Lipid segregation and IgE receptor signaling: A decade of progress

http://www.elsevier.com/locate/bba

Biochimica et Biophysica Act

Review

Lipid segregation and IgE receptor signaling: A decade of progress

David Holowka, Julie A. Gosse, Adam T. Hammond, Xuemei Han, Prabuddha Sengupta,

Norah L. Smith, Alice Wagenknecht-Wiesner, Min Wu, Ryan M. Young, Barbara Baird*

Department of Chemistry and Chemical Biology, Cornell University, Baker Laboratory, Ithaca, NY 14853-1301, USA

Received 12 April 2005; received in revised form 11 June 2005; accepted 15 June 2005

Available online 11 July 2005

Abstract

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of

liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating

dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with

the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment.

Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream

signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other

results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and

other cell surface receptors.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Lipid raft; Immunoreceptor signaling; Tyrosine kinase and phosphatase; Cytoskeleton; Membrane domain

Plasma membrane domains that are implicated in

particular cell functions and depend on physical properties

of lipids have received much attention during the past dozen

years [1]. Chief among these are membrane compartments,

commonly referred to as lipid rafts, that participate in

membrane trafficking and receptor-mediated signaling,

among other essential cellular processes. Lipid rafts were

originally defined in terms of their resistance to solubiliza-

tion by non-ionic detergents such as Triton X-100 (TX-100;

[2]). This property was first demonstrated by Brown and

Rose while investigating differential membrane trafficking

in polarized epithelial cells [3]. They showed that when cells

are lysed under standard conditions with TX-100 and

fractionated on sucrose density gradients, a characteristic

subset of cellular phospholipids, including most sphingo-

myelin derivatives, as well as GPI-linked proteins, are not

solubilized, but rather they float as low density membrane

0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2005.06.007

* Corresponding author. Tel.: +1 607 255 4095; fax: +1 607 255 4137.

E-mail address: [email protected] (B. Baird).

vesicles containing relatively small amounts of cellular

proteins. Subsequent biophysical studies on model mem-

branes [4,5] and plasma membrane vesicles [6,7] or

reconstituted plasma membrane lipids [8] showed that these

lipid rafts also have the properties of a liquid ordered (Lo)

phase, in which cholesterol-dependent lipid packing

increases phospholipid acyl chain order while allowing free

lateral diffusion of membrane components. Mass spectro-

metric analysis of detergent-resistant membranes confirmed

a composition characterized by phospholipids with mostly

saturated fatty acid chains, including sphingomyelin [9].

Detergent insolubility and flotation on density gradients

has served as a valuable correlative tool for identifying lipid

rafts; we use it in this manner in the work described below.

The definition of lipid rafts has been broadened by some

laboratories to include, more generally, low-density mem-

brane fractions that are isolated in the absence of detergents

[10,11]. This alternative method of membrane fractionation

suffers from a lack of discriminating physical criteria for the

putative membrane domains isolated and will not be

considered further in this review. Although useful as a first

a 1746 (2005) 252 – 259

D. Holowka et al. / Biochimica et Biophysica Acta 1746 (2005) 252–259 253

approximation, it is clear that the criterion of detergent

insolubility is limited in revealing the true nature of lipid

rafts. Their compositional heterogeneity of lipids and

proteins and their dynamic nature in the living cells requires

that more precise tools be applied collectively to character-

ize these critical membrane compartments as related to the

particular cellular functions that they mediate.

Our studies on IgE receptor (FcERI) signaling in mast

cells pointed to the importance of plasma membrane

domains in these stimulated processes. A number of

different types of experiments have led to two general

conclusions concerning lipid rafts in cells. One is that

segregation of certain proteins in ordered lipid rafts from

other proteins in disordered regions of the plasma mem-

branes serves a fundamental role in orchestrating cell

signaling processes. The second conclusion is that the

membrane-associated cytoskeleton plays a substantial role

in regulating lipid segregation at the plasma membrane of

mammalian cells. These general themes of lipid-mediated

protein segregation and cytoskeletally-regulated lipid seg-

regation will be developed in succeeding paragraphs citing

specific examples from studies in our laboratory and from

other recent literature.

