Báo cáo khoa học: Differential membrane compartmentalization of Ret by PTB-adaptor engagement pdf

Differential membrane compartmentalization of Ret
by PTB-adaptor engagement
T. K. Lundgren, Moritz Luebke, Anna Stenqvist and Patrik Ernfors
Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
The Ret receptor tyrosine kinase (RTK) has many dif-
ferent functions during development and in adult life.
Ret is activated upon engagement of its ligands glial
cell line-derived neurotrophic factor (GDNF), neurtu-
rin, persephin, and artemin, and the coreceptors
GDNF family receptors (GFRs) a1–4. Dysregulated
Ret is implicated in diseases such as the multiple endo-
crine neoplasia (MEN) syndromes (MEN2a and
MEN2b), as well as in agangliosis of the colon, one of
the most common developmental defects in young chil-
dren [1]. In the developing nervous system, neural crest
cell migration depends on Ret ligands produced in the
surrounding tissue, which guide the migrating cells
to a correct position within the embryo [2]. In the
Keywords
fractionation; Frs2; lipid rafts; PTB adaptors;
Ret
Correspondence
P. Ernfors, Division of Molecular
Neurobiology, Department of Medical
Biochemistry and Biophysics, Karolinska
Institute, 171 77 Stockholm, Sweden
Fax: +468 341960
Tel: +468 52487659
E-mail:
(Received 14 December 2007, revised 23
February 2008, accepted 26 February 2008)

doi:10.1111/j.1742-4658.2008.06360.x
Glial cell line-derived neurotrophic factor family ligands act through the
receptor tyrosine kinase Ret, which plays important roles during embryonic
development for cell differentiation, survival, and migration. Ret signaling
is markedly affected by compartmentalization of receptor complexes into
membrane subdomains. Ret can propagate biochemical signaling from
within concentrates in cholesterol-rich membrane microdomains or lipid
rafts, or outside such regions, but the mechanisms for, and consequences
of, Ret translocation between these membrane compartments remain lar-
gely unclear. Here we investigate the interaction of Shc and Frs2 phos-
photyrosine-binding domain-containing adaptor molecules with Ret and
their function in redistributing Ret to specialized membrane compartments.
We found that engagement of Ret with the Frs2 adaptor results in an
enrichment of Ret in lipid rafts and that signal transduction pathways and
chemotaxis responses depend on the integrity of such rafts. The competing
Shc adaptor did not promote Ret translocation to equivalent domains, and
Shc-mediated effects were less affected by disruption of lipid rafts. How-
ever, by expressing a chimeric Shc protein that localizes to lipid rafts, we
showed that biochemical signaling downstream of Ret resembled that of
Ret signaling via Frs2. We have identified a previously unknown mecha-
nism in which phosphotyrosine-binding domain-containing adaptors, by
means of relocating Ret receptor complexes to lipid rafts, segregate diverse
signaling and cellular functions mediated by Ret. These results reveal the
existence of a novel mechanism that could, by subcellular relocation of
Ret, work to amplify ligand gradients during chemotaxis.
Abbreviations
CO, cholesterol oxidase; CTB, cholera toxin B; DRG, dorsal root ganglia; DRM, detergent-resistant membrane; E, embryonic day; eGFP,
enhanced green fluorescent protein; ERK, extracellular signal-related kinase; GFR, glial cell line-derived neurotrophic factor family receptor;
GNDF, glial cell line-derived neurotrophic factor; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; MCD, methyl- b -cyclodextrin;
MEN, multiple endocrine neoplasia; MLS, membrane localization signal; PI3K, phosphoinositide-3-kinase; PTB adaptor, phosphotyrosine-

binding domain-containing adaptor; PVDF, poly(vinylidene difluoride); RTK, receptor tyrosine kinase; SUP, detergent-soluble supernatant;
TfR, transferrin receptor.
FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2055
developing and adult mouse, Ret is also instrumental
in promoting survival of neurons, including parasym-
pathetic neurons and dopaminergic neurons of the sub-
stantia nigra pars compacta [3–5].
Many of the functions regulated by Ret activation
depend on an intact Tyr1062 in the intracellular
domain [6,7]. Upon Ret phosphorylation, this tyrosine
mediates biochemical signal transduction via interac-
tion with adaptor proteins. Several phosphotyrosine-
binding domain-containing adaptors (PTB adaptors)
compete for interaction with Tyr1062, but only one
adaptor may interact at any given time [8]. The dis-
tinct functional outcome of Ret activation is corre-
lated with the different PTB adaptors interacting with
Ret [9,10]. We have shown previously that expression
of Ret mutants, selective for binding to either the
adaptor protein Shc or Frs2 to Tyr1062, results in
distinctly different patterns of plasma membrane
localization of Ret. Frs2 recruitment resulted in a
concentration of Ret into membrane foci [4,11]. The
Ret receptor has recently been shown to signal from
within different cellular compartments. The oncogenic
precursor of Ret (Men2B) can be activated and
induce downstream signaling from within the endo-
plasmic reticulum [12]. Ret can also be recruited to
specialized lipid raft domains in the plasma mem-
brane, where it can be phosphorylated, interact with

