1Departments of Physiology and Medicine, Yokohama City University School of Medicine, and 2Department of Medical Pathophysiology, Yokohama City University College of Nursing, Yokohama 236-0004, Japan; and 3Cardiovascular Research Institute, Departments of Medicine and Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
Submitted 16 September 2002 ; accepted in final form 7 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
cholesterol; PC-12 cells
Lipid rafts, or caveolae in some tissues, are the plasma membrane
microdomains that are enriched in cholesterol and sphingolipids. These
microdomains are separated by insolubility in detergent, such as Triton X-100,
and low density in sucrose gradient centrifugation from all cell types
(27,
28). Caveolae domains are
specialized lipid rafts that are plasma membrane invaginations with a diameter
of 50100 nm. Caveolins are the defining and principal component
proteins of caveolae and are expressed mainly in endothelia, adipocytes, and
myocytes. The low-density, detergent-insoluble domains without caveolin are
lipid rafts. Lipid rafts exist in all cell types, including neuronal cells, in
which no caveolin protein or mRNA can be detected. However, the functional
difference between the two has not been well determined. Both lipid rafts and
caveolae accommodate specific signaling molecules, such as small and
heterotrimeric G proteins, G protein-coupled receptors, receptor tyrosine
kinases, Src family tyrosine kinases, protein kinase C, and nitric oxide
synthase. It is proposed that the clusters of these molecules within lipid
rafts contribute to the regulation of various signal transduction pathways. A
recent study reported that nAChR7 can also be found within
lipid rafts (5). However, the
role of nAChR
7 in lipid rafts and its interaction with the
other signaling molecules remain mostly unknown.
We hypothesized that nAChR subunits are localized to distinct subcellular
microdomains, i.e., lipid rafts of neuronal cells. For certain subunits, such
as 7, subcellular localization may be important for
interaction with molecules that are regulated by Ca2+.
We demonstrate here that nAChR
7 not only localizes to this
microdomain but also regulates the function of adenylyl cyclase (AC)
coexisting in lipid rafts.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell fractionation by sucrose gradient centrifugation. Lipid rafts were obtained by the sodium carbonate-based detergent-free method (29). Briefly, four 10-cm plates of PC-12 cells with 80% confluence were homogenized in a solution containing 0.5 M sodium carbonate (pH 11) and protease inhibitors with three 10-s bursts of a Polytron tissue grinder and four 20-s bursts of a sonicator. The homogenates were adjusted to 45% sucrose by adding 90% sucrose in 25 mM MES (pH 6.5)-0.15 M NaCl (MBS) and placed at the bottom of an ultracentrifugation tube. A 535% discontinuous sucrose gradient was formed above and centrifuged at 39,000 rpm at 4°C for 16 h in a Beckman SW-41Ti rotor. From the top of the tube, 13 fractions (equal volume from each) were collected and subjected to immunoblotting. Protein concentrations were determined by the method of Bradford (4) with bovine serum albumin as a standard.
Preparation of particulate fraction. PC-12 cells were lysed in 50 mM Tris buffer (pH 8.0) containing (in mM) 1 EDTA, 1 dithiothreitol, and 200 sucrose with protease inhibitors, followed by disruption with four 20-s bursts of a sonicator. Unbroken cells were removed by centrifugation at 500 g for 5 min. The particulate fraction was prepared by centrifugation at 100,000 g at 4°C for 35 min. The supernatant was discarded, and the pellet was resuspended in the same buffer, followed by immunoblotting.
Immunoblotting. Each fraction obtained from sucrose gradient
centrifugation or the particulate fraction from PC-12 cells was separated by
SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to
polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h
with 5% nonfat dry milk and 5% bovine serum albumin in Tris-buffered saline
and 0.1% Tween 20 (TBS-T) at room temperature. Polyclonal antibodies against
nAChR5 (no. SC-9345),-
7 (no. SC-1447), and
-
2 (no. SC-11372) and AC type 3 (AC3) (no. SC-588) and AC5/6
(no. SC-590) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Polyclonal or monoclonal antibodies against caveolin-1 [nos. C37120
[GenBank]
and C13630
[GenBank]
from Transduction Laboratories (Lexington, KY) and no. SC-894 from Santa Cruz
Biotechnology] and that against caveolin-2 (no. C57820
[GenBank]
) from Transduction
Laboratories were used (13,
23). Monoclonal antibody
against flotillin-1 (no. F65020
[GenBank]
) was purchased from Transduction Laboratories.
The membranes were incubated with specific primary antibodies for 2 h at room
temperature, followed by visualization with horseradish peroxidase-conjugated
specific secondary antibodies. Immunoreactive bands were detected with a
SuperSignal substrate kit (Pierce, Rockford, IL).
Immunoprecipitation. PC-12 cells were lysed in a lysis buffer (10 mM Tris · HCl pH 8.0, 0.15 M NaCl, 5 mM EDTA, 60 mM octylglucoside, 1% Triton X-100, and protease inhibitors) at 4°C for 30 min. The lysates were incubated with primary antibodies for 12 h. Immune complexes were formed by incubation with protein G-Sepharose for 2 h. The immune complexes were washed with washing buffer (10 mM Tris · HCl pH 8.0, 0.15 M NaCl, 5 mM EDTA, 0.1% Triton X-100) three times, solubilized, and subjected to immunoblotting.