1. Lipid-mediated protein segregation

Despite hundreds of studies documenting the involve-

ment of lipid rafts in signal transduction, very few of these

provide direct evidence for specific role(s) that lipid rafts

play in these processes. A common, misguiding assumption

is that lipid rafts represent a small fraction of the membrane

surface, such that association with lipid rafts would confer

concentration of signaling components. This assumption is

usually based on the relatively low abundance of proteins in

lipid rafts. The assumption is incorrect, as a number of

observations reveal that ordered lipids represent a majority

of the plasma membrane lipids [1]. We recently addressed

this question by measuring fluorescence resonance energy

transfer between carbocyanine lipid probes in the outer

leaflet of the plasma membrane of live RBL cells (Sengupta,

P., Holowka, D. and Baird, B. manuscript in preparation)

and obtained strong evidence for nanoscopic phase separa-

tion between Lo and Ld regions. These results further

indicate that about 65% of the plasma membrane lipids are

in the more ordered phase, consistent with recent electron

spin resonance (ESR) measurements in live cells [12].

Previous measurements of lipid order in plasma membrane

vesicles from RBL cells using fluorescence anisotropy [6]

and ESR [7] indicated that at least 40% of the lipids in these

membranes are in an Lo-like environment.

In earlier studies, we found that ligand-mediated cross-

linking of IgE receptors on RBL mast cells causes their

association with detergent-resistant lipid rafts. This associ-

ation correlates with the initiation of tyrosine phosphoryla-

tion of FcERI and the signaling cascade that emanates from

this initiating event [13,14]. Depletion of cholesterol from

RBL mast cells using methyl h-cyclodextrin results in

selective loss of both crosslinked IgE receptors and Lyn

kinase from lipid rafts in parallel with the loss of Lyn-

mediated phosphorylation of FcERI ITAM sequences in the

h and g subunits of this receptor [14]. Outer leaflet

components, including a ganglioside and a GPI-linked

protein, are not lost from lipid rafts under these conditions.

These results indicate that localization of either Lyn or

crosslinked FcERI or both to detergent-resistant lipid rafts is

necessary for the initiation of tyrosine phosphorylation in

these cells.

These early studies on the role of lipid rafts in FcERI

signaling relied heavily on sensitivity to solubilization by

TX-100 to define lipid raft association. Because of the

possible pitfalls of this single approach, we also evaluated

the sensitivity of interactions between crosslinked IgE

receptors and Lyn to cholesterol depletion with confocal

fluorescence microscopy of intact cells with results that

were parallel to those obtained with sucrose gradients of

TX-100-lysed cells [14]. A limitation of these imaging

experiments is that long-term incubations at cold temper-

atures was required for visualizing the clustered receptors

and coalesced lipid rafts. Nonetheless, close correspondence

of the results from these two independent methods provided

early support for the hypothesis that lipid rafts are integrally

involved in IgE-receptor mediated signaling. As described

below, more recent studies take advantage of new tech-

nologies and microscopy of living cells in real time to probe

these interactions.

To determine whether the lipid raft environment regulates

the activity of Lyn kinase, we took a biochemical approach

and compared the specific activity of Lyn isolated from both

raft (ordered) and non-raft (disordered) regions of the

plasma membrane. Using two different measures of kinase

activity, we found that Lyn kinase from lipid rafts has a 4- to

5-fold higher specific activity than that from disordered

regions of unstimulated mast cells [15]. Interestingly,

stimulation via FcERI was found to cause a time-dependent

decrease in Lyn kinase activity, rather than an increase, and

this decrease has been shown to correlate with a stimulation-

dependent increase in C-terminal tyrosine phosphorylation

of Lyn by Csk kinase [16]. We found that the higher specific

activity of Lyn from lipid rafts corresponds to its tyrosine

phosphorylation in the active site loop, which occurs on

<20% of the Lyn kinases in lipid rafts and is undetectable on

Lyn from disordered regions of the plasma membrane [15].

