BACKGROUND
the wnt signaling pathway is largely conserved from drosophila to humans and has been shown to regulate diverse embryonic processes including axis specification and organogenesis.
wnt signaling events have also been implicated in a number of cancers, bone density phenotypes, and neurological conditions in adulthood  <cit> .
wnts are a family of secreted proteins which are glycosylated and lipidated, exerting their predominant physiological effects through receptors of the frizzled and arrow/low-density lipoprotein  receptor-related protein 5/ <dig>  families.
frizzleds are seven transmembrane domain-containing proteins that are the primary receptors for wnts, whereas lrps  are single pass membrane proteins that serve as co-receptors.
in the "canonical wnt pathway," wnts bind to their cognate receptors and stimulate the cytoplasmic stabilization of β-catenin through the inhibition of an elaborate degradation complex consisting of apc, axin, and glycogen synthase kinase  <dig> .
stabilized β-catenin translocates to the nucleus where it interacts with transcriptional regulators of the t cell factor/lymphoid enhancer factor  family of proteins to mediate transcription of wnt target genes  <cit> .
recently, there has been evidence suggesting that heterotrimeric g proteins may have a role in wnt signaling  <cit> .
while wnts, their receptors, and numerous downstream components have been identified through genetic and biochemical approaches, the upstream molecular events occurring directly after frizzled activation have not been well-characterized.
importantly, it has not been established whether the majority of wnt signal transduction occurs at the plasma membrane or on some intracellular organelle.
there have been very few studies examining wnt trafficking to intracellular compartments, and those demonstrating internalization of wg/frizzled <dig> in drosophila  <cit>  and wnt5a/frizzled <dig> in mammalian cells  <cit>  have primarily suggested a role for wnt trafficking in degradation of the ligand/receptor complex following signal transduction.
while endocytosis of ligand/receptor pairs has traditionally been considered a longer-term mechanism for termination of a signaling pathway, recent evidence with receptor tyrosine kinases  and g protein-coupled receptors  has suggested that internalization facilitates certain aspects of cellular signaling.
indeed, both epidermal growth factor- and β-adrenergic receptor-mediated activation of mitogen-activated protein kinase have been demonstrated to be dependent upon clathrin-mediated internalization  <cit> .
however, wnt signaling requires two kinds of receptors, the frizzleds and the lrps, and it would therefore be important to establish the cellular compartment where signaling takes place.
RESULTS
here we have examined a potential role for endocytosis in wnt signal transduction.
both wnt-3a  and wg  stimulate high levels of β-catenin accumulation in l cells , suggesting the utility of this cell line for dissecting the role of trafficking in the wnt pathway.
incubation of l cells for  <dig> hour at 37°c with wg-conditioned medium, but not control-conditioned medium , leads to the appearance of a punctate immunostaining pattern as assessed by confocal microscopy .
suggestive of internalization into vesicles, this punctate staining pattern is not observed when cells are incubated with wg at 4°c , a temperature known to inhibit vesicular trafficking pathways.
we next sought to determine whether the internalization of wnt is mediated by clathrin-coated vesicle formation, by far the most well-characterized mechanism for regulated endocytosis  <cit> .
there are a variety of ways to perturb clathrin-mediated internalization, including the use of small molecules  and chlorpromazine ), osmotics , dominant-interfering mutant proteins , and loss of function reagents .
mdc is believed to inhibit clathrin-mediated endocytosis by stabilizing nascent clathrin-coated vesicles and preventing uncoating  <cit> , presumably reducing the availability of free clathrin to assemble at the plasma membrane.
hypertonic sucrose has been shown to prevent the proper assembly of clathrin lattices at the plasma membrane  <cit> .
moreover, cpz blocks clathrin-mediated endocytosis through a mechanism whereby adaptor complex  <dig>  and clathrin are redistributed away from the plasma membrane, making clathrin unavailable for assembly at the cell surface  <cit> .
dynamin, a gtpase recruited to clathrin-coated pits, is an additional component of endocytosis which is thought to regulate the final step of vesicle biogenesis by catalyzing the scission of budding plasma membrane  <cit> .
mutation of lysine  <dig>  has been shown to decrease the affinity of dynamin for gtp, and expression of this mutant in cells dramatically inhibits endocytosis from the plasma membrane without affecting vesicular transport processes elsewhere in the cell  <cit> .