adaptor proteins and induce downstream signaling
[12–14].
Lipid rafts are membrane microdomains, rich in
sphingolipids and cholesterol, that form lateral assem-
blies in the plasma membrane [15]. Lipid rafts seques-
ter a number of different proteins that are
palmitoylated or contain a number of other lipid
anchors, which may be regulated by the selective
interaction with these domains. In this way, the
recruitment of Ret to lipid rafts can lead to the acti-
vation of distinct signaling pathways, due to the com-
partmentalized cell signaling events. However, the
mechanism for directing Ret into lipid rafts remains
largely unknown. We report here that the PTB adap-
tor Frs2 functions to translocate Ret to membrane
subdomains of the lipid raft type. Interactions of Ret
with the Shc adaptor, which, in contrast to Frs2,
lacks a palmitoylation tail that confers attachment to
lipid rafts, did not result in redistribution of Ret to
lipid rafts. Targeting the Shc adaptor to lipid rafts by
engineering the adaptor with a raft targeting tail led
to altered biochemical signaling resembling, in several
respects, signal transduction downstream of Frs2. We
show that the distinct biological outcomes of Ret acti-
vation largely depends on the targeting to, and signal-
ing from within, lipid rafts.
Results
A PTB-adaptor-dependent membrane relocation
of Ret receptors
To investigate whether Tyr1062 is important for Ret

translocation into lipid rafts, we performed crude
fractionations of neuronal SK-N-MC cells transfected
with the MEN2a version of Ret (
2a
Ret). The MEN2a
mutation C634R is found in more than 85% of
patients with MEN syndrome, and renders Ret con-
stitutively active, thus omitting the need for ligand
for its activation. Lipid rafts and nonraft membranes
were isolated according to their resistance to and sol-
ubilization by detergent. Cells expressing
2a
Ret or a
2a
Ret
Y1062F
mutant that is incapable of adaptor inter-
action with the phosphorylated Tyr1062 were har-
vested in detergent, and the detergent-resistant
membrane (DRM) fraction was separated from the
detergent-soluble supernatant (SUP) fraction by cen-
trifugation. Each fraction was subjected to PAGE
and transferred to poly(vinylidene difluoride) (PVDF)
membranes. Immunoblotting against Ret revealed that
the majority of Ret partitioned in the DRM fraction.
This was in contrast to the
2a
Ret
Y1062F
mutant, where

the majority of Ret was found in the SUP fraction
(Fig. 1A). To test whether Ret distribution was
affected by interaction with either the Frs2 or the Shc
adaptor, we overexpressed Shc or Frs2 constructs
together with
2a
Ret in SK-N-MC cells. Lysates from
cells overexpressing Shc or Frs2 were divided into
equal halves, and each half was immunoprecipitated
for Shc or Frs2 and immunoblotted against Ret.
Nearly all Ret precipitated with the overexpressed
adaptor, showing that overexpression of one adaptor
leads to outcompetition of the other with respect to
Ret interaction (Fig. 1B). Next, we fractionated
lysates of cells expressing
2a
Ret or
2a
Ret
Y1062F
together with overexpressed Frs2 or Shc into DRM
and SUP fractions. Adaptor overexpression led to
high amounts of Ret in the DRM fraction in both
Frs2 and Shc conditions, whereas
2a
Ret
Y1062F
,as
before, displayed a much lower partitioning into
DRMs (Fig. 1C). Ret localization to the supernatant

fraction was largely unaffected by the Y1062 muta-
tion in the presence of Frs2 or Shc. To confirm the
relative purity of raft and nonraft membranes, DRM
and SUP fractions were immunoblotted towards the
lipid raft marker flotillin-1 and the transferrin recep-
tor (TfR), the latter of which is excluded from raft-
like domains. As expected, flottillin-1 was found
nearly exclusively in the DRM fraction, whereas TfR
was mainly found in the SUP farction (Fig. 1C).
Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.
2056 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS
Furthermore, the DRM and SUP fractions were im-
munoblotted towards the adaptor proteins themselves.
Both Shc and Frs2 were found predominantly in the
DRM fractions (Fig. 1D). These results suggest that
Ret localization to nonraft membrane regions occurs
independently of adaptor engagement, whereas its
localization to lipid rafts depends on Shc or Frs2
interactions with Ret Tyr1062.
To examine whether Ret and adaptor partitioning
is affected by disruption of lipid-ordered regions, we
exposed cells to methyl-b-cyclodextrin (MCD), an
agent that is commonly used to extract cholesterol
from cell membranes in order to disrupt lipid-ordered
regions in the plasma membrane. Cells were treated
for 30 min with 8 mm MCD or vehicle prior to frac-
tionation and subsequent immunoblotting towards
Ret. The amount of Ret present in the DRM fraction
was significantly reduced, with a corresponding
increase of Ret in the SUP fraction, when cells were

treated with MCD (Fig. 1D). This indicates that the
integrity of cholesterol-rich regions is necessary for
Ret partitioning into DRMs, agreeing with previous
data [14].
The insoluble material recovered in the DRM frac-
tion represents a collection of many discrete lipid-
ordered structures, and is not exclusively composed of
lipid rafts [16]. Previous studies have found that Ret
partitions into DRM fractions upon ligand induction
[14], and that interaction of Ret with the Frs2 adap-
tor, but not the Shc adaptor, occurs in such fractions
[13]. To further investigate whether the previously
determined localization of Ret to membrane foci,
depending on adaptor engagement [11], had any rela-
tion to lipid rafts, we expressed Ret tagged with
enhanced green fluorescent protein (eGFP) (Ret
eGFP
)
in cells. Fusion of eGFP to Ret allows direct visuali-
zation of the subcellular distribution of Ret. Ret
eGFP
was expressed with Shc or Frs2 in SK-N-MC neuro-
nal cells that were stained for visualization of the
membrane lipid rafts using a fluorescently conjugated
cholera toxin B (CTB) subunit that binds to the pen-
tasaccharide chain of plasma membrane ganglioside
GM1, which selectively partitions into lipid rafts [17].
Using confocal microscopy, we found that there was
a consistent punctuate pattern of lipid rafts with high
abundance in neurites after 30 min of ligand stimulus.

