via death effector domain ( ded ) , recruits the inactive pro - forms of the initiator caspases pro - caspase-8 and -10 . proximity - induced self - cleavage of pro - caspases within the disc leads to their activation and subsequent dissociation from the membrane - localized receptor complexes . besides death receptors , trail can also bind to two additional plasma membrane - bound receptors , trail - r3 and trail - r4 . trail - r3 is a glycosylphosphatidylinositol - anchored receptor lacking transmembrane and cytoplasmatic domains and is therefore not capable of inducing intracellular signaling . trail - r4 is more homologous to the trail death receptors , but because of a truncated dd , it is unable to induce apoptosis . efficiency of the initiator caspase activation may be influenced at the disc level , for example , by variation of its composition . it has been shown that the complex formation between death receptors trail - r1 and trail - r2 and the apoptosis - incompetent receptors , trail - r3 or trail - r4 , leads to impaired disc function . in addition , recruitment of the proteolytically inactive caspase homolog cflip ( cellular flice - like inhibitory protein ) represents an apoptosis - inhibiting mechanism . in some cells , activated initiator caspases may directly activate the effector caspases 3 , 6 and 7 , which in turn cleave a broad range of protein substrates and execute cell death . such cells have been classified as the so - called type i cells with respect to apoptosis induction . in contrast , type ii cells use the loop way via engagement of the intrinsic apoptosis pathway to amplify the death signal generated at the disc . in these cells , caspase-8-mediated cleavage of the bh3-only protein bid generates its truncated form ( tbid ) that , by association with bax and bak , leads to the mitochondrial outer membrane permeabilization ( momp ) and to the release of pro - apoptotic mitochondrial proteins like cytochrome c , smac / diablo ( second mitochondria - derived activator of caspase / direct iap binding protein with low pi ) and htra2/omi ( high temperature requirement protein a2/omi ) into the cytosol . cytosolic cytochrome c together with atp and apaf-1 ( apoptotic protease activating factor-1 ) form a second caspase - inducing platform , the apoptosome , that mediates the activation of caspase-9 . an alternative trail - induced apoptosis pathway has been described in hepatocytes ( figure 1 ) . in these cells , trail - r2 is internalized upon trail stimulation by endocytosis , targeted via vesicular transport to lysosomes that become permeabilized and release cathepsin b , subsequently promoting cell demise . lysosomal membrane permeabilization ( lmp ) as a precondition for the execution of apoptosis in these cells requires the pacs-2 ( phosphofurin acidic cluster sorting protein 2)-mediated recruitment of bim and bax to lysosomal membranes . under conditions when caspases are inhibited , plasma membrane - expressed trail death receptors may also induce an alternative death pathway known as necroptosis . for this pathway , a pivotal role of the kinases rip-1 ( receptor - interacting protein-1 ) , rip-3 as well as pseudo - kinase mlkl ( mixed lineage kinase domain - like ) has been shown , but the exact molecular mechanisms of trail - induced necroptosis are still not fully understood . over the past years , it has become increasingly evident that trail - r1 and trail - r2 , besides death signaling pathways , can also activate multiple non - cell death signal transduction pathways like nf-b ( nuclear factor -light - chain - enhancer of activated b cells ) , pkc ( protein kinase c ) , pi3k ( phosphatidylinositide 3-kinases ) , akt / pkb , src ( rous sarcoma virus oncogene cellular homolog ) and tak1 ( transforming growth factor- activated kinase 1 ) , as well as different members of the map - kinase family like erk1/erk2 ( extracellular regulated kinases ) , jnk ( c - jun n - terminal kinase ) and p38 ( figure 1 ) . regarding the molecular determinants for trail - mediated activation of cell death or non - apoptotic pathways , the engagement of different protein complexes has been proposed . in this respect , non - apoptotic pathways have been suggested to be activated within a secondary signaling complexes formed downstream of the disc . besides fadd and pro - capase-8 , the secondary signaling complex also include specific adaptor proteins like rip1 , traf2 and nemo . different localizations of aggregated trail receptors within specific plasma membrane domains have also been proposed to determine the formation and activity of ligand - induced trail - r1/r2 signaling complexes . hence , aggregated trail receptors localized in lipid