internalization of wg is markedly reduced when cells are incubated in the presence of mdc , hypertonic sucrose , and cpz , with most of the staining in these cases being confined to the cell surface.
further, while l cells transiently transfected with either gfp alone or gfp in combination with wild-type dynamin internalize wg into intracellular vesicles, cells expressing gfp in combination with the k44e mutant dynamin display predominantly cell surface staining .
by contrast, wg internalizes quite robustly in adjacent non-transfected cells surrounding those harboring the k44e-mutated dynamin .
whereas approximately 70–80% of cells transfected with gfp alone or in combination with the wild-type dynamin internalize wg, only 20–25% of those expressing the k44e mutant protein exhibit comparable levels of endocytosis .
consequently, the internalization of wg into l cells occurs on a time-frame consistent with regulated endocytosis , and is sensitive to various manipulations which inhibit clathrin assembly and dynamin-mediated scission of budding plasma membrane.
we next sought to characterize the subcellular localization of internalized wg.
to identify potential intracellular compartments containing the trafficked wnt, we performed wg internalization assays in the presence of cy3-conjugated transferrin.
receptors for transferrin constitutively cycle among the plasma membrane, early sorting endosomes, and perinuclear recycling endosomes, and have served as a classic system for the study of clathrin-mediated endocytosis  <cit> .
incubation of l cells with transferrin for  <dig> hour at 37°c leads to a steady-state distribution which is both punctate and perinuclear , suggestive of the constitutive cycling of the protein from the plasma membrane via vesicles to perinuclear recycling endosomes.
internalized wg exhibits partial overlap with transferrin, particularly in the perinuclear region of the cell .
by contrast, co-incubation of cells with wg and transferrin at 4°c leads to an overlapping accumulation of the two proteins at the cell surface .
hence, both wg and transferrin not only internalize through similar endocytic pathways, but also have a similar steady-state localization in a perinuclear recycling endosome.
the lack of complete co-localization of wg and transferrin suggest that while the two proteins enter the cell through similar compartments, they ultimately have somewhat distinct fates.
indeed, it has previously been demonstrated that wg transits through multivesicular bodies to the lysosomes for degradation following signal transduction  <cit> .
given that wg is internalized within a time-frame relevant to wnt signaling in l cells, we sought to determine whether the two processes are functionally coupled.
wnt signaling is typically measured through wnt-stimulated accumulation of cytoplasmic β-catenin, as well as downstream target gene expression.
l cells express very low basal levels of β-catenin and are quite responsive to exogenous mammalian wnt-3a and drosophila wg , making them a powerful system for measuring wnt-induced increases in β-catenin.
we assayed wnt-stimulated accumulation of β-catenin in the presence or absence of the same mechanistically-distinct inhibitors which abolish wg endocytosis.
as demonstrated in figure 3a, both wnt-3a and wg induce substantial stabilization of β-catenin over a 3-hour time-course, without altering the levels of a control cytoplasmic protein .
by contrast, incubation of cells with mdc , hypertonic sucrose , and cpz  abrogates the wnt-3a-stimulated increase in β-catenin.
however, none of these inhibitors alter the levels of the control protein gsk3β.
to assess the potential requirement for endocytic trafficking of the more downstream process of wnt target gene expression, we measured wnt-3a-stimulated expression of a luciferase reporter fused to a promoter containing tcf/lef binding sites .
as depicted in figure 3e, vehicle-treated l cells stably expressing reporter constructs yield an approximately 50-fold increase in topflash activity relative to co-expressed lacz following a 5-hour stimulation with wnt-3a.
by contrast, pre-incubation of these cells with mdc, hypertonic sucrose, or cpz completely abolishes wnt-induced reporter gene activity .
to order the endocytic trafficking step in the pathway, we assessed the sensitivity of various downstream pathway activators to these endocytosis inhibitors.
activation of the pathway through lithium-mediated inhibition of gsk3β is completely impaired when endocytosis is blocked , a surprising observation which will be discussed later in this report.
by contrast, the constitutive expression of cytoplasmic β-catenin in sw <dig> cells, which harbor an inactive version of apc, is resistant to perturbation of endocytosis .