Interestingly, extensive localization of Ret
eGFP
to lipid
rafts was seen only in Frs2-expressing cells, and not
in cells expressing Shc (Fig. 2A,C). The distinct recep-
tor localization was also correlated with morphologi-
cal differences between Shc- and Frs2-expressing cells;
Frs2-expressing cells often contained markedly more
cell processes and neurites than Shc-expressing cells.
Frs2 is exclusively localized to lipid rafts [18], whereas
Shc has been shown to inducibly localize to lipid-
ordered regions in some instances, e.g. in T-cell recep-
tor signaling [19]. To examine whether lipid raft
targeting of Shc could mediate translocation of Ret
to a subcellular localization similar to that mediated
by Frs2, we expressed an Shc construct containing
the Ras membrane localization signal (MLS)
(Shc
MLS
), which permanently localizes Shc to lipid
rafts [20]. This Shc construct was shown in previous
work to activate the mitogen-activated protein kinase
(MAPK) pathway constitutively [20]. Intriguingly,
sustained activation of MAPK is one prominent dis-
tinguishing feature of Ret signaling by Frs2 recruit-
ment, in contrast to Ret signaling via Shc [4]. When
Shc
MLS
was overexpressed in cells along with
Ret

eGFP
, the membrane localization of Ret was lar-
gely confined to lipid raft regions, similar to that of
Ret
eGFP
- and Frs2-expressing cells (Fig. 2B), indicat-
ing that lipid raft targeting of Shc leads to a redistri-
bution and enrichment of Ret in lipid rafts.
A
C
D
B
Fig. 1. Tyr1062 is necessary for Ret partitioning into lipid rafts. (A)
Mutation of Tyr1062 results in a loss of
2a
Ret partitioning to the
DRM fraction.
2a
Ret
WT
or
2a
Ret
Y1062F
was expressed in SK-N-MC
cells. Cells were harvested in 1% Triton buffer, separated into
DRM or SUP fractions, separated on polyacrylamide gels, and
transferred to PVDF membranes, with subsequent blotting for Ret
(n = 3 with similar results). (B) Overexpression of Shc or Frs2 adap-
tors forces

2a
Ret to interact with either adaptor at the expense of
the other. Lysates of SK-N-MC cells were separated, and each half
was immunoprecipitated for Shc or Frs2 and immunoblotted
against Ret (n = 2). (C) Overexpression of Shc or Frs2 adaptors
results in
2a
Ret
WT
but not
2a
Ret
Y1062F
partitioning into DRM frac-
tions. SK-N-MC cells were treated and harvested as in (A), and blot-
ted for detection of Ret (n = 3). (D) Shc and Frs2 partitioning of Ret
to the DRM fraction depends on intact lipid rafts. SK-N-MC cells
expressing
2a
Ret
WT
and
2a
Shc or Frs2 were treated with MCD or
vehicle, harvested, and separated into DRM and SUP fractions.
Immunoblot towards Ret and Frs2 or Shc as indicated (n = 3).
T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts
FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2057
A critical role of Ret membrane localization
in downstream signaling events in cell lines

and primary cells
We next examined the intracellular signaling down-
stream of Ret in cells expressing the Shc, Shc
MLS
or
Frs2 adaptors. Phosphorylated (active) Akt or p42,44
extracellular signal-related kinase (ERK) MAPK pro-
tein levels were examined by immunoblotting at differ-
ent time points after Ret ligand stimuli. Shc
MLS
expression together with Ret resulted in signal activa-
tion resembling the Ret ⁄ Frs2 coexpression characteris-
tics. Specifically, a higher and sustained ERK p42,44
MAPK level was also observed at 12 h in cells express-
ing Shc
MLS
as compared to Shc (Fig. 3A). Further-
more, Ret signaling via Shc
MLS
did not lead to as
robust activation ⁄ phosphorylation of Akt as did Shc,
but levels were higher than for signaling via Frs2
(Fig. 3C). To investigate the importance of rafts for
downstream ERK p42,44 MAPK activation, we
applied cholesterol oxidase (CO) to cells during ligand
stimuli. CO was chosen because cells cannot withstand
MCD treatment for long time periods, and CO has
been shown to disrupt the biochemical effects of mem-
brane-localized Ret as well as other receptor complexes
in a fashion similar to what is accomplished by using

MCD [11,21,22]. In the presence of CO, ERK p42,44
MAPK activation was markedly reduced both in cells
expressing Ret ⁄ Shc
MLS
as well as in cells expressing
Ret ⁄ Frs2 at all time points, whereas activation of
ERK p42,44 MAPK downstream of Shc was much less
affected (Fig. 3A). The specificity of cholesterol species
for phosphorylated ERK p42,44 MAPK was also
important in MEN2a versions of Ret when expressed
with Shc or Frs2. In cells expressing
2a
Ret, phosphory-
lated ERK p42,44 MAPK levels were high when Shc
and Frs2 were coexpressed. CO treatment attenuated
phosphorylated ERK p42,44 MAPK levels down-
AA'
BB'
CC'
Fig. 2. Labeling of plasma membrane gan-
glioside GM1 lipid rafts shows extensive
colocalization of Frs2-associated but not
Shc-associated Ret to lipid rafts. (A–C)
SK-N-MC cells expressing Ret
eGFP
(green)
together with Shc (A,A¢), Shc
MLS
(B,B¢)or
Frs2 (C, C¢). Actin was stained with Alexa-