rafts were shown to be involved in the apoptotic signaling , whereas those localized outside lipid rafts form complexes capable of non - apoptotic signaling . alternative to the mapks activation via secondary protein complexes , their stimulation via effector caspases was described in prostate cancer cells . here , two different proteolytic fragments of mst1 ( mammalian sterile 20-like kinase 1 ) generated upon cleavage by caspases-3 or -7 selectively activate erk or jnk and p38 , respectively . this multiplicity and heterogeneity of trail - induced pathways , as briefly outlined , is responsible for the observed broad cellular effects of this cytokine ranging from the induction of cell death , establishment of a death - resistant phenotype and expression of pro - inflammatory molecules promoting cell proliferation , migration and invasion ( figure 1 ) . particularly in apoptosis - resistant tumor cells , trail - induced pro - inflammatory pathways may result in enhanced invasion and metastasis in vivo . a comprehensive overview of trail - induced non - apoptotic signaling pathways and their functional relevance in different cellular systems is provided in the recent review by azijli et al . in addition to their plasma membrane expression , trail - r1 and trail - r2 are also often found in the cytoplasm of several cell types ( tables 1 and 2 ) . however , in most cases the cytoplasmic localization of trail death receptors was not resolved in detail . thus , it still remained unclear whether the intracellular staining in cancer cells observed by immunohistochemistry reflects localization of trail receptors in the membranes of organelles within the secretory and/or endocytotic pathway or whether a non - membraneous cytosolic localization of the receptors was also detected in these cases . the presence of trail - r1 and trail - r2 in soluble cytosolic fractions of diverse tumor cells was demonstrated recently . as the subcellular fractionation was performed without the use of any detergent , it can be concluded that these receptors are not anchored in intracellular membranes , but are present in the non - membraneous cytosolic compartment . the function of these soluble trail death receptors remains to be shown . in colon carcinoma , the cytoplasmic staining of trail death receptors was interpreted as the presence of these receptors in the trans - golgi network , a finding previously described for melanoma cells . the study on apoptosis - resistant breast cancer cells demonstrated the presence of trail receptors in autophagosomes rather than the plasma membrane . treatment of these cells with different inhibitors of autophagy led to a re - appearance of both trail death receptors on the cell surface and sensitized these cells toward trail - mediated death . a correlation of apoptosis resistance with intracellular presence of death receptors in breast cancer cells was also proposed in another study . finally , as internalization of trail death receptors in response to trail treatment has been demonstrated in cell lines , the cytoplasmic localization of trail receptors in tumor tissue could be caused by an autocrine or paracrine activation of their internalization by stroma and/or tumor cell - derived trail . summing up , despite the variety of intracellular trail death receptor locations , the frequently observed loss of trail death receptors at the cell surface in tumors seems to be a general principle for the acquisition of trail resistance in cancer cells . in addition , high cytoplasmic levels of these receptors as well as their presence in soluble form suggest specific pro - tumoral functions in this location . few relatively recent studies show the localization of trail receptors in the nucleus ( tables 1 and 2 ) . an early report on the melanoma cell line , mel - fh , demonstrated trail - r1 as predominantly expressed in the nucleus , where it colocalized with trail - r3 and trail - r4 . nuclear presence of trail - r1 and trail - r2 has also been shown for normal and psoriatic skin , with ntrail - r2 being more strongly expressed in psoriatic lesions and in the dermis of psoriatic skin compared with healthy skin . in the same study , cd4 + and cd8 + cells of the inflammatory infiltrate showed exclusive nuclear staining for trail - r1 . nuclear presence of trail - r2 was also described for nonmalignant , normal lung bronchial epithelial cells in basal nuclei . however , staining of these cell nuclei was in general weak and restricted to a small number of cells . only very few studies have addressed the functional relevance of ntrail - r2 . again , as for the presence of trail - r1/r2 in the cytoplasm , the nuclear sequestration has been proposed as an