taken together, these results suggest that wnt signaling is heavily dependent upon clathrin-mediated endocytosis, and that the explicit internalization event occurs between the steps of gsk3β inhibition and apc inactivation.
in addition to perturbing clathrin assembly at the plasma membrane, we also examined the effect of the dynamin mutant on wnt signaling.
we measured both wnt-3a- and wg-stimulated accumulation of β-catenin on a cell-by-cell basis by immunofluorescence microscopy .
wnt-3a-stimulated β-catenin stabilization was measured in l cells transfected with gfp alone  or gfp in combination with either wild-type  or k44e-mutated dynamin .
as observed in the western blot experiments , very little β-catenin is detected in the l cells in the absence of wnt-3a .
addition of wnt-3a, by contrast, leads to prominent immunostaining of nuclear β-catenin in cells expressing gfp alone or in combination with wild-type dynamin .
however, as demonstrated in figure 4k, wnt-3a-stimulated accumulation of β-catenin is completely abolished in cells expressing the k44e-mutated dynamin , whereas neighboring non-transfected cells stabilize β-catenin comparably to control cells .
as depicted in the quantitations , cells expressing gfp alone or in combination with wild-type dynamin demonstrate wnt-dependent increases in β-catenin in approximately 70–90% of cells, with only approximately 20–30% of the k44e dynamin-expressing cells conferring a similar level of responsiveness.
taken together with the previous results using mdc, hypertonic sucrose, and cpz, the ability of k44e-mutated dynamin to block wnt signaling suggests that endocytosis is a key mechanistic step in the pathway.
the various methods used to inhibit clathrin-mediated endocytosis, including small molecules, osmotics, and dominant interfering mutants, all potently abolish wnt internalization and signal transduction.
in order to further examine the requirement of internalization in wnt signaling, we have performed loss of function experiments using sirnas directed against clathrin heavy chain.
although clathrin is composed of both light and heavy chains, it is the latter which is truly critical for establishing the lattice coat necessary for membrane budding, as sirna-mediated silencing of this subunit has previously been shown to greatly attenuate transferrin endocytosis  <cit> .
consequently, we generated sirna oligonucleotides corresponding to the region of clathrin heavy chain which has previously been shown to silence the protein  <cit> .
as illustrated in figure 5a, sirnas against clathrin heavy chain selectively ablate this protein without affecting levels of the control protein gsk3β.
moreover, sirna-mediated silencing of clathrin heavy chain substantially attenuates wnt-3a-stimulated reporter activity .
discussion
we demonstrate an essential role for endocytic trafficking in wnt signaling.
through the use of various small molecule, dominant-interfering, and loss of function inhibitors of distinct stages of endocytosis, we define an explicit requirement for internalization in wnt signaling.
our observation that β-catenin stabilization by both mammalian  and drosophila  wnt proteins is sensitive to blockade of endocytosis suggests that this requirement may be universal for the wnt pathway, and not merely limited to one particular wnt.
after submission of this manuscript, seto and bellen  <cit>  showed that wg signaling in vivo is also dependent on internalization.
the differential sensitivity of β-catenin stabilization to blockade of endocytosis in l cells treated with lithium and the apc mutant sw <dig> cells indicates that the key endocytic step may reside between gsk3β and apc in the pathway.
indeed, it was quite surprising that the stabilization of β-catenin effected by lithium stimulation  required an intact endocytic pathway, given traditional models of the wnt pathway scheme.
however, recent studies have implicated gsk3β in the regulation of endocytic trafficking, perhaps independent of its role in the wnt pathway  <cit> .
doronin et al.
<cit>  demonstrates that stimulation of cells with lithium causes rapid endocytosis of the β2-adrenergic receptor, and pelkmans et al.