648 phalloidin and Alexa-546 (red) conju-
gated to CTB to mark lipid rafts (n = 5).
Arrows indicate colocalization of Ret
eGFP
with lipid rafts. Scale bar = 25 lm.
Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.
2058 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS
stream of Shc and led to an almost complete absence
of phosphorylated ERK p42,44 MAPK downstream of
Frs2 (Fig. 3B).
Most studies on receptor signaling from within rafts
have been performed on immortalized cell lines in cul-
ture or by using artificial membranes. We attempted to
see whether the PTB adaptors determine Ret subcellu-
lar distribution and signaling in primary neurons of a
more complex system. To this end, we electroporated
Ret
eGFP
and either adaptor into the neural tube of
chicken embryos. DNA was injected in ovo into the
neural tube of embryos at Hamburger–Hamilton
stage 11 [approximately embryonic day (E)2], and elec-
troporation was performed by placing electrodes along
the rostral–caudal axis of the neural tube. The egg was
then closed and placed in an incubator until the
embryos had grown to stage 24 (E4.5). At this stage,
the expression of transfected Ret
eGFP
was found in the
developing spinal cord and in dorsal root ganglia

(DRG), and the transfected tissue was dissected under
fluorescent light (supplementary Fig. S1). eGFP-posi-
tive spinal cord segments were pooled and weighed,
and equal amounts of tissue were trypsinized into sin-
gle cells and immediately incubated with Ret ligands
for 30 min. After ligand stimulation, the cells were
fractionated into DRM and SUP fractions. In accor-
dance with the results obtained with neuronal SK-N-
MC cells, immunoblotting against Ret revealed that it
was predominantly located in DRM fractions, regard-
less of which adaptor (Shc, Shc
MLS
, or Frs2) was con-
comitantly overexpressed (Fig. 3D). Immunoblotting
of DRM and SUP fractions against the adaptor pro-
teins showed that Shc
MLS
and Frs2 were nearly exclu-
sive to the DRM fraction, whereas Shc was present
also in the SUP fractions (Fig. 3E).
We investigated the activation of MAPK and Akt
pathways in the developing chick embryo. Previous
studies have found that the ERK MAPK pathway [and
to a lesser extent, also the phosphoinositide-3-kinase
(PI3K) ⁄ Akt pathway] can be temporally and quantita-
tively affected by activation within or outside DRM
fractions [23]. To examine the role of the PTB adaptors
in the activation of the MAPK and Akt pathways,
spinal cords electroporated with Ret together with
either Shc, Shc

MLS
or Frs2 were separated into DRM
and SUP fractions, as above. Phosphorylated Akt lev-
els were generally low for all PTB adaptors in the
DRM fraction, showing that phosphorylated Akt
A
C
DF
G
E
B
Fig. 3. The raft-targeting adaptors Frs2 and Shc
MLS
show enhanced ERK phophorylation, which is dependent on the integrity of cholesterol-
rich domains. (A) SK-N-MC cells expressing Ret and either Shc, Shc
MLS
or Frs2 adaptors were lysed after Ret ligand stimulation for indicated
times and subsequently immunoblotted towards phosphorylated ERK and all ERK. CO was applied to cells as indicated. (B) Experiments as
in (A) with MEN2a versions of Ret. (C) Experiments as in (A) with immunoblotting towards phosphorylated Akt and Akt. (D) E2 chicken
embryos were electroporated in ovo with Ret and adaptor constructs as indicated, and allowed to develop to E4.5. At E4.5, positively trans-
fected spinal cord segments were dissected out and pooled to equal amounts. After dissociation into single cells and stimulation with Ret
ligands for 30 min cells, were lysed and fractionated into DRM and SUP fractions. Fractions were immunoblotted for detection of Ret. (E)
Experiments as in (C) with immunoblotting for the hemagglutinin (HA)-tag of Shc and Shc
MLS
or for Frs2. (F, G) Experiments as in (C) with
immunoblots towards phosphoryated Akt or phosphorylated ERK.
T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts
FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2059
signaling predominantly takes place in cellular struc-
tures partitioning into the SUP fraction (Fig. 3G).

Phosphorylated Akt levels were greater in the SUP
fraction when Shc or Shc
MLS
was expressed with Ret
as compared to Frs2, where levels were overall lower
and more similar between the DRM and SUP fractions
(Fig. 3G). Phosphorylated ERK p42,44 MAPK immu-
noblotting revealed the strongest signal when Frs2 was
expressed with Ret in both the DRM and the SUP
fractions (Fig. 3F). Shc
MLS
resulted in activation of
ERK p42,44 MAPK in the DRM. Shc, on the other
hand, was very poor in mediating ERK p42,44 MAPK
activation in the DRM fraction (Fig. 3G). These results
therefore agree with the results obtained with SK-N-
MC cells, and further support the idea that phos-
phory lated ERK p42,44 MAPK is often localized to
complexes of PTB adaptors with Ret in lipid rafts and
that phosphorylated Akt is mostly found outside rafts.
To characterize the Ret distribution in greater detail
than is permitted by DRM ⁄ SUP fractionation, we used
a recently published fractionation protocol that omits
the need for detergent and is highly specific with
regard to molecular distribution within and outside
lipid raft fractions [24]. Dissected spinal cords from
chick embryos were lysed in detergent-free buffer
and fractionated by ultracentrifugation in OptiPrep
gradients. Seven fractions were aspirated from
columns, and the protein content within each fraction

