Báo cáo khoa học: Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells docx

Data concerning the structural and mechanistic aspects required for Keywords epidermal growth factor receptor; ErbB2; GM3; HC11 cells; lipid rafts Correspondence M.. In addition, analysi

rafts in mammary epithelial HC11 cells

Elena Sottocornola1, Roberta Misasi2, Vincenzo Mattei2,3, Laura Ciarlo2, Roberto Gradini2,4,

Tina Garofalo2,3, Bruno Berra1, Irma Colombo1and Maurizio Sorice2,3

1 Institute of General Physiology and Biological Chemistry, University of Milan, Italy

2 Department of Experimental Medicine and Pathology, University of Rome ‘La Sapienza’, Italy

3 Laboratory of Experimental Medicine and Environmental Pathology, Rieti, Italy

4 INM Neuromed, Pozzilli, Italy

Gangliosides, ubiquitous components of eukaryotic

membranes, are not uniformly distributed within the

outer leaflet of the plasma membrane, but segregate,

together with cholesterol,

glycosylphosphatidylinositol-anchored proteins and signaling-transduction

mole-cules, into unique, more or less stable clusters or

microdomains called as ‘glycosphingolipid-enriched

microdomains’ (GEM), which contribute to membrane

structure, organization and, more importantly,

func-tion Indeed, GEM are viewed as a dynamic and

pref-erential association of sphingolipids and cholesterol

into moving platforms, termed lipid rafts, which can

selectively incorporate or exclude proteins [1] and

con-tribute to lipid-mediated protein trafficking and signal

transduction [2]

The growth factor receptor tyrosine kinase ErbB2

is a 185 kDa transmembrane glycoprotein intensively investigated because of its important role in normal mammary gland development and in the deregulation

of growth displayed by cancer cells, including breast and ovarian tumor cells [3,4] A ligand which binds directly and specifically to ErbB2 has not been identi-fied to date, but it can be activated in trans by ligands binding to epidermal growth factor receptor (EGFR), such as epidermal growth factor (EGF) and transform-ing growth factor a1 [3] Indeed, in cells coexpresstransform-ing both ErbB2 and EGFR, EGF preferentially stimulates the formation of ErbB2⁄ EGFR heterodimers in which cross-phosphorylation occurs [5,6] Data concerning the structural and mechanistic aspects required for

Keywords

epidermal growth factor receptor; ErbB2;

GM3; HC11 cells; lipid rafts

Correspondence

M Sorice, Department of Experimental

Medicine and Pathology, University of Rome

‘La Sapienza’, viale Regina Elena 324,

Rome 00161, Italy

Fax: +39 6 445 4820

Tel: +39 6 499 72675

E-mail: maurizio.sorice@uniroma1.it

(Received 24 December 2005, revised 15

February 2006, accepted 27 February 2006)

doi:10.1111/j.1742-4658.2006.05203.x

We analyzed the role of gangliosides in the association of the ErbB2 recep-tor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells Scanning confocal microscopy experiments revealed a strict ErbB2– GM3 colocalization in wild-type cells In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epi-dermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched

in lipid rafts together with gangliosides Blocking of endogenous ganglioside synthesis by (+⁄ –)-threo-1-phenyl-2-decanoylamino-3-morpho-lino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells The results point out the key role of ganglioside GM3

in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts

Abbreviations

[D]-PDMP, (+ ⁄ –)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; GEM, glycosphingolipid-enriched microdomains; HRP, horseradish peroxidase;

Rf, retardation factor; RTK, receptor tyrosine-kinase; TX-100, Triton X-100.

EGF-dependent ‘trans activation’ of EGFR and ErbB2

in ErbB2⁄ EGFR heterodimers, as well as data

con-cerning the overall effects induced by changes in the

relative expression levels of EGFR and ErbB2, are

growing rapidly and several lines of evidence have

shown that ErbB2 is associated with lipid

microdo-mains [7–10] By contrast, the role of cell-membrane

components, such as gangliosides, for determining the

plasma membrane distribution and relative densities of

receptors has not yet been investigated thoroughly

Recently, we reported the first evidence that

ganglio-side depletion associates with increased levels of the

activated ErbB2 and EGFR, whereas increased

gan-glioside GM3 content correlates with the

downregula-tion of both receptors [11]