<cit>  report that sirna-mediated silencing of gsk3β causes internalized transferrin to accumulate in early endosomes.
incorporating these recent findings  <cit>  with our epistasis data could suggest the following scenario: inhibition of gsk3β in response to a wnt signal  facilitates rapid internalization of the signaling machinery in a manner which sequesters it from the apc protein, thereby leading to the stabilization and nuclear translocation of β-catenin.
over the past few years, a number of studies have suggested that endocytosis may have a facilitating role in signal transduction.
for example, activation of erk1/ <dig> directly through rtks, or through certain gpcrs, has been shown to require endocytosis  <cit> .
indeed, activated rtks and gpcrs have been found to be present on populations of clathrin-coated vesicles containing a number of intermediate proteins in the erk1/ <dig> pathway including ras, raf, mek, and shc  <cit> .
further, it was recently discovered that clathrin-mediated endocytosis and subsequent trafficking of tgfβ receptors to endosomes is required for productive signaling  <cit> .
these studies have contributed to a "signaling endosome hypothesis," suggesting that this organelle may serve as a nexus for a number of signal transduction pathways.
indeed, partial co-localization of internalized wnt with transferrin further implicates the clathrin-mediated pathway in this process and suggests that a perinuclear recycling endosome functions as a compartment for wnt signal transduction.
in contrast to the rtk, gpcr, and tgfβ pathways, very little is known about the subcellular localization of wnt signaling.
the few studies examining the potential endocytosis and trafficking of wnt proteins have considered this as a mechanism for the formation of the wnt morphogen gradient in the embryo or the termination of the pathway  <cit> .
to date, no previous studies have suggested that internalization is explicitly required for propagation of wnt signaling cascades.
in an elegant study in the drosophila embryo, a horseradish peroxidase-conjugated wg was shown to be degraded by receptive cells in a manner sensitive to chloroquine and dependent upon deep orange , suggesting that the ligand is trafficked to the lysosomes  <cit> .
CONCLUSIONS
taken together with our results, these data indicate that clathrin-mediated endocytosis may serve two purposes: promotion of wnt signaling in an endosome, followed by ligand degradation and receptor down-regulation in the lysosomes.
the critical balance between an endosomal localization conducive for productive wnt signaling and lysosomal trafficking for attenuation of the signal may involve an explicit intracellular sorting step.
nevertheless, in the context of previous work on the internalization of wg  <cit>  and wnt-5a  <cit> , our study delineates a previously unappreciated role for endocytosis in wnt signal transduction.

 the wnt signaling pathway is largely conserved from drosophila to humans and has been shown to regulate diverse embryonic processes including axis specification and organogenesis. wnt signaling events have also been implicated in a number of cancers, bone density phenotypes, and neurological conditions in adulthood. wnts are a family of secreted proteins which are glycosylated and lipidated, exerting their predominant physiological effects through receptors of the frizzled and arrow/low-density lipoprotein receptor-related protein 5/ families. frizzleds are seven transmembrane domain-containing proteins that are the primary receptors for wnts, whereas lrps are single pass membrane proteins that serve as co-receptors. in the "canonical wnt pathway," wnts bind to their cognate receptors and stimulate the cytoplasmic stabilization of β-catenin through the inhibition of an elaborate degradation complex consisting of apc, axin, and glycogen synthase kinase. stabilized β-catenin translocates to the nucleus where it interacts with transcriptional regulators of the t cell factor/lymphoid enhancer factor family of proteins to mediate transcription of wnt target genes. recently, there has been evidence suggesting that heterotrimeric g proteins may have a role in wnt signaling. while wnts, their receptors, and numerous downstream components have been identified through genetic and biochemical approaches, the upstream molecular events occurring directly after frizzled activation have not been well-characterized. importantly, it has not been established whether the majority of wnt signal transduction occurs at the plasma membrane or on some intracellular organelle. there have been very few studies examining wnt trafficking to intracellular compartments, and those demonstrating internalization of wg/frizzled in drosophila and wnt5a/frizzled in mammalian cells have primarily suggested a role for wnt trafficking in degradation of the ligand/receptor complex following signal transduction. while endocytosis of ligand/receptor pairs has traditionally been considered a longer-term mechanism for termination of a signaling pathway, recent evidence with receptor tyrosine kinases and g protein-coupled receptors has suggested that internalization facilitates certain aspects of cellular signaling. indeed, both epidermal growth factor- and β-adrenergic receptor-mediated activation of mitogen-activated protein kinase have been demonstrated to be dependent upon clathrin-mediated internalization. however, wnt signaling requires two kinds of receptors, the frizzleds and the lrps, and it would therefore be important to establish the cellular compartment where signaling takes place.  