was concentrated by precipitation, loaded on poly-
acrylamide gels, and transferred to PVDF membranes.
The separation of raft fractions from nonraft fractions
was confirmed by immunoblotting against flotillin-1
and TfR (Fig. 4). As determined by immunoblotting
against Ret, this more sensitive method revealed that
the fractional distribution of Ret varied with the adap-
tor expressed in the embryos. Frs2 overexpression led
to Ret being directed towards lower-density fractions
(corresponding to lipid rafts), peaking in fraction 2.
Shc overexpression resulted in significantly less Ret
partitioning into the fractions of lowest density, with
little or no Ret in fraction 2 and peak levels in frac-
tion 3 (Fig. 4). Overexpression of the raft-localizing
Shc
MLS
construct resulted in a significant portion of
Ret being partitioned into both fraction 2 and frac-
tion 3. Immunoblotting against the adaptors them-
selves showed that whereas both Shc and Shc
MLS
were
present in both low-density and high density fractions,
Frs2 was nearly exclusive to the same fraction as Ret
(fraction 2) (Fig. 4).
Functional consequences of signaling from raft
and nonraft membrane compartments
Association of Ret with the different adaptors may
result in distinct cellular responses to Ret ligand stimu-
lation [4,9]. In functional terms, the chemotactic prop-

erties of Ret signaling via Frs2 are much greater than
those of Ret signaling via Shc [11]. Ret signaling via
recruitment of Shc, on the other hand, is necessary for
Ret-mediated cell survival when neuronal cells are pre-
sented to toxic agents, and also for neurite formation
in certain cell types [4,9]. We investigated how disrup-
tion of lipid-ordered domains affected these functional
aspects, and whether the Shc
MLS
adaptor is similar to
Frs2 or Shc in terms of cellular response to Ret stimu-
lation. Chemotactic migration towards Ret ligands was
examined by seeding neuronal SK-N-MC cells express-
ing Ret receptors that are selective for binding of only
Frs2 (Ret
Frs+
) or Shc (Ret
Shc+
) to the region of
Tyr1062 [4]. Ret mutants binding to Frs2 showed a
nearly three-fold higher migrational capacity than did
Ret
Shc+
, and this effect was ligand-dependent, as all
conditions displayed a similar low level of random cell
migration without ligand being supplied to the lower
culture compartment (Fig. 5A). Cells expressing
Ret
Shc+
⁄ Shc did not show any statistically significant

ligand-induced migration. Cells expressing Shc
MLS
together with Ret
Shc+
displayed a two-fold increase of
migration towards Ret ligand (Fig. 5A). The same
experiment was then performed in the presence of CO.
A
Fig. 4. Density fractionation of the membrane localizes Frs2 and
Shc partly to different membrane compartments. (A) E2 chicken
embryos were electroporated in ovo and allowed to develop to E5.
At E5, positively transfected spinal cord segments were dissected
out and pooled to equal amounts of input. After dissociation into
single cells and stimulation with Ret ligands for 30 min, cells were
harvested in detergent-free buffer and subjected to ultracentrifuga-
tion in OptiPrep density gradients. Fractions were taken out after
centrifugation, and concentrated protein from each fraction was
immunoblotted against Ret or HA-tag for Shc and Shc
MLS
or
against Frs2, as indicated. Fraction 1 is the less dense fraction, and
fraction 7 is the fraction with the highest density. The bottom panel
shows immunoblotting for the lipid raft marker flotillin-1 and the
nonraft marker TfR.
Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.
2060 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS
CO treatment of cells led to a large decrease in the
number of migrating cells, almost reaching baseline
levels in all conditions (Fig. 5A), indicating that the
integrity of cholesterol-rich membrane domains is nec-

essary for the occurrence of directional migration
towards Ret ligand.
The ability of Ret selective mutants to promote cell
survival was next examined. SK-N-MC cells treated
with anisomycin to induce apoptosis were rescued by
supplementing the medium with Ret ligands. To detect
the apoptotic response to anisomycin with high sensi-
tivity, single cells were examined in the comet assay. In
this system, fragmented DNA moves out of the cell
soma in the shape of comet tails when cells are embed-
ded in agarose and subjected to an electric field. After
staining of DNA with SYBR-green, images were cap-
tured using fluorescence microscopy. The comet-tail
moment was determined by measuring pixel intensity
in the comet head and tail, and calculating the
momentum of the comet tails [25]. In accordance with
what was previously found [4], Ret ligands resulted in
a much greater cell survival effect for cells expressing
Ret
Shc+
than for those expressing Ret
Frs+
(Fig. 5B,C).
When Shc
MLS
was coexpressed with Ret
Shc+
to
enforce Shc signaling in lipid rafts, an intermediate
survival-promoting effect was seen (Fig. 5B,C). Dis-