This small percentage of Lyn that is highly active because of

its active site phosphorylation [17], both before and after

stimulation, is also predicted from mathematical modeling

of this experimental system by Wofsy and colleagues [18].

It is interesting to compare the role of lipid rafts in

regulating Lyn kinase to that for the T cell-specific Src-

family kinase, Lck. Rogers and Rose found that Lck is

hyperphosphorylated at its negative regulatory site, Tyr505,

in lipid rafts of unstimulated T cells due to exclusion of the

D. Holowka et al. / Biochimica et Biophysica Acta 1746 (2005) 252–259254

positive regulatory tyrosine phosphatase, CD45 [19]. This

results in relatively low activity for Lck in lipid rafts in the

absence of stimulation. Brdicka et al. found that this

negative regulation of Lck in lipid rafts is also promoted

by constituative phosphorylation of the raft-associated

adaptor protein, PAGE/Cbp, which mediates lipid raft

association of Csk [20]. Furthermore, they found that

stimulation via T cell receptors causes dephosphorylation

of PAGE/Cbp and consequent dissociation of Csk, which

results in the activation of Lck in lipid rafts [21]. These

results suggest a more complex regulation of Lck activity in

lipid rafts of unstimulated T cells compared to that for Lyn

in RBL mast cells.

Our experimental results for Lyn in mast cells suggested

that its active site tyrosine is more protected from

dephosphorylation by membrane-associated phosphatases

in a raft environment than Lyn in a disordered membrane

environment. We decided to test this hypothesis using a

CHO fibroblast reconstitution system previously character-

ized by Metzger and colleagues [22,23]. We used CHO cells

with stably expressed FcERI, and we transiently transfected

these with Lyn alone or with Lyn together with a tyrosine

phosphatase. Consistent with previous observations [23], we

found that expression of Lyn by itself in these cells resulted

in a high level of FcERI tyrosine phosphorylation in the

absence of stimulation, and only a small further increase due

to antigen-mediated crosslinking of IgE-FcERI. Correspond-

ingly, we found that Lyn expressed in these cells had high

specific activity due to enhanced phosphorylation at its

active site [24]. We tested the effects of co-expression of

several different tyrosine phosphatases, and we found that

one in particular, protein tyrosine phosphatase a (PTPa),

can control the basal activity of Lyn kinase while allowing

robust stimulation of FcERI. Two other phosphatases tested

had different effects: SHP-1, a cytosolic protein that is

recruited to phosphorylated proteins via its SH2 domain

[25], was very effective at suppressing Lyn kinase activity,

but did not allow stimulated phosphorylation of FcERI.

CD45, a transmembrane phosphatase expressed in many

cells of hematopoietic lineage, had a modest stimulatory

effect on Lyn kinase activity, both before and after

stimulation by antigen, consistent with its predominantly

positive effects in T and B cell signaling [26].

PTPa is a transmembrane phosphatase that is ubiqui-

tously expressed, but its endogenous expression in CHO

cells is less than in other cells, including RBL mast cells

[24]. As for most transmembrane proteins in the plasma

membrane, it is excluded from detergent-resistant lipid rafts.

To test the significance of membrane localization in its

capacity to reconstitute stimulated FcERI phosphorylation in

CHO cells, we constructed a chimeric version of this

phosphatase that is anchored to the plasma membrane via

two saturated fatty acid chains, replacing in this manner its

normal transmembrane sequence. As expected, this palmi-

toyl, myristoyl-anchored phosphatase efficiently associated

with lipid rafts. At levels of functional expression similar to

the transmembrane parent, the lipid-anchored chimera was

equally effective at suppressing the high level of sponta-

neous Lyn kinase activity, but it did not allow antigen-

stimulated FcERI phosphorylation [24].