apoptosis - resistance mechanism . trophoblast cells of the human placenta were found resistant to trail - mediated apoptosis and expressed high levels of trail - r2 that was mainly localized in the nucleus . following stimulation with tnf , trail - r2 relocated to the plasma membrane and the cells became trail sensitive . another study demonstrated that inhibition of the nuclear import of trail - r2 sensitized hela and hepg2 cells to trail - induced apoptosis . two very recent studies provide hints for surprizing and entirely novel functions of ntrail - r . in one report , the pattern of intracellular distribution of trail - r during the development of the mouse cns was studied . the authors demonstrated that the expression of trail - r was not only restricted to proliferating zones but , importantly , was mainly located in the cell nuclei , suggesting a specific , proliferation - associated function of ntrail - r . another study investigating human nuclear trail receptors finally uncovered specific functions of ntrail - r2 connecting this receptor to the process of mirna maturation ( figure 2 ) . briefly , in several different tumor cells , ntrail - r2 interacts with the core microprocessor complex drosha / dgcr8 and with several of its accessory proteins , p68 , nf45 and hnrnpa1 . through this interaction , ntrail - r2 inhibits maturation of the mirna let-7 and consequently increases the levels of let-7 targets lin28b and hmga2 . hmga2 and lin28b proteins are highly expressed in embryonic tissues , downregulated in differentiated tissues and , because of low expression of let-7 , re - expressed in tumors , where their levels positively correlate with tumor progression . in this scenario , inhibition of let-7-maturation by ntrail - r2 enhances the malignancy of tumor cells . in line with this notion , it was observed that trail - r2 was expressed at higher levels in cell nuclei of pdac tissue than in nuclei of peritumoral normal duct cells . moreover , high ntrail - r2 correlated with high nuclear hmga2 expression in the same tissue and with worse prognosis of patients with early - stage pdac . furthermore , knockdown of trail - r2 resulted in decreased growth of experimental orthotopic tumors in mice , and progressive decrease of trail - r2 expression as well as its exclusion from the nuclei accompanied the differentiation of pancreatic tumor cells into three - dimensional duct - like structures in vitro . the correlation of nuclear localization of trail receptors and survival of cancer patients was analyzed , to the best of our knowledge , in only one additional report . in a study on nsclc tumors from patients treated with chemotherapy , all four trail receptors were detected in the nuclei and cytoplasm . in these patients , however , high nuclear and cytoplasmic expression of trail - r2 correlated with improved survival . however , it is well known that chemotherapeutic agents upregulate trail - r2 in cells with wild - type p53 . unfortunately , the p53 status was not analyzed in this study and it may be possible that the observed positive correlation simply reflects the better prognosis of patients with wild - type p53 . in agreement with this interpretation , other studies on untreated lung cancer patients , as well as patients suffering from other cancers , have instead found negative correlations between high overall expression of trail - r2 and the survival time of the patients . the nuclear presence of trail death receptors represents a new example of plasma membrane receptors that can localize to the nucleus , where they elicit functions distinct from their canonical signal transduction pathways that originate at the plasma membrane . most prominently , the receptor tyrosine kinases ( rtks ) , and among them the members of the epidermal growth factor receptor ( egfr ) family , have been found to shuttle to the nucleus in response to ligand binding ( reviewed by carpenter and liao and wang et al . ) . here , via interaction with classical transcription factors or with rna helicase a ( rha ) , in addition , via interaction with rna - polymerase i , egfr2 increases rrna expression and cellular translation . egfrs may also phosphorylate nuclear proteins such as pcna , and dna - dependent protein kinase ( dna - pk ) , thus influencing a variety of other nuclear processes like dna replication and dna damage repair . other rtks found in the nucleus are fibroblast growth factor receptors ( fgf - r1 , -r2 and -r3 ; reviewed by bryant and stow ) vascular endothelial growth factor receptors ( vegf - r1 , see cai et al . and vegf - r2 , insulin receptor , cmet , trka , growth hormone receptor and receptor tyrosine kinase - like orphan receptor 1 ( ror1 ) . particularly well understood are functions of nuclear fgf - r1 that , via its interaction with 90-kda ribosomal s6 kinase ( rsk1 ) , activates the transcription factor creb , thus regulating the transcription of genes involved in proliferation and differentiation . as rsk1 phosphorylates several transcription factors as well as histone h3 , the ability to activate rsk1 enables nuclear fgf - r1 to regulate a plethora of biological processes . besides rtks , other types of cell surface transmembrane receptors have also been described as being localized in the nucleus . type i transforming growth factor  receptor ( tr1 ) can localize to the nucleus following tgf- treatment , where it can interact with hnrnpa1 , thus stimulating the alternative splicing of pre - mrna for egfr1 . it is noteworthy that hnrnp a1 , besides its known regulatory role in splicing , was also described as an accessory protein of the microprocessor complex interacting with ntrail - r2 in this context . however , in contrast to the findings for trail - r2 , no association of tr1 with mirna maturation has been described till now . cd40 and the b cell - activating factor receptor ( baff - r ) , both members of the tnf receptor superfamily , also localize to the nucleus in normal and malignant b cells , in addition to their plasma membrane expression . in the nucleus , these receptors interact with c - rel and enhance transcription of several nf-b target genes . nuclear baff - r , in addition , interacts with ikk- and histone h3 , leading to increased ikk--mediated phosphorylation of histone h3 . the surprising role of ntrail - r2 in regulating let-7-maturation strongly demands future research on the mechanisms of intracellular trafficking of this receptor . regarding the mechanism by which full - length plasma membrane receptors passage to the nucleus , the intracellular trafficking of rtks might serve as a model . following ligand binding at the cell surface , egfrs are internalized via endocytosis , moved by retrograde transport in endosomal vesicles into the er and subsequently translocated through the er membrane into the cytosol by action of the sec61 translocon . free cytosolic full - length receptors can then be bound at their tripartite nuclear localization motif ( nls ) by -importin and finally imported into the nucleus through the nuclear pore complex . an alternative model for the entry of full - length egfr family members into the nucleus involving a passage of these receptors into the nucleoplasm through a sec61 translocon in the inner nuclear membrane has also been proposed . for fgf - r1 it was shown that soluble cytoplasmic and nuclear forms and , on the other hand , plasma membrane receptors represent separated pools of different origin and with distinct functions . it has been proposed that fgf - r1 is synthesized at er - attached polyribosomes and either enters golgi , becomes extensively glycosylated and reaches the plasma membrane or , in non - glycosylated form , exits pre - golgi vesicles and translocates as soluble cytoplasmic form complexed with rsk1 into the nucleus via nuclear pores . the origin of the soluble form of ntrail - r2 as well as the mode of its nuclear translocation is currently completely unknown . the receptors may either originate from the plasma membrane or from the non - membraneous soluble cytoplasmic receptor pool . the latter pathway seems to be more likely , as neither inhibition of endogenous secreted or plasma membrane expressed trail via neutralizing antibodies nor the treatment of cells with recombinant trail influenced the nuclear levels of trail - r2 . it has been reported recently that mutation of these sequences or knockdown of importin- resulted in a diminished nuclear presence of trail - r2 and , in parallel , enhanced expression at the plasma membrane . in contrast to this study , experiments performed in our laboratory using the same nls - mutated trail - r2 form did not reveal any impact of these nls sequences on the nuclear import of the recombinant trail - r2 in panc1 and mda - mb-231 cells ( a. trauzold , unpublished observations ) . however , we can not rule out that the overexpressed nls - mutated trail - r2 interacts with endogenous trail death receptors and utilize them as trojan horses for nuclear import in these cells . further studies are necessary to clarify the exact mechanism of the translocation of trail - r2 to the nucleus . another important question concerns the mechanism(s ) by which ntrail - r2 inhibits the drosha - mediated processing activity toward pri - let-7 . drosha and dgcr8 constitute the core of the microprocessor complex that associates with multiple accessory proteins as hnrnpa1 , p68 , ksrp , nf45 and