ruption of cholesterol-rich membrane compartments
with CO had no effects on cell survival (Fig. 5B,C).
Furthermore, CO alone without anisomycin did not
result in any detectable cell injury (Fig. 5B,C), suggest-
ing that CO at the concentrations used does not have
an effect on cell survival. Thus, these data suggest that
Ret-mediated cell migration, but not cell survival,
requires intact lipid rafts.
Discussion
The present study was conducted to determine whether
PTB adaptors determine Ret localization to different
membrane compartments, and whether pathway-spe-
cific signaling and functional outcomes are affected by
signaling from within and outside rafts. It has previ-
ously been shown that Ret localizes to lipid rafts in
a glycophosphatidylinositol-bound GFRa1 coreceptor-
dependent fashion [26]. However, more recent results
have demonstrated that both soluble and glycophos-
A
B
C
Fig. 5. Frs2- dependent chemotaxis but not Shc-dependent cell
survival depends on raft integrity. (A) SK-N-MC cells expressing Ret
mutants were analyzed in transwell chemotaxis assays after 14 h,
with Ret ligands supplied below the membrane and CO supplied
above and below the membrane. Two-way
ANOVA compared to the
maximally migrating Ret
Frs+
⁄ Frs2 condition, ***P < 0.001 (n = 3).

(B) SK-N-MC cells were treated with anisomycin and CO as indi-
cated, and subjected to the comet assay. (C) Quantification of (B).
The cell-rescuing effect of Ret ligands against the apoptosis-induc-
ing agent anisomycin was measured by quantifying apoptosis ⁄ DNA
damage expressed as comet-tail momentum. Two-way
ANOVA
compared to the maximally rescuing Ret
Shc+
⁄ Shc condition,
**P < 0.01, *P < 0.05 (n = 3). Scale bar in (B) = 50 lm.
T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts
FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2061
phatidylinositol-anchored GFRa1 increase Ret distri-
bution to rafts [13]. In the same study, Frs2 and Shc
adaptor engagement with Tyr1062 was found to occur
predominantly within and outside rafts, respectively.
We report here that PTB adaptor engagement is criti-
cal for Ret to localize to lipid rafts, suggesting that
upon ligand engagement, the PTB adaptor association
results in a movement of Ret from nonraft to raft
membrane regions. Consistently, phosphorylated ERK
p42,44 MAPK signaling by a MEN2a form of Ret,
which signals even in the absence of ligands and
GFRa1, was more sensitive to disruption of choles-
terol-rich membranes by CO upon Frs2 as compared
to Shc recruitment. Our data also suggest that recruit-
ment of Ret to rafts by PTB adaptors results in dis-
tinct signal transduction patterns. The biochemical
integrity of Ret signaling depends on undisrupted
lipid-ordered domains when Ret assembles together

with adaptors localizing to rafts, but less so when the
complex is localized outside rafts. Furthermore, a
modified version of the Shc adaptor that localizes to
lipid rafts in a similar way to Frs2 results in signaling
resembling Frs2 recruitment by Ret.
Our results show that Ret resides largely in DRM
fractions both in cell lines and in vivo in the chick
spinal cord. This localization was critically dependent
on Tyr1062 and its interaction with Frs2 and Shc,
because eliminating interactions at this site resulted in
a clear loss of Ret in the DRM fraction and an
increase in the SUP fraction. Cyclodextrins such as
MCD effectively remove cholesterol from the plasma
membrane [27]. This property has led to the extensive
use of MCD to study the function of lipid rafts, which
are membrane microdomains whose integrity depends
on the presence of cholesterol. In this article, we show
that cholesterol depletion by MCD results in a loss of
both Frs2- and Shc-induced increases of Ret in the
DRM fraction.
Analysis of the DRM and SUP fractions suggested
that both Shc and Frs2 reside largely in the DRM
fraction, and that signaling from Ret by means of Shc
and Frs2 might be initiated from within lipid raft
membrane compartments. However, with the use of a
more sensitive density-dependent fractionation, it was
clear that Ret associated with Frs2 and Shc resides in
different membrane compartments, with Ret interac-
tions with Frs2 being more strongly associated with
the flottilin-containing fractions, which are believed to

include lipid rafts [28]. These experiments also showed
that, unlike Shc, which is present in very low amounts
in the Frs2-associated Ret fractions, Shc
MLS
is located
in both raft and nonraft fractions. Details of the lipid
distribution of the sphingolipid- and cholesterol-rich
lipid rafts are not well characterized. Clearly, there is a
great spatial and functional heterogeneity of these
membrane domains. Recently, it was found that
plasma membrane sphingomyelin-rich domains are
spatially distinct from ganglioside GM1-rich mem-
brane domains in Jurkat T cells, and may form distinct
and unique signaling platforms [29]. Distinct cellular
localization of Shc and Frs2 with Ret ⁄ Frs2 but not
Ret ⁄ Shc localized to GM1-rich lipid rafts was also
confirmed using CTB labeling. In this experiment, a
clear colocalization of Ret
eGFP
to lipid rafts in the
presence of Frs2 but not Shc was evident.
We noticed a high baseline activity without ligand in
Frs2 conditions throughout our study. This is consis-
tent with previous results on ERK MAPK and on
other downstream effector proteins activated via Frs2,
and is most likely due to the sustained interaction of
Frs2 with Ret and also other RTKs [4,30]. In a recent
study, we have shown that selective interaction of Ret
with Shc results in the activation of AKT to a much
greater extent than when Ret signals by recruitment of