These results demonstrate that exclusion of a specific

transmembrane phosphatase from ordered lipid raft

domains provides a means by which the raft environment

can protect the cognate kinase from negative regulation by

that phosphatase (Fig. 1A). Because uncrosslinked IgE

receptors reside largely outside of lipid rafts, they are not

effectively phosphorylated by active Lyn within the raft

environment. Crosslinking of these receptors enhances

their association with lipid rafts and thus puts them in

proximity to active Lyn kinase, promoting their phosphor-

ylation and possibly protecting them from dephosphoryla-

tion by the same phosphatases that inactivate Lyn (Fig.

1B). Lipid raft association of crosslinked FcERI is a

dynamic process that is readily reversed by agents such as

monovalent hapten that disrupt crosslinks, [27], and these

manipulations cause a rapid reversal of the phosphorylated

state [28,29]. Undoubtedly, other phosphatases, such as

SHP-1, also contribute to the negative regulation of Lyn

kinase activity in mast cells, and the net result is a

dynamic balance of receptor phosphorylation and dephos-

phorylation that is perturbed in a positive manner when

crosslinking drives the receptors to increased association

with lipid rafts.

The structural basis for crosslink-dependent lipid raft

association of FcERI remains incompletely understood, as

for other structurally related multichain immune recognition

receptors [30]. An earlier study ruled out an essential role

for the h subunit of FcERI in this process and pointed

toward possible roles for transmembrane segments of FcERI

a and/or g [31]. To examine the structural basis for this lipid

raft association and for immunoreceptor signaling, we

expressed a panel of single chain chimeric IgE receptors

containing the extracellular domain from human FcERI a,

the cytoplasmic domain of T cell receptor ~, and six differenttransmembrane sequences. The results were striking: chi-

meric receptors containing transmembrane sequences that

did not enable crosslink-dependent association with lipid

rafts mediated only tiny amounts or no tyrosine phosphor-

ylation and Ca2+ responses, whereas those that exhibited

crosslink-dependent lipid raft association caused significant,

albeit variable, amounts of signaling leading to stimulated

degranulation [32]. For one chimeric receptor, disulfide-

mediated oligomerization was found to enhance the signal-

ing response, and, for another, palmitoylation of a cysteine

residue in its transmembrane sequence was found to be

critical for lipid raft association leading to signal trans-

duction. These results strengthen the paradigm of lipid raft-

dependent signal initiation for multichain immune recog-

nition receptors, and they demonstrate that relatively subtle

changes in lipid raft partitioning can make dramatic differ-

ences in the signaling capacity of immunoreceptors. The

prevailing message from these studies is that Lo/Ld phase

Fig. 1. Graphic representation of protein segregation by lipid rafts that is relevant to IgE receptor signal initiation. (A) Distributions of relevant membrane

components in unstimulated cells. (B) Distributions of relevant components following signal initiation by antigen-mediated IgE receptor crosslinking.


separation in biological membranes can serve to segregate

proteins from each other in a manner that facilitates ligand-

stimulated signal transduction.

2. Regulation of lipid segregation by the cytoskeleton

Giant unilamellar vesicles (GUVs) composed of lipid

mixtures designed to simulate the composition of the plasma

membrane outer leaflet have been shown to exhibit large-

scale (micron-sized) Lo/Ld phase separations [8,33,34], and

we recently found that cholera toxin B, a pentameric ligand

for the ganglioside GM1, can drive such phase separations

in GUVs with compositions near a phase boundary [35].

Lipids isolated from plasma membranes [8] or from total

cellular lipids [36] can also undergo large-scale Lo/Ld phase

separations when reconstituted into GUVs. However,

optically resolvable phase separations are not observed in

the plasma membrane of intact cells under most circum-

stances [1,37,38]. An exception to this generalization is the

situation where lipid raft-associated proteins or lipids are

extensively crosslinked to form large patches or caps, often

at lower temperatures to prevent endocytosis and to

suppress other signaling-induced cellular changes such as

membrane ruffling. Under these conditions, various lipid

raft components, defined by detergent resistance, are often

observed to undergo co-redistribution with other crosslinked

lipid raft components [39–42]. Non-raft associated proteins

and lipids show little or no co-redistribution, suggesting the

possibility that a large-scale phase separation occurs.