here we have examined a potential role for endocytosis in wnt signal transduction. both wnt-3a and wg stimulate high levels of β-catenin accumulation in l cells, suggesting the utility of this cell line for dissecting the role of trafficking in the wnt pathway. incubation of l cells for hour at 37°c with wg-conditioned medium, but not control-conditioned medium, leads to the appearance of a punctate immunostaining pattern as assessed by confocal microscopy. suggestive of internalization into vesicles, this punctate staining pattern is not observed when cells are incubated with wg at 4°c, a temperature known to inhibit vesicular trafficking pathways. we next sought to determine whether the internalization of wnt is mediated by clathrin-coated vesicle formation, by far the most well-characterized mechanism for regulated endocytosis. there are a variety of ways to perturb clathrin-mediated internalization, including the use of small molecules and chlorpromazine ), osmotics, dominant-interfering mutant proteins, and loss of function reagents. mdc is believed to inhibit clathrin-mediated endocytosis by stabilizing nascent clathrin-coated vesicles and preventing uncoating, presumably reducing the availability of free clathrin to assemble at the plasma membrane. hypertonic sucrose has been shown to prevent the proper assembly of clathrin lattices at the plasma membrane. moreover, cpz blocks clathrin-mediated endocytosis through a mechanism whereby adaptor complex and clathrin are redistributed away from the plasma membrane, making clathrin unavailable for assembly at the cell surface. dynamin, a gtpase recruited to clathrin-coated pits, is an additional component of endocytosis which is thought to regulate the final step of vesicle biogenesis by catalyzing the scission of budding plasma membrane. mutation of lysine has been shown to decrease the affinity of dynamin for gtp, and expression of this mutant in cells dramatically inhibits endocytosis from the plasma membrane without affecting vesicular transport processes elsewhere in the cell. internalization of wg is markedly reduced when cells are incubated in the presence of mdc, hypertonic sucrose, and cpz, with most of the staining in these cases being confined to the cell surface. further, while l cells transiently transfected with either gfp alone or gfp in combination with wild-type dynamin internalize wg into intracellular vesicles, cells expressing gfp in combination with the k44e mutant dynamin display predominantly cell surface staining. by contrast, wg internalizes quite robustly in adjacent non-transfected cells surrounding those harboring the k44e-mutated dynamin. whereas approximately 70–80% of cells transfected with gfp alone or in combination with the wild-type dynamin internalize wg, only 20–25% of those expressing the k44e mutant protein exhibit comparable levels of endocytosis. consequently, the internalization of wg into l cells occurs on a time-frame consistent with regulated endocytosis, and is sensitive to various manipulations which inhibit clathrin assembly and dynamin-mediated scission of budding plasma membrane. we next sought to characterize the subcellular localization of internalized wg. to identify potential intracellular compartments containing the trafficked wnt, we performed wg internalization assays in the presence of cy3-conjugated transferrin. receptors for transferrin constitutively cycle among the plasma membrane, early sorting endosomes, and perinuclear recycling endosomes, and have served as a classic system for the study of clathrin-mediated endocytosis. incubation of l cells with transferrin for hour at 37°c leads to a steady-state distribution which is both punctate and perinuclear, suggestive of the constitutive cycling of the protein from the plasma membrane via vesicles to perinuclear recycling endosomes. internalized wg exhibits partial overlap with transferrin, particularly in the perinuclear region of the cell. by contrast, co-incubation of cells with wg and transferrin at 4°c leads to an overlapping accumulation of the two proteins at the cell surface. hence, both wg and transferrin not only internalize through similar endocytic pathways, but also have a similar steady-state localization in a perinuclear recycling endosome. the lack of complete co-localization of wg and transferrin suggest that while the two proteins enter the cell through similar compartments, they ultimately have somewhat distinct fates. indeed, it has previously been demonstrated that wg transits through multivesicular bodies to the lysosomes for degradation following signal transduction. given that wg is internalized within a time-frame relevant to wnt signaling in l cells, we sought to determine whether the two processes are functionally coupled. wnt signaling is typically measured through wnt-stimulated accumulation of cytoplasmic β-catenin, as well as downstream target gene