Frs2, and conversely that, unlike Shc signaling, Frs2
signaling leads to ERK p42,44 MAPK activation at
high levels. Interestingly, the difference in signaling was
reflected not only by more robust activation, but also
by a significantly sustained activation from 5 min to at
least 12 h. Signaling by Ret via Shc activates ERK to a
lesser degree, and peaks at about 30 min [4]. Several
lines of evidence suggest that the sustained activation
of ERK is dependent on the raft context rather than it
being PTB adaptor-specific. Signaling via Frs2 after
disruption of the rafts by CO resulted in a marked
attenuation ERK MAPK signaling, with a duration of
minutes instead of hours. Furthermore, recruitment of
Shc to lipid rafts by introduction of the Ras membrane
localization signal resulted in elevated and sustained
ERK p42,44 MAPK activation, similar to that seen for
Frs2. Whereas activation of ERK p42,44 MAPK by
the normal Shc adaptor was less attenuated by lipid
raft disruption, ERK p42,44 MAPK activation by
Shc
MLS
was dependent on intact rafts, and when the
rafts were disrupted, ERK p42,44 MAPK activation
was almost completely absent. On the basis of these
results, we conclude that sustained ERK p42,44
MAPK signaling by Ret depends on intact lipid rafts.
It is interesting to note from our in vivo data result-
ing from the chick experiments that Shc activates Akt
almost exclusively outside of rafts, with there being
little ERK p42,44 MAPK activation either within or

outside of rafts, as seen by its partitioning into the
DRM fraction. In contrast, whereas Shc
MLS
activates
Akt mostly outside of rafts, its activation of ERK
p42,44 MAPK was almost exclusively within the
Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.
2062 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS
DRM raft fraction. Frs2 activation of Akt was overall
very weak, whereas ERK p42,44 MAPK activation
was strong in both the DRM fraction and in the non-
raft SUP fraction. Because our density-dependent
fractionation and colabeling of Ret with lipid rafts in
cellular staining both suggest that Ret associated with
Frs2 and Shc resides in different membrane compart-
ments, our results suggest that Akt activation may
result largely from nonraft activation of Shc by Ret.
Unlike Akt, ERK p42,44 MAPK, which is activated
downstream of Frs2, appeared to localize both to raft
membranes and to nonraft membrane fractions.
Because Ret association with Frs2 is almost exclusive
to the lipid rafts, this suggests that ERK MAPK par-
titioning into nonraft fractions is presumably initiated
from within the raft. It is interesting that ERK p42,44
MAPK signaling resulting from Shc
MLS
association
with Ret takes place exclusively in the DRM fraction,
suggesting that, unlike signaling downstream of Frs2,
components of the ERK MAPK signaling pathway

stay associated with the raft-localized Shc
MLS
also
after activation.
Both Shc and Frs2 have been implicated in cell
migration, and we cannot exclude a role also for
PI3K ⁄ Akt signaling from Ret. This pathway has previ-
ously been implicated in such cellular functions
mediated by Ret in, for example, kidney epithelial
Madin–Darby canine kidney cells [31]. However, our
findings suggest that PI3K⁄ Akt signaling is not suffi-
cient for cell migration by itself in the absence of
MAPK ERK signaling via Frs2. Directional signaling
onto RTKs at the leading edge appears to be critical
for chemotaxis, and defines the direction of actin poly-
merization and subsequent cell migration. It is not
clear how the cells measure gradients of RTK ligands
resulting in gradients of receptor activation along the
surface that are translated into polarization of the
cytoskeleton, extension of cell processes, and eventu-
ally translocation of the cell body. Our results show
that Ret-mediated chemotaxis is critically dependent
on lipid rafts, whereas cell survival signaling via Shc is
not significantly affected by CO. This is consistent with
the conclusion that Shc activation by Ret may take
place in nonraft membrane regions, similar to what
happens with many other tyrosine kinase receptors
[13,32], whereas Frs2, which is necessary for Ret-elic-
ited directional migration, is activated in raft-like foci
[11,18]. Our results do not allow us to distinguish

whether the effects of CO on migration result directly
from the loss of ERK signaling or from the physical
localization of Ret to the growing axons, as seen by
the Ret colocalization with CTB. As for other RTKs,
Ret-stimulated migration is dependent on ERK signal-
ing, as blocking of this pathway prevents Ret-induced
migration [11,33]. However, this does not exclude the
possibility that that a raft-dependent localization of
Ret to filopodia ⁄ lamellipodia may also be important
for directional migration and axonal extension. Consis-
tent with this hypothesis are data showing that local
stimulation of cells expressing the epidermal growth fac-
tor receptor with a bead soaked in epidermal growth
factor leads to ERK activation spreading throughout
the cell, whereas actin polymerization remains local [34].
Our results open the possibility that Frs2-dependent
recruitment of Ret receptors to lipid rafts upon ligand
engagement may participate in increasing receptor levels
in the direction of increasing ligand concentrations. This
may provide a molecular mechanism for cellular amplifi-
cation of ligand gradients that could play important
roles in directed cell migration.
Experimental procedures
Cell culture, DNA constructs, and mutagenesis
All Ret mutants were harbored and expressed in PJ7W plas-
mids and subcloned into peGFP vectors (Clonetech Inc.,
Mountain View, CA, USA) to make fluorescent constructs,
as described previously [11]. SK-N-MC cells were main-
tained in DMEM supplemented with 10% fetal bovine
serum, 2% horse serum and 1 mm glutamine. Starvations