In this study, we provide evidence of ErbB2–GM3

association on the plasma membrane of mouse

mam-mary epithelial HC11 cells and demonstrate that

gan-gliosides, and particularly GM3, play a key role in

retaining ErbB2 and proteins correlated with its

signal-transduction pathway in lipid rafts

Results

ErbB2–GM3 colocalization in mammary epithelial

HC11 cells

In order to evaluate ErbB2 distribution and its

pos-sible association with GM3, we performed

immunoflu-orescence labeling, followed by scanning confocal

microscopy analysis Cells were labeled with

anti-ErbB2 polyclonal serum and then with anti-GM3

monoclonal serum

Analysis of GM3 expression and distribution in

untreated HC11 cells (Fig 1A) revealed that GM3

staining appeared uneven over the cell surface, similar

to that seen on ErbB2 molecule fluorescence A

merged image of the two stainings clearly revealed

orange areas, resulting from the overlap of green and

red fluorescence, which corresponded to colocalization

areas

To analyze the effect of ganglioside depletion on

the association of ErbB2 with ganglioside GM3, we

preliminary treated the cells with (+⁄

–)-threo-1-phe-nyl-2-decanoylamino-3-morpholino-1-propanol

hydro-chloride ([D]-PDMP), which blocks endogenous

ganglioside biosynthesis, resulting in the almost

com-plete disappearance of all ganglioside species [11]

Vir-tually no staining was observed in cells labeled with

anti-GM3 serum (Fig 1B) The lack of

immunolabe-ling demonstrates the effect of [D]-PDMP on the

depletion of gangliosides and the specificity of the

anti-GM3 serum The distribution of ErbB2 appeared more

diffuse compared with control untreated cells No colocalization areas between GM3 and ErbB2 were detected

By contrast, overlain areas were reverted when exo-genous GM3 was added to ganglioside-depleted HC11 cells (Fig 1C)

Scatter-plot diagrams showed how the dual labels are colocalized Figure 1D shows a colocalization area that is evident in untreated HC11 cells In cells treated

A

B

C

D

Fig 1 Scanning confocal microscopy analysis of GM3–ErbB2 association on HC11 cells Cells were fixed with 4% paraformalde-hyde, permeabilized with 0.5% TX-100 and then incubated with anti-ErbB2 polyclonal serum, followed by the addition of FITC-conju-gated goat anti-(rabbit IgG) serum Cells were then labeled with anti-GM3 monoclonal serum (GMR6), followed by the addition of Texas red-conjugated anti-(mouse IgM) serum Merge: dual immuno-labeling of anti-GM3 (red) and anti-ErbB2 (green) Colocalization areas are stained in orange (A) Untreated HC11 cells (B) HC11 cells treated with 30 l M [D]-PDMP for 5 days (C) HC11 cells

treat-ed with 30 l M [D]-PDMP for 5 days and then with 125 l M GM3 for 5 min (D) Two-dimensional scatter plot analysis of the dual-labeled fluorochromes (pseudocolor) GM3–ErbB2 Diagrams show the pixel intensity distribution of a dual-channel section The x-axis represents intensity from the red channel; the y-axis represents intensity from the green channel; a colocalization area is evident in untreated HC11 cells In cells treated with [D]-PDMP and then with GM3 a major colocalization index is evident because the blue area

is larger and more directed towards the diagonal line Figure shows analysis of about 40 cells.

with [D]-PDMP and then with GM3 a major

colocali-zation index is evident, because the blue area is larger

and more directed towards the diagonal line

Effect of ganglioside depletion on cholesterol and

caveolin-1 distribution in HC11 cells

In order to verify that [D]-PDMP does not interfere

with the membrane distribution of known lipid-raft

markers, we investigated the cholesterol and caveolin-1

content of membrane fractions obtained by a 5–30%

linear sucrose gradient from HC11 cells in the absence

or presence of treatment with [D]-PDMP

As expected, cholesterol was present mainly in

frac-tions 4–6 (Fig 2A), which, under our experimental

conditions, correspond to lipid-raft fractions About

90% of the cholesterol content of the total cell extract

was recovered in fractions 4–6 Virtually the same

cho-lesterol distribution was observed in cells treated with

[D]-PDMP

Similar findings were found in western blot analysis

of caveolin-1 distribution in the sucrose gradient

frac-tions The analysis was performed loading fraction

samples by volume Because the protein content of

Triton X-100 (TX-100)-soluble fractions 10 and 11 was higher than that of TX-100-insoluble fractions 4–6 (not shown) [12], we can observe that caveolin-1 was consistently enriched in TX-100-insoluble fractions

in untreated, as well as in [D]-PDMP-treated, cells (Fig 2B)