expression. l cells express very low basal levels of β-catenin and are quite responsive to exogenous mammalian wnt-3a and drosophila wg, making them a powerful system for measuring wnt-induced increases in β-catenin. we assayed wnt-stimulated accumulation of β-catenin in the presence or absence of the same mechanistically-distinct inhibitors which abolish wg endocytosis. as demonstrated in figure 3a, both wnt-3a and wg induce substantial stabilization of β-catenin over a 3-hour time-course, without altering the levels of a control cytoplasmic protein. by contrast, incubation of cells with mdc, hypertonic sucrose, and cpz abrogates the wnt-3a-stimulated increase in β-catenin. however, none of these inhibitors alter the levels of the control protein gsk3β. to assess the potential requirement for endocytic trafficking of the more downstream process of wnt target gene expression, we measured wnt-3a-stimulated expression of a luciferase reporter fused to a promoter containing tcf/lef binding sites. as depicted in figure 3e, vehicle-treated l cells stably expressing reporter constructs yield an approximately 50-fold increase in topflash activity relative to co-expressed lacz following a 5-hour stimulation with wnt-3a. by contrast, pre-incubation of these cells with mdc, hypertonic sucrose, or cpz completely abolishes wnt-induced reporter gene activity. to order the endocytic trafficking step in the pathway, we assessed the sensitivity of various downstream pathway activators to these endocytosis inhibitors. activation of the pathway through lithium-mediated inhibition of gsk3β is completely impaired when endocytosis is blocked, a surprising observation which will be discussed later in this report. by contrast, the constitutive expression of cytoplasmic β-catenin in sw cells, which harbor an inactive version of apc, is resistant to perturbation of endocytosis. taken together, these results suggest that wnt signaling is heavily dependent upon clathrin-mediated endocytosis, and that the explicit internalization event occurs between the steps of gsk3β inhibition and apc inactivation. in addition to perturbing clathrin assembly at the plasma membrane, we also examined the effect of the dynamin mutant on wnt signaling. we measured both wnt-3a- and wg-stimulated accumulation of β-catenin on a cell-by-cell basis by immunofluorescence microscopy. wnt-3a-stimulated β-catenin stabilization was measured in l cells transfected with gfp alone or gfp in combination with either wild-type or k44e-mutated dynamin. as observed in the western blot experiments, very little β-catenin is detected in the l cells in the absence of wnt-3a. addition of wnt-3a, by contrast, leads to prominent immunostaining of nuclear β-catenin in cells expressing gfp alone or in combination with wild-type dynamin. however, as demonstrated in figure 4k, wnt-3a-stimulated accumulation of β-catenin is completely abolished in cells expressing the k44e-mutated dynamin, whereas neighboring non-transfected cells stabilize β-catenin comparably to control cells. as depicted in the quantitations, cells expressing gfp alone or in combination with wild-type dynamin demonstrate wnt-dependent increases in β-catenin in approximately 70–90% of cells, with only approximately 20–30% of the k44e dynamin-expressing cells conferring a similar level of responsiveness. taken together with the previous results using mdc, hypertonic sucrose, and cpz, the ability of k44e-mutated dynamin to block wnt signaling suggests that endocytosis is a key mechanistic step in the pathway. the various methods used to inhibit clathrin-mediated endocytosis, including small molecules, osmotics, and dominant interfering mutants, all potently abolish wnt internalization and signal transduction. in order to further examine the requirement of internalization in wnt signaling, we have performed loss of function experiments using sirnas directed against clathrin heavy chain. although clathrin is composed of both light and heavy chains, it is the latter which is truly critical for establishing the lattice coat necessary for membrane budding, as sirna-mediated silencing of this subunit has previously been shown to greatly attenuate transferrin endocytosis. consequently, we generated sirna oligonucleotides corresponding to the region of clathrin heavy chain which has previously been shown to silence the protein. as illustrated in figure 5a, sirnas against clathrin heavy chain selectively ablate this protein without affecting levels of the control protein gsk3β. moreover, sirna-mediated silencing of clathrin heavy chain substantially attenuates wnt-3a-stimulated reporter activity. discussion we demonstrate an essential role for endocytic trafficking in wnt signaling. through the use of various small molecule, dominant-interfering, and loss of function inhibitors of distinct stages of endocytosis, we define an explicit requirement for internalization in wnt signaling. our observation that β-catenin stabilization by both mammalian and drosophila wnt proteins is sensitive to blockade of endocytosis suggests that this requirement may be universal for the wnt pathway, and not merely limited to one particular wnt. after submission of this manuscript, seto and bellen showed that wg