were done in DMEM containing 0.5% total serum. All
ligand stimulations were performed for 30 min unless stated
otherwise, using 50 ngÆmL
)1
recombinant human GDNF
and 100 ngÆmL
)1
recombinant human GFRa1 ⁄ FC chimera
(R&D Systems, Minneapolis, MN, USA). CO (Sigma,
Munich, Germany) was used at 8 mm, as previously
described [21]; specifically, cultured cells were incubated with
CO at 1.8 UÆmL
)1
for 1 h prior to and during ligand stimu-
lation. Anisomycin (Sigma) was applied to cells 30 min
before ligand application at a final concentration of
12 lgÆmL
)1
, and cells were incubated for another 2 h before
examination. Transfections were performed using Lipofecta-
mine LTX (Invitrogen, Karlsruhe, Germany), according to
the manufacturer’s instructions. Growth medium was
replaced approximately 7 h after transfection. Transfection
efficiency was continuously monitored by eGFP fluores-
cence.
Antibodies and reagents
Antibodies against Ret (Ret H-300), and phosphotyrosine
(PY99) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Antibodies against HA tags were
from BD Biosciences (CA, USA). Antibodies against Frs2

were from Sigma. Antibodies against flotillin-1 were from
Transduction Labs (Lexington, KY, USA). Antibodies
T. K. Lundgren et al. Ret PTB-adaptor translocation to rafts
FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS 2063
against TfR were from Zymed (San Francisco, CA, USA).
Antibodies against Akt, phosphorylated Akt Ser473,
p44 ⁄ 42 MAPK and phosphorylated p44 ⁄ 42 MAPK were
from Cell Signalling (Hitchin, UK). Alexa-546-conjugated
GM1-CTB was from Molecular Probes (Leiden, the Neth-
erlands), and was used according to the manufacturer’s
instructions. After staining with GM1-CTB, cells were
fixed in 4% paraformaldehyde and examined on
Zeiss LSM 5 exciter microscopes with axiovision software
(Zeiss, Karlsruhe, Germany).
Immunoblotting and SDS ⁄ PAGE
Cells were lysed in Laemmli buffer for phospho-protein
blots. Precipitated proteins from fractionations were eluted
by boiling in Laemmli buffer. Proteins were fractionated on
polyacrylamide gels and immobilized on PVDF membranes
(GE Healthcare, Uppsala, Sweden). Western blot detection
was carried out by the enhanced chemiluminescence method
(GE Healthcare), according to standard darkroom proce-
dures. Quantifications were done using imagej software
( />Preparation of DRMs and lipid raft fractionation
DRM and SUP fractions were prepared by harvesting cells
in 1% Triton X-100 buffer for SK-N-MC cells and 0.9%
Triton X-100 buffer for chick spinal cord cells, as previously
described [14]. Detergent-free fractionation of chick spinal
cord was done by electroporation of E2 (approximately
stage 11) chicken embryos in ovo. Electroporator settings

were as described previously [35]. The embryos were incu-
bated until E5. At E5, positively transfected spinal cord and
DRG segments were dissected out and pooled to equal
amounts of input (eight embryos were routinely needed per
experiment and condition). After dissociation into single
cells and stimuli with Ret ligands for 30 min, cells were har-
vested in detergent-free buffer and subjected to ultracentrif-
ugation in 0–20% OptiPrep density gradients as previously
described [24]. Seven fractions of 0.68 mL each were taken
from columns after centrifugation, and concentrated protein
from each fraction was immunoblotted against proteins as
indicated in the figure legends.
Vertical cell migration assays
Cell migration response towards Ret ligands was assessed
using transwell cell culture inserts (Falcon, Europe) with
12 lm pores. Transfected cells were seeded (1–5 · 10
5
cells)
in the chambers and allowed to migrate towards ligands, as
indicated in the figures. Quantification was done under a
microscope by counting cells in three visual fields after
removal of stationary cells on the upper side of membranes
using a cotton-tip.
Comet assay
SK-N-MC cells were treated as indicated in the figures.
Cells were spun down in ice-cold NaCl ⁄ P
i
and immediately
combined with low-melt agarose to a final concentration of
0.9% agarose. The samples were immobilized on precoated

agarose slides (Trevigen, Gaithersburg, MD, USA) and
allowed to settle for 20 min at 4 °C. The slides were
immersed in lysis solution (Trevigen) for 55 min at 4 °C.
Slides were submersed in alkaline solution (NaOH ⁄
EDTA ⁄ H
2
O) with a pH > 13 for 50 min at room tempera-
ture. After equilibration in 1 · TBE buffer three times for
5 min each, the slides were placed in a horizontal electro-
phoresis chamber with a voltage of 0.9 VÆcm
)1
for 14 min.
DNA was fixed in MeOH and EtOH and dried at room
temperature. Examination was performed by staining slides
with SYBR-green, and images were captured at 200· mag-
nification. Quantification was done on digital images of 25
random cells per condition using tritek comet scoring
software bridged to Mac OSX software via a Parallel Win-
dows emulator.
Acknowledgements
This work was supported by the Swedish Cancer Soci-
ety, the Swedish Foundation for Child Cancer, the
Swedish Medical Research Council and the Swedish
Foundation for Strategic Research (CEDB and
DBRM grants) for P. Ernfors, the Karolinska Institute
MD-PhD programme for T. K. Lundgren and the
LERU Graduate Programme for A. Stenqvist.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Experimental procedure for exogenous DNA
expression in embryonal chick spinal cord.
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from
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sponding author for the article.
Ret PTB-adaptor translocation to rafts T. K. Lundgren et al.
2066 FEBS Journal 275 (2008) 2055–2066 ª 2008 The Authors Journal compilation ª 2008 FEBS