nf90 . the core complex is capable of pri - mirna processing in the absence of cofactors , whereas the associated factors in the larger complex may facilitate or alter the selectivity of nuclear mirna processing . accordingly , pri - let-7 is processed in a complex manner involving stimulatory factors ( p68 and ksrp ) as well as inhibitors ( hnrnpa1 , nf45 and nf90 ; figure 3 ) . it seems likely that ntrail - r2 modulates the pri - let-7 processing by interaction with nf45 , hnrnpa1 and p68 . this may be facilitated by either sequestration of activators or stabilization of an inhibitory complex . it can be assumed that via its interaction with aforementioned proteins , ntrail - r2 might also influence the maturation of other mirnas in addition to let-7 . in this respect it is worth mentioning that p68 has been implicated in the recognition of a subset of pri - mirnas targeting these for drosha - mediated processing . analysis of global changes of mirna levels in cells with knockdown of trail - r2 indicates the existence of further trail - r2-regulated mirna , besides the members of the let-7-family being most prominently represented . however , the array results have to be independently verified and , in addition , the question of whether these mirnas are regulated by ntrail - r2 or by plasma membrane - expressed trail - r2 remains to be answered . further investigations should also tackle the structural basis for the interactions of ntrail - r2 with proteins of the microprocessor complex . as the core components drosha and dgcr8 were found to interact with trail - r2 in an indirect manner , attempts should be made to delineate the physical contact sites between trail - r2 and the accessory proteins , that is , hnrnpa1 , p68 , nf45 and nf90 . insights into the structural constraints of the interaction sites might allow the design of pharmacological inhibitors of these interactions , opening opportunities for targeted therapeutic interventions in the future . in spite of more than a decade of research on trail death receptors , until very recently only their canonical signaling functions at the plasma membrane or within the endosomal / lysosomal compartment have been investigated . however , a number of reports also pointed to the intracellular presence of trail - r1 and trail - r2 . in intracellular compartments , trail death receptors are likely to neither contribute to canonical apoptosis signaling nor to non - apoptotic signal transduction , regardless of whether they are soluble within cytosol , trapped within the trans - golgi network , in autophagosomes or are present in the nucleus . the elevated intracellular expression of trail death receptors in cancer and the emerging evidence for their compartmentalization - dependent functions make clear that it is not sufficient to determine the general receptor expression in cells or tissues in a given experimental context . instead , it is important to perform differentiated analyses of the subcellular distribution of receptors for the understanding of both the molecular biology and the disease - related relevance of trail death receptors , especially when conclusions on the impact of these receptors for disease prognosis are drawn . now , a first study has demonstrated a functional impact of the nuclear localization of trail death receptors in cancer cells . trail - r2 was found to associate with components of the large microprocessor complex inhibiting the processing of pri - let-7 mirna . such an effect of ntrail - r2 on mirna processing is clearly distinct from the known nuclear functions of other cell surface receptors as egfr family members , other rtks or cytokine receptors that can modulate dna - associated processes like replication , transcription or dna damage repair and rna splicing . thus , these novel findings will stimulate the search for unknown trail - r2-specific nuclear functions , among them regulation of other mirnas and possibly also rna- and dna - targeting mechanisms . this will further broaden the range of possibly affected nuclear regulatory processes of plasma membrane receptors that emerge as important regulators in the cell nucleus . the findings on a specific function of ntrail - r2 should also promote the search for possible unknown nuclear functions of other members of the death receptor family . in this respect , the capability of trail - r1 and trail - r4 to specifically interact with trail - r2 in the nucleus also raises the question of the capacity of these receptors to modulate the nuclear functions of trail - r2 . finally , these recent insights into trail receptor biology might open new perspectives for anti - cancer therapies targeting processes regulated by ntrail - r2 .