signaling in vivo is also dependent on internalization. the differential sensitivity of β-catenin stabilization to blockade of endocytosis in l cells treated with lithium and the apc mutant sw cells indicates that the key endocytic step may reside between gsk3β and apc in the pathway. indeed, it was quite surprising that the stabilization of β-catenin effected by lithium stimulation required an intact endocytic pathway, given traditional models of the wnt pathway scheme. however, recent studies have implicated gsk3β in the regulation of endocytic trafficking, perhaps independent of its role in the wnt pathway. doronin et al. demonstrates that stimulation of cells with lithium causes rapid endocytosis of the β2-adrenergic receptor, and pelkmans et al. report that sirna-mediated silencing of gsk3β causes internalized transferrin to accumulate in early endosomes. incorporating these recent findings with our epistasis data could suggest the following scenario: inhibition of gsk3β in response to a wnt signal facilitates rapid internalization of the signaling machinery in a manner which sequesters it from the apc protein, thereby leading to the stabilization and nuclear translocation of β-catenin. over the past few years, a number of studies have suggested that endocytosis may have a facilitating role in signal transduction. for example, activation of erk1/ directly through rtks, or through certain gpcrs, has been shown to require endocytosis. indeed, activated rtks and gpcrs have been found to be present on populations of clathrin-coated vesicles containing a number of intermediate proteins in the erk1/ pathway including ras, raf, mek, and shc. further, it was recently discovered that clathrin-mediated endocytosis and subsequent trafficking of tgfβ receptors to endosomes is required for productive signaling. these studies have contributed to a "signaling endosome hypothesis," suggesting that this organelle may serve as a nexus for a number of signal transduction pathways. indeed, partial co-localization of internalized wnt with transferrin further implicates the clathrin-mediated pathway in this process and suggests that a perinuclear recycling endosome functions as a compartment for wnt signal transduction. in contrast to the rtk, gpcr, and tgfβ pathways, very little is known about the subcellular localization of wnt signaling. the few studies examining the potential endocytosis and trafficking of wnt proteins have considered this as a mechanism for the formation of the wnt morphogen gradient in the embryo or the termination of the pathway. to date, no previous studies have suggested that internalization is explicitly required for propagation of wnt signaling cascades. in an elegant study in the drosophila embryo, a horseradish peroxidase-conjugated wg was shown to be degraded by receptive cells in a manner sensitive to chloroquine and dependent upon deep orange, suggesting that the ligand is trafficked to the lysosomes.  taken together with our results, these data indicate that clathrin-mediated endocytosis may serve two purposes: promotion of wnt signaling in an endosome, followed by ligand degradation and receptor down-regulation in the lysosomes. the critical balance between an endosomal localization conducive for productive wnt signaling and lysosomal trafficking for attenuation of the signal may involve an explicit intracellular sorting step. nevertheless, in the context of previous work on the internalization of wg and wnt-5a, our study delineates a previously unappreciated role for endocytosis in wnt signal transduction. 
while endocytosis has traditionally been considered a principal mechanism for receptor down-regulation and termination of signaling pathways, we demonstrate that interfering with clathrin-mediated endocytosis actually blocks wnt signaling at the level of β-catenin accumulation and target gene expression. the wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. while numerous wnts, their cognate receptors of the frizzled and arrow/lrp5/ families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation. we show here that wnt proteins are rapidly endocytosed by a clathrin- and dynamin-mediated process. a necessary component of wnt signaling occurs in a subcellular compartment distinct from the plasma membrane. wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood.
 the wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. while numerous wnts, their cognate receptors of the frizzled and arrow/lrp5/ families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.  we show here that wnt proteins are rapidly endocytosed by a clathrin- and dynamin-mediated process. while endocytosis has traditionally been considered a principal mechanism for receptor down-regulation and termination of signaling pathways, we demonstrate that interfering with clathrin-mediated endocytosis actually blocks wnt signaling at the level of β-catenin accumulation and target gene expression.  a necessary component of wnt signaling occurs in a subcellular compartment distinct from the plasma membrane. moreover, as internalized wnts transit partially through the transferrin recycling pathway, it is possible that a "signaling endosome" serves as a nexus for activated wnt pathway components. 