ErbB2 preferential association with lipid-raft fractions in HC11 cells

To evaluate the distribution of ErbB2 in raft fractions

of HC11 cells, treated or not with [D]-PDMP, EGF or [D]-PDMP and EGF, all fractions obtained by sucrose gradient were analyzed by western blot (Fig 3) The results revealed that in non-EGF-stimulated cells ErbB2 was present mainly in fractions 5 and 6, but also in fractions 7–11 (Fig 3A), indicating that ErbB2

is preferentially present in raft fractions EGF stimulat-ion did not seem to appreciably modify this distribut-ion (Fig 3B)

Interestingly, [D]-PDMP treatment induced a drastic cell-surface redistribution of ErbB2 (Fig 3C) Indeed, the receptor became completely Triton soluble and was present exclusively in fractions 10 and 11 Identical profile redistribution of ErbB2 was also evident in

A

B

Fig 2 (A) Densitometric analysis of cholesterol content in HC11

sucrose-gradient membrane fractions HC11 cells, either untreated

or treated with 30 l M [D]-PDMP for 5 days, were lyzed in lysis

buf-fer and the supernatant (postnuclear fraction) was subjected to

sucrose density gradient separation After centrifugation, the

gradi-ent was fractionated and free cholesterol of each fraction was

anal-yzed by TLC and quantified by densitometric scanning analysis (B)

western blot analysis of caveolin-1 distribution in HC11 sucrose

gradient membrane fractions Lysates from HC11 cells, either

untreated or treated with 30 l M [D]-PDMP for 5 days, were

anal-yzed by western blot with anti-(caveolin-1) polyclonal serum,

fol-lowed by incubation with an HRP-conjugated anti-(rabbit IgG)

serum, as a secondary antibody.

A

B

C

D

E

Fig 3 ErbB2 distribution in HC11 sucrose gradient membrane fract-ions HC11 cells were lyzed in lysis buffer and the supernatant (postnuclear fraction) was subjected to sucrose density gradient separation After centrifugation, the gradient was fractionated and each fraction was analyzed by western blotting with anti-ErbB2 polyclonal serum, followed by incubation with a HRP-conjugated anti-(rabbit IgG) serum, as a secondary antibody (A) Untreated HC11 cells (B) HC11 cells treated with 10 n M EGF for 15 min (C) HC11 cells treated with 30 l M [D]-PDMP for 5 days (D) HC11 cells treated with 30 l M [D]-PDMP for 5 days and then with 10 n M EGF for 15 min (E) HC11 cells treated with 30 l M [D]-PDMP for 5 days and then with 125 l M GM3 for 5 min.

HC11 cells treated with [D]-PDMP and EGF

(Fig 3D), indicating that EGF is not determinant in

defining the retention of ErbB2 into the lipid rafts

The effect of [D]-PDMP treatment was partially

abolished by addition of exogenous GM3 to

ganglio-side-depleted HC11 cells In fact, after [D]-PDMP

incubation, followed by GM3 treatment, a significant

proportion of ErbB2 returned to fractions 4 and 5

(Fig 3E)

In order to better clarify the functional role of the

association between ErbB2 and lipid rafts, we analyzed

the distribution of phospho-ErbB2 in sucrose-gradient

fractions obtained from HC11 cells in the absence or

presence of treatment with EGF, [D]-PDMP and

[D]-PDMP⁄ GM3 Although, as expected, virtually no

phosphorylated ErbB2 was detected in all the fractions

obtained from control cells (Fig 4A), after triggering

with EGF (Fig 4B), phosphorylated ErbB2 was found

in both the TX-100-insoluble fractions and the

TX-100-soluble fractions Interestingly, in cells treated

with [D]-PDMP and EGF (Fig 4D) a band

corres-ponding to phosphorylated ErbB2 was detected in

fractions 10 and 11, whereas in ganglioside-depleted

cells and cells treated with [D]-PDMP and GM3 no

ErbB2 phosphorylation was observed (Fig 4C,E

respectively) These findings support the view that

GM3 is mainly involved in retaining ErbB2 in

lipid-raft domains, but that it is not involved in ErbB2

phosphorylation

EGFR and Shc-p66 preferential association with lipid-raft fractions in HC11 cells