However, it was found that the actin cytoskeleton also

dramatically co-redistributes with lipid raft components

under these conditions [43–45], suggesting that the

structural basis for this large-scale separation of Lo/Ld

components involves interactions more complex than

simply Lo/Ld phase separation in live cells.


More recent studies also indicate a role for the

membrane-associated cytoskeleton in the regulation of

lipid raft structure. Kusumi and colleagues use ultrafast

cameras to study single molecule lateral diffusion on live

cells, and they conclude that the membrane skeleton

provides barriers to long-range diffusion of both raft and

non-raft membrane components [46]. Although they detect

little or no difference in the lateral mobilities of raft vs.

non-raft components in the absence of ligand-mediated

crosslinking, they see a more substantial restriction of la-

teral mobility for lipid raft components after crosslinking

[47,48]. This may be related to the earlier reports of actin

cytoskeleton accumulation under large-scale lipid raft

patches described above. Other studies by Kenworthy and

colleagues also found only small differences between the

lateral diffusion of raft and non-raft proteins in the absence

of crosslinking using more conventional fluorescence

photobleaching recovery methods [49]. This is consistent

with expectations from lateral diffusion studies on model

membranes of Lo vs. Ld compositions [8,50], indicating

that ordering of lipids by cholesterol has no dramatic effect

on lateral mobility on the lipid and protein probes tested. In

the cell studies, however, depletion of cholesterol caused

significant alterations in the mobility of both raft and non-

raft proteins, suggesting that perturbation of membrane

composition by this strategy has larger effects on mem-

brane structure. Consistent with this view, Edidin and

colleagues showed that cholesterol depletion substantially

reduces F-actin association with the plasma membrane and,

in parallel, causes increased exposure of phosphatidylino-

sitol 4,5-bisphosphate [51]. These results, taken together,

indicate an integral but complex relationship between

cholesterol-dependent membrane structure and plasma

membrane-cytoskeleton coupling.

Our laboratory employs patterned lipid bilayers contain-

ing a specific ligand for IgE that enables spatially defined

clusters of IgE–receptor complexes on cells at room

temperature and 37 -C. Recent studies revealed a surprising

segregation of inner and outer leaflet lipid raft components

[52]. In this experimental situation, the micron-sized patches

of ligand-clustered receptors stimulate rapid, spatially

localized tyrosine phosphorylation (¨1 min) that is fol-

lowed by a slower recruitment of Lyn kinase (¨10 min) and

other inner leaflet components of lipid rafts (10–30 min).

Under these conditions, PIP2 bound to GFP-PLCy PH does

not co-redistribute with these patterned features, nor do

outer leaflet lipid raft markers, including DiIC16 and FITC-

cholera toxin B bound to ganglioside GM1. Recruitment of

Lyn and other inner leaflet components to the patterned

bilayers is strictly dependent on F-actin polymerization, as it

is prevented by micromolar cytochalasin D. These results

point to complex regulation by the actin cytoskeleton of

lipids and proteins that are organized in rafts. Furthermore,

they demonstrate that inner leaflet raft components can

segregate independently from outer leaflet raft components

in live cells.

The view of an intimate relationship between the actin

cytoskeleton and lipid raft structure in cells is further

strengthened by other approaches. We first showed with

mass spectrometry that antigen stimulation of RBL mast

cells changes the lipid composition of detergent-resistant

lipid rafts [9], and our extended examination revealed that

this change is caused by antigen-stimulated actin depolyme-

rization. We observe an increase in polyunsaturated phos-

pholipids that fractionate as detergent-resistant lipids after

antigen stimulation. This shift can be mimicked by treat-

ment of the RBL mast cells with low concentrations of

cytochalasin D; furthermore, antigen-stimulated changes

can be inhibited by the F-actin stabilizer, jasplakinolide

(Han, X., Smith, N., Holowka, D., McLafferty, F. and Baird,

B. manuscript in preparation). These results indicate that the

composition of Lo domains in live cells is tightly regulated

by the actin cytoskeleton, as small perturbations of this

cytoskeleton dramatically affect the structure and composi-

tion of these domains. They suggest that Lo/Ld phase

separation detected by energy transfer depends on the

membrane skeleton that orchestrates plasma membrane

structure in live cells. Understanding the dynamic organ-

ization of lipids and proteins within rafts that facilitates cell

function and the specific molecular mechanisms that

underlie raft regulation by the cytoskeleton are goals of

ongoing studies in this and other laboratories.