Intracellular Ca2+ concentration measurements. The intracellular Ca2+ concentration ([Ca2+]i) of individual cells was examined as previously described (14) with some modifications. PC-12 cells were incubated for 1 h at 37°C in a basal salt solution (in mM: 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 HEPES pH 7.3, and 5.5 glucose) containing 4 µM fura 2-AM (Dojin, Kumamoto, Japan) and 0.025% Pluronic F-127 (Molecular Probes, Eugene, OR). After cells were washed, changes in [Ca2+]i were measured by the fluorescence emitted at 510 nm resulting from alternate excitation at 340 and 360 nm with an ARGUS-50/CA apparatus (Hamamatsu Photonics, Hamamatsu, Japan). Conversion of fluorescence signals into absolute [Ca2+]i values was performed with ARGUS-50/CA analysis software.
cAMP accumulation assay. cAMP accumulation in intact cells was
measured according to the method of Kawabe et al.
(20) with a modification. The
PC-12 cells were incubated in normal growing medium containing
3H-labeled adenine for 24 h. The cells were washed three times with
HEPES-buffered serum-free DMEM (pH 7.4). Nicotine and -bungarotoxin
(
-BTX) were added and incubated for 30 min with 20 µM
N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide
(H89). The cells were incubated in the presence of 0.5 mM
3-isobutyl-1-methylxanthine (IBMX) for 10 min before reactions. Reactions were
started by adding 1 µM forskolin at 37°C and terminated after 10 min by
the addition of cold 12% trichloroacetic acid, 0.25 mM ATP and 0.25 mM cAMP.
3H-labeled ATP and 3H-labeled cAMP were separated
according to the method of Alvarez and Daniels
(1). cAMP production was
calculated by [3H]cAMP/([3H]cAMP + [3H]ATP)
x 103. All assays were performed four to six times, and the
values are shown as percentage of control in RESULTS. Student's
t-test was used for statistical analysis.
Cholesterol depletion. To deplete cholesterol from plasma membrane
in PC-12 cells, cells were washed twice with PBS and incubated with DMEM
containing 10 mM methyl--cyclodextrin (M
CD) at 37°C for 1 h
(11). In some experiments,
cholesterol (16 µg/ml) was applied in addition to M
CD
(12). The cells were then
harvested and used for immunoblotting, immunoprecipitation, and cAMP
accumulation assays.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Because caveolin was not detectable in our PC-12 cells, we examined the expression and distribution of flotillin, a marker of cholesterol-enriched lipid rafts in tissues that do not express caveolin (3). Figure 1B demonstrates that flotillin was present in PC-12 cells whereas caveolin was not. Figure 1C demonstrates the subcellular localization of flotillin in our PC-12 cells. Flotillin was found mainly in fractions 46, indicating that these fractions represented lipid rafts in our PC-12 cells.
To determine whether nAChRs exist within or out-side of lipid rafts, 13
fractions obtained from sucrose gradient centrifugation were subjected to
immunoblotting. Polyclonal antibodies against specific nAChRs were used for
immunoblotting, which found that nAChR7 was localized to
fractions 46 but that nAChR
5 and
-
2 were found mainly in fractions 913
(Fig. 1C). Doublet
bands of nAChR
7 were detected as shown in previous studies
(5,
10). This distinct subcellular
localization of
7 relative to that of the other subunits
might represent functional significance of this subunit. We thus investigated
whether
7-subunit may interact with the other molecules
located in the same lipid rafts.
Interaction of nAChR7 with AC.
nAChR
7 is
-BTX sensitive and has a higher
Ca2+ permeability than the other subunits. A previous
study demonstrated that nicotine induced the elevation of
[Ca2+] in PC-12 cells
(14). Accordingly, the high
Ca2+ permeability of nAChR
7 may
regulate Ca2+-sensitive molecules within lipid rafts. AC
is the enzyme that produces cAMP from ATP. At least nine isoforms of AC
(AC1AC9) have been cloned and characterized in mammals
(18). AC5 and AC6 are directly
inhibited by low concentrations of Ca2+
(15,
22). It is possible that
nicotinic receptor agonists may regulate the activity of AC5 or -6 through
increasing the entry of Ca2+. In this regard, we
previously demonstrated (25)
that molecules involved in cAMP signaling such as AC and G proteins are
located in caveolar microdomains.
We first examined the subcellular distributions of AC in PC-12 by sucrose
gradient centrifugation. The fractionated PC-12 cellular proteins were
subjected to immunoblotting with polyclonal antibodies for AC3 and AC5/6
(17). It should be noted that
the polyclonal antibody against AC5/6 potentially detects both AC5 and AC6.
However, this antibody detected a single AC species in PC-12, which is most
likely AC6 because the mRNA expression of AC6, but not AC5, was readily
detected (data not shown). We also examined the protein expression of AC1, -2,
and -4, but they were not readily detected by immunoblotting (data not shown).
Figure 2A shows that
both AC3 and AC6 were in fractions 46, i.e., lipid rafts. This
suggests that both AC and nAChR7 exist within the lipid
rafts. Consequently, to determine whether nAChR
7 directly
interacts with AC, coimmunoprecipitation assays were performed. As shown in
Fig. 2B,
left, reciprocal immunoprecipitation assays with polyclonal
antibodies against AC6 or nAChR
7 followed by immunoblotting
for nAChR
7 or AC6, respectively, demonstrated the
association between nAChR
7 and AC6. Similarly, reciprocal
immunoblotting assays of the supernatants with antibodies against AC6 or
nAChR
7 after immunoprecipitation with
nAChR
7 or AC6 antibodies, respectively, showed that most,
but not all, AC6 and nAChR
7 were associated with each other.
In contrast, AC3 had little association with nAChR
7
(Fig. 2B,
right). Our results suggest that nAChR
7 and AC6 are
colocalized and associated in lipid rafts.
|
Nicotinic stimulation and cAMP accumulation. To examine whether
the activation of nAChRs leads to changes in
[Ca2+]i, and thus altered