Because in cells coexpressing both ErbB2 and EGFR,

as is the case of HC11 cells, the two proteins strictly interact and EGF preferentially stimulates the forma-tion of ErbB2⁄ EGFR heterodimers [5,6], in the same raft fractions of HC11 cells analyzed previously, we also examined the distribution of EGFR EGFR was present in fractions 5 and 6, but also in fractions 7–11 (Fig 5A) In EGF-stimulated cells, movement of the receptor to TX-100-soluble fractions was observed (Fig 5B) After [D]-PDMP treatment, the receptor became completely Triton soluble and was present exclusively

in fractions 10 and 11 (Fig 5C) After [D]-PDMP incu-bation, followed by GM3 treatment, a proportion of EGFR returned to fractions 4–6 (Fig 5D)

Because the Shc proteins are translocated into the lipid rafts of the plasma membrane after phosphoryla-tion by ErbB2 and EGFR receptors [13], we also ana-lyzed the distribution of Shc-p66 in the same fractions (Fig 6) The results revealed that in control cells only

a small amount of Shc-p66 was detectable in fractions

5 and 6, corresponding to lipid rafts; a higher amount

of Shc-p66 was detected in TX-100-soluble fractions (mainly 10 and 11) (Fig 6A) In cells treated with EGF the higher amount of Shc-p66 was detected in the Triton-insoluble fractions (Fig 6B), indicating that

A

B

C

D

E

Fig 4 Analysis of the distribution of phosphorylated ErbB2 in

HC11 sucrose gradient membrane fractions HC11 cells were lyzed

in lysis buffer and the supernatant (postnuclear fraction) was

sub-jected to sucrose density gradient separation After centrifugation,

the gradient was fractionated and each fraction was analyzed by

western blotting with anti-(phospho-ErbB2) polyclonal serum,

fol-lowed by incubation with an HRP-conjugated anti-(rabbit IgG)

serum, as a secondary antibody (A) Untreated HC11 cells (B)

HC11 cells treated with 10 n M EGF for 15 min (C) HC11 cells

treat-ed with 30 l M [D]-PDMP for 5 days (D) HC11 cells treated with

30 l M [D]-PDMP for 5 days and then with 10 n M EGF for 15 min.

(E) HC11 cells treated with 30 l M [D]-PDMP for 5 days and then

with 125 l M GM3 for 5 min.

A

B

C

D

Fig 5 EGFR distribution in HC11 sucrose gradient membrane fract-ions HC11 cells were lyzed in lysis buffer and the supernatant (postnuclear fraction) was subjected to sucrose density gradient separation After centrifugation, the gradient was fractionated and each gradient fraction analyzed by western blotting with anti-EGFR polyclonal serum, followed by incubation with an HRP-conjugated anti-(rabbit IgG), as a secondary antibody (A) Untreated HC11 cells (B) HC11 cells treated with 10 n M EGF for 15 min (C) HC11 cells treated with 30 l M [D]-PDMP for 5 days (D) HC11 cells treated with 30 l M [D]-PDMP for 5 days and then with 125 l M GM3 for

5 min.

EGF induced Shc-p66 recruitment to lipid rafts.

Importantly, [D]-PDMP treatment caused a significant

modification of its membrane distribution, inducing,

as for ErbB2 and EGFR, an almost complete shift

of Shc-p66 to Triton-soluble fractions 10 and 11

(Fig 6C), which was partially reverted in

ganglioside-depleted HC11 cells by the addition of GM3 and EGF

(Fig 6D)

Association of ErbB2 with Shc-p66

To verify whether Shc-p66 may interact with activated

ErbB2, lysates from TX-100-insoluble fractions

tions 4–6 pooled) and TX-100-soluble fractions (frac-tions 10 and 11 pooled), obtained from EGF-treated and untreated cells, were immunoprecipitated with the anti-Shc-p66 Ab, followed by protein G–acrylic beads The results in Fig 7A show that, in control unsti-mulated cells, ErbB2 was slightly associated with Shc-p66 mainly in TX-100 soluble fractions By contrast, after triggering with EGF, a significant proportion of Shc-p66 also became associated with ErbB2 in the TX-100-insoluble fractions, suggesting that, after EGF stimulation, Shc-p66 may associate with activated ErbB2 within lipid rafts

No bands were detected after EGF stimulation in control immunoprecipitation experiments with a rabbit IgG having irrelevant specificity Immunoprecipitation was verified by western blot (Fig 7B)