3. Future directions

As summarized above, IgE receptor signaling clearly

involves participation of the plasma membrane, and inves-

tigation of the mechanism has provided a useful platform

from which to observe and understand roles for lipid

segregation in cell function. An important role is lipid-

mediated protein segregation to regulate signal transduction

that is stimulated by an external event, such as antigen

crosslinking of IgE-receptors. Other findings point to a

direct coupling between the actin cytoskeleton and deter-

gent-resistant lipid rafts that is integral to this regulation, but

the molecular bases for the underlying structural organiza-

tion remain to be defined. Outstanding questions include

whether the membrane cytoskeleton facilitates Lo/Ld phase

separation of lipids in the plasma membrane at sub-micron

spatial resolution, and how ligand-mediated receptor clus-

tering might perturb such structural organization at the

nanometer scale that translates to functional consequences at

micron and longer length scales. New and improved

electron microscopy methods are needed to probe nano-

scopic structural organization of both proteins and lipids,

and recent efforts in this regard have provoked new

questions concerning membrane structural organization

and its relationship to signal transduction [53,54].

New ways to perturb membrane structure under con-

trolled conditions are also needed to develop a more

complete understanding of plasma membrane architecture


and its dynamic regulation. Increasing the cholesterol

content of biological membranes has been shown to be a

useful strategy in this regard [55], and the use of other

sterols with differing effects on Lo structure holds promise

for future studies in cellular systems [56]. Short chain

ceramides have been shown to disrupt Lo structure in

biological membranes, with consequent perturbations of

certain signal transduction pathways [57]. Long chain

ceramides are natural substitutes for cholesterol in the Lo

phase, and they have the interesting property of restricting

lateral diffusion of proteins and lipids in such Lo environ-

ments [58]. Alteration of phospholipid acyl chain compo-

sition by expression of a sterol desaturase [59,60] pioneers

molecular genetic approaches to perturbation of membrane

structure, which will provide important new directions for

detailed inquiry.

This review has focused on the participation of ordered

membrane domains in signal transduction. Roles for Lo

structure in intracellular membrane trafficking processes,

sometimes related to signaling, are receiving increasing

attention. For example, endosomal vesicles with apparent

large-scale phase separations have been reported [61]. We

found that receptor-stimulated outward trafficking of chol-

era toxin B bound to GM1 in recycling endosomes is strictly

dependent on normal cholesterol content in RBL mast cells

[62]. Other studies have indicated aberrant endocytic

trafficking under conditions of altered cholesterol content

[60,63,64]. The dynamic relationship between the plasma

membrane and internally trafficked membranes, and the role

played by ordered lipid domains in related cell processes, is

an intriguing and complicated problem. Understanding the

structural and mechanistic bases for these processes will

require a combination of multidisciplinary approaches and

further development of new technologies. It is clear from the

studies of the past decade on roles for lipid segregation in

cell function that this is a new frontier in cell biology and

much remains to be learned.

Acknowledgments

This work was supported by NIH grants R01-AI22449

and R01-AI18306 and in part by the Nanobiotechnology

Center (NSF: ECS9876771). We acknowledge the pioneer-

ing work on lipid rafts in our laboratory by Drs. K. A. Field,

L.M. Pierini, E.D. Sheets, E.K. Fridriksson, P.S. Pyenta, and

A. Gidwani. We are grateful to our collaborators at Cornell

University: W.W. Webb, F.W. McLafferty, J.H. Freed, G.W.

Feigenson, H.G. Craighead, and their co-workers.

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Lipid segregation and IgE receptor signaling: A decade of progress

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