Profile distribution of raft markers in plasma membrane fractions of HC11 cells

Because gangliosides are well-known markers of lipid-raft domains, we examined the ganglioside profile of sucrose-gradient fractions from HC11 cells Ganglio-sides were extracted in chloroform⁄ methanol ⁄ water and separated by HPTLC Resorcinol-positive bands were identified on the basis of their HPTLC mobility, compared with standard reference molecules Three main resorcinol positive bands, having a retardation factor (Rf) analogous to GM3, GM2 (the most prom-inent) and GD1a, respectively, were detected (Fig 8A) The observation that the main band comi-grates with GM2 is not surprising, because this mole-cule is the main ganglioside constituent in these cells,

as reported previously [11] All the ganglioside bands were exclusively detectable in fractions 4–6, which, under our experimental conditions, correspond to lipid rafts

A

B

C

D

Fig 6 Shc-p66 distribution in HC11 sucrose gradient membrane

fractions HC11 cells were lyzed in lysis buffer and the supernatant

(postnuclear fraction) was subjected to sucrose density gradient

separation After centrifugation, the gradient was fractionated and

each gradient fraction was analyzed by western blotting with

anti-Shc polyclonal serum, followed by incubation with an

HRP-conju-gated anti-(rabbit IgG), as a secondary antibody (A) Untreated

HC11 cells (B) HC11 cells treated with 10 n M EGF for 15 min (C)

HC11 cells treated with 30 l M [D]-PDMP for 5 days (D) HC11 cells

treated with 30 l M [D]-PDMP for 5 days and then with 125 l M

GM3 for 5 min plus 10 n M EGF for 15 min.

A

B

Fig 7 HC11 cells, treated or not with EGF (10 n M for 5 min at 37
C), were lyzed in lysis buffer and the supernatant (postnuclear fraction) was subjected to sucrose density gradient separation After centrifugation, the gradient was fractionated and coimmunoprecipitation of Shc-p66 with ErbB2 was performed in TX-100-insoluble fractions (4–6 pooled together) or in TX-100-soluble fractions (10 and 11 pooled together) with an anti-Shc-p66 specific serum The immunoprecipitates were analyzed by western blot with anti-ErbB2 (A) and anti-Shc-p66 (B) sera.

To further confirm the correct sucrose density

gradi-ent separation of TX-100-insoluble and TX-100-soluble

fractions, we also analyzed the distribution pattern of

the known raft protein flotillin-2

The analysis was performed loading fraction samples

by volume Because the protein content of

TX-100-soluble fractions 10 and 11 was much higher than that

of TX-100-insoluble fractions 4–6 (not shown) [12], we

can observe that flotillin-2 was consistently enriched in

rafts (TX-100-insoluble fractions) (Fig 8B)

Discussion

In this study we analyzed primarily the localization of

ErbB2 in lipid rafts of mouse mammary epithelial

HC11 cells Laser scanning confocal microscopy

obser-vations revealed colocalization areas between GM3, a

well-known marker of lipid rafts [14], and ErbB2 This

finding is in agreement with and extends previous

observations about the surface distribution of ErbB2,

which is mostly excluded from clathrin-coated pits on

the cell plasma membrane [15], and it gives further

support to the conclusions of Nagy et al [9], who

hypothesized the association of ErbB proteins (ErbB2

and ErbB3) with these microdomains by quantitative

fluorescence microscopy in SKBR-3 breast cancer cells

In addition, in CHO-K1 cells, expression of GD3

affected, to some extent, the plasma membrane

distri-bution of endogenous ErbB2 [16] The preferential

distribution of ErbB2 in lipid rafts was clearly

demon-strated by our membrane fractionation experiments,

which also revealed that EGF is not able to modify

the receptor localization However, analysis revealed

that ErbB2 is not exclusively associated with the raft

fractions This finding is consistent with the observa-tions of Hommelgaard et al [10], prompting us to hypothesize that ErbB2 is in dynamic equilibrium with lipid rafts in the membrane protrusions so that, at a single time point, only a fraction of ErbB2 is directly interacting with the raft gangliosides This transient ErbB2–gangliosides interaction could potentially regu-late the function of ErbB2 (heterodimerization, signa-ling and metabolic fate) [10]

We therefore analyzed the role of gangliosides in the association of ErbB2 with lipid rafts in mammary epi-thelial HC11 cells The key role played by gangliosides

in defining the distribution of ErbB2 into signaling specialized plasma membrane domains was shown by treatment of HC11 cells with [D]-PDMP Ganglioside depletion, due to the inhibition of endogenous ganglio-side synthesis, was shown to have striking effects upon the plasma membrane localization of ErbB2 Indeed, ErbB2 underwent complete redistribution within the high-density TX-100-soluble fractions of the plasma membrane, indicating, by a novel approach, that gan-gliosides play a key role in the retention of this protein

in lipid rafts These findings are strongly supported by the observation that [D]-PDMP does not destroy the organization of lipid rafts, because cholesterol as well

as caveolin-1 could still be detected in TX-100-insol-uble fractions after treatment with [D]-PDMP In addi-tion, treatment of ganglioside-depleted HC11 cells with exogenous ganglioside GM3 induced the return of a significant proportion of ErbB2 in raft fractions How-ever, both ErbB2 localized in TX-100-soluble and TX-100-insoluble fractions were phosphorylated These data, together with previous results [11], strengthen the view that GM3 plays an important role in ErbB2 membrane localization but not in its phosphorylation, suggesting that gangliosides might influence the signa-ling-transduction pathways after EGF stimulation by compartmentalizing the receptor in different membrane domains

Because in cells coexpressing ErbB2 and EGFR, like HC11 cells, ligand stimulation largely favors the for-mation of ErbB2⁄ EGFR heterodimers [5,6], EGFR distribution in the same plasma membrane fractions was also analyzed Conflicting results have been repor-ted in the literature on the presence of EGFR within lipid rafts [7,17–21] It may depend on the cell type, the use of different detergents [17,18] and, mainly, the ganglioside composition of the cells The latter may also be influenced by cell cycle and⁄ or cell density [19] Zurita et al [16] found EGFR mainly in TX-100-soluble fractions in CHO-K1 cells, although they observed that EGFR and GD3 colocalized on the cell surface In the same vein, Wang et al demonstrated

GM3

GM1

GD1a GD1b GT1b

1

Flotillin

45 kDa

A

B

Fig 8 Ganglioside distribution in HC11 sucrose gradient membrane

fractions HC11 cells were lyzed in lysis buffer and the supernatant

(postnuclear fraction) was subjected to sucrose density gradient

separation After centrifugation, 11 gradient fractions were

recov-ered (A) Gangliosides were extracted in chloroform ⁄ methanol ⁄

water from each fraction and analyzed by HPTLC St: standard

gan-gliosides GM3, GM2, GM1, GD1a, GD1b, GT1b (B) Western

blot-ting with anti-(flotillin-2) polyclonal serum in the same fractions.

that endogenous overexpression of GM3 promotes

co-immunoprecipitation of GM3 with EGFR [20]

How-ever, it has been shown that GM3 can specifically

interact with the purified recombinant extracellular

domain of EGFR [22] and that this tyrosine kinase

receptor contains a structural domain with targeting

information for lipid domains [23] Our results

dis-played variations in the distribution profiles rather

similar to that of ErbB2 Indeed, in control cells,

EGFR is mainly enriched in TX-100-insoluble

fractions, whereas treatment with [D]-PDMP and

[D]-PDMP⁄ EGF shifts the receptor towards the

TX-100-soluble fractions, confirming the direct correlation

between the two receptors [3,5] However, after

stimu-lation with EGF, we observed a movement of EGFR

to Triton-soluble fractions, in agreement with previous

studies showing that EGFR is initially concentrated in

caveolae within lipid rafts, but rapidly moves out of

this membrane domain in response to EGF [7] The

inefficient movement of ErbB2 out of these

micro-domains may be related to its impaired internalization

by clathrin-coated pits [24]

ErbB receptors, and ErbB2 in particular, are able to

activate the ras⁄ MAP kinase signaling pathway via the

Shc proteins [25] In order to elucidate whether this

sig-nal transduction pathway triggered by ErbB2 may take

place inside rafts in HC11 cells, we investigated the

presence of Shc-p66 in raft fractions Our findings from

fractionation experiments showed a preferential

associ-ation of Shc-p66 with lipid rafts after EGF stimulassoci-ation

These data were also confirmed by

coimmunoprecipitat-ion experiments, in which a consistent proportcoimmunoprecipitat-ion of

Shc-p66 coimmunoprecipitates with ErbB2 in the

lipid-raft fractions, suggesting that actually Shc-p66 is

recruited by ErbB2 after triggering via EGF

In conclusion, we demonstrated a key role for

gan-gliosides in the association of ErbB2 with lipid rafts

in mammary epithelial HC11 cells This finding was

strongly supported by the observation that addition of

GM3 after [D]-PDMP treatment induced a marked

redistribution of ErbB2 and proteins correlated with

the its signal transduction pathway (i.e Shc-p66) to

Triton-insoluble fractions

Experimental procedures

Cell culture and treatments

Mouse mammary epithelial HC11 cells were a gift from

E Garattini (Institute for Pharmacological Research ‘M

Negri’, Milan, Italy) Cells were maintained in RPMI-1640

(Gibco-BRL, Life Technologies Italia srl, Italy),

supplemen-ted with 10% heat-inactivasupplemen-ted newborn bovine serum,

8 mm glutamine, 50 lgÆmL)1 gentamycin and 5 lgÆmL)1 insulin from bovine pancreas (Sigma, St Louis, MO), in a humidified 5% CO2atmosphere at 37
C

As described previously [11], total ganglioside depletion was obtained by treating cells for 5 days at 37
C with

30 lm [D]-PDMP (Sigma), a competitive inhibitor of gluco-sylceramide synthetase, resulting in ganglioside biosynthesis inhibition

Exogenous ganglioside GM3 treatment of ganglioside-depleted HC11 cells was performed by incubating the cells at

37
C for 5 min with 125 lm GM3 (Alexis, S Diego, CA), dissolved in routine medium without serum EGF stimulation was carried out by incubating the cells, treated or not with [D]-PDMP, with 10 nm EGF (Sigma) for 15 min at 37
C

Analysis of ErbB2–GM3 colocalization by scanning confocal microscopy

HC11 cells, treated or not with [D]-PDMP and GM3, were fixed in situ with 4% paraformaldehyde in NaCl⁄ Pi for

30 min at room temperature and then permeabilized with 0.5% TX-100 in NaCl⁄ Pifor 30 min at room temperature Cells were labeled with rabbit anti-ErbB2 polyclonal serum (C18, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h

at 4
C, followed by the addition (30 min at 4
C) of fluo-rescein isothiocyanate (FITC)-conjugated anti-(rabbit IgG) serum (Calbiochem, La Jolla CA) After three washes in NaCl⁄ Pi, cells were incubated with GMR6 anti-GM3 monoclonal serum (Seikagaku Corp., Chuo-ku, Tokyo, Japan) [26] for 1 h at 4
C, followed by three washes in NaCl⁄ Pi and the addition (30 min at 4
C) of Texas red-conjugated goat anti-(mouse IgM) serum (Sigma) In parallel experiments, cells were stained with anti-GM3 monoclonal serum before fixing the cells Alternatively, control experiments were performed omitting the monoclo-nal antibody from the immunolabeling procedure After washing as above, cells were mounted upside down onto a glass slide in 5 lL of glycerol⁄ Tris ⁄ HCl (6 : 4, v : v), pH 9.2

As a control, cells were mounted in glycerol⁄ NaCl ⁄ Pi(6 : 4,

v : v), pH 7.4 and the results were virtually the same The images were acquired using a high-resolution ·63 objective through a confocal laser scanning microscope Zeiss LSM

510 (Zeiss, Oberkochen, Germany) equipped with argon and HeNe ion lasers The green (FITC) and red (Texas Red) fluorophores were excited simultaneously at 488 and

543 nm Acquisition of single FITC-stained samples in dual-fluorescence scanning configuration did not show con-tribution of green signal in red Images were collected at

512· 512 pixels

Isolation and analysis of lipid-raft fractions GEM fractions from HC11 cells, treated or not with EGF (10 nm for 15 min at 37
C), [D]-PDMP (30 lm for 5 days

at 37
C), [D]-PDMP and EGF, [D]-PDMP and GM3

(125 lm for 5 min at 37
C), or [D]-PDMP and GM3 plus

EGF, were isolated as described previously [27] Briefly,

2· 108

cells were suspended in 1 mL of lysis buffer,

con-taining 1% TX-100, 10 mm Tris⁄ HCl pH 7.5, 150 mm

NaCl, 5 mm EDTA, 1 mm Na3VO4, and 75 U aprotinin,

and allowed to stand for 20 min at 4
C The cell

suspen-sion was mechanically disrupted by Dounce

homogeniza-tion (10 strokes) The lysate was centrifuged for 5 min at

1300 g to remove nuclei and large cellular debris The

supernatant fraction (postnuclear fraction) was subjected to

sucrose density gradient centrifugation, i.e the fraction was

mixed with an equal volume of 85% sucrose (w⁄ v) in lysis

buffer (10 mm Tris⁄ HCl pH 7.5, 150 mm NaCl, 5 mm

EDTA) The resulting diluent was placed at the bottom of

a linear sucrose gradient (5–30%) in the same buffer and

centrifuged at 200 000 g for 16–18 h at 4
C in a SW41

rotor (Beckman Institute, Palo Alto, CA) After

centrifuga-tion, the gradient was fractionated, and 11 fractions were

collected starting from the top of the tube All steps were

performed at 0–4
C The amount of protein in each

frac-tion was first quantified by Bio-Rad protein assay (Bio-Rad

Laboratory GmbH, Munchen, Germany)

Finally, fractions were subjected to cholesterol analysis,

western blot, immunoprecipitation experiments or

ganglio-side extraction

Analysis of cholesterol content

All the fractions obtained as reported above from HC11

cells, treated or not with [D]-PDMP, were subjected to

cho-lesterol analysis The amount of chocho-lesterol was evaluated

as described previously [28] Free cholesterol was quantified

from TLC plates by densitometric scanning The density of

the bands used to quantitate cholesterol concentration fell

within the linear range of compound concentration vs

absorbance

Immunoblotting analysis of plasma membrane

fractions

All the fractions obtained as reported above were subjected

to 7.5 or 10% SDS⁄ PAGE Equal volumes of each fraction

were loaded in SDS⁄ PAGE, according to Parolini et al

[12] The proteins were electrophoretically transferred to

nitrocellulose membrane (Bio-Rad, Hercules, CA) and then,

after blocking with NaCl⁄ Pi containing 1% albumin,

probed with rabbit anti-(ErbB2 IgG) polyclonal serum,

rab-bit anti-(phospho ErbB2) polyclonal serum (Sigma), rabrab-bit

anti-(EGFR IgG) polyclonal serum, clone 1005 (Santa Cruz

Biotechnology), rabbit anti-(Shc IgG) polyclonal serum

(Transduction Laboratories, Lexington, KY) or, as

con-trols, rabbit anti-(caveolin-1) polyclonal serum (N-20, Santa

Cruz Biotechnology) or goat anti-(flotillin-2) polyclonal

serum (C-20, Santa Cruz Biotechnology) Bound antibodies

were visualized with horseradish peroxidase (HRP)-conju-gated anti-(rabbit IgG) or anti-(goat IgG) serum (Sigma) and immunoreactivity was assessed by chemiluminescence reaction using the ECL western blotting detection system (Amersham, UK) As a control for nonspecific reactivity, parallel blots were performed as above, using an anti-(rabbit IgG) serum (Sigma)

Immunoprecipitation experiments Briefly, TX-100-insoluble (fractions 4–6) or TX-100-soluble (fractions 10–11) fractions from HC11 cells, untreated or treated with 10 nm EGF (Sigma) for 15 min at 37
C were lyzed in lysis buffer (10 mm Tris⁄ HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluor-ide, 10 lg of leupeptinÆmL)1) Cell-free lysates were mixed with protein G–acrylic beads and stirred by a rotary shaker for 2 h at 4
C to preclear nonspecific binding After cen-trifugation (500 g for 1 min), the supernatant was immuno-precipitated with the rabbit polyclonal anti-(Shc IgG) serum (Transduction Laboratories) plus protein G–acrylic beads A rabbit IgG isotypic control (Sigma) was employed

Immunoprecipitates were subjected to western blot anal-ysis with the rabbit anti-ErbB2 polyclonal serum (Santa Cruz Biotechnology) Immunoreactivity was assessed by chemiluminescence reaction using the ECL western blotting detection system (Amersham)

Ganglioside extraction and analysis by HPTLC Ganglioside extraction was performed according to the method of Svennerholm & Fredman [29] with minor modi-fications Briefly, glycosphingolipids were extracted twice in chloroform⁄ methanol ⁄ water (4 : 8 : 3 v ⁄ v ⁄ v) and subjected

to Folch partition by the addition of water resulting in

a final chloroform⁄ methanol ⁄ water ratio of 1 : 2 : 1.4 (v⁄ v ⁄ v) The upper phase, containing polar glycosphingo-lipids, was purified of salts and low molecular mass contaminants using Bond Elut-C18 columns, 3 mL (Superchrom, Harbor City, CA), according to the method

of Williams & McCluer [30] The eluted glycosphingolipids were dried and separated by HPTLC, using silica gel 60 HPTLC plates (Merck, Darmstadt, Germany) Chromato-graphy was performed in chloroform⁄ methanol ⁄ 0.25% aqueous KCl (5 : 4 : 1 v⁄ v ⁄ v) Plates were then air-dried and gangliosides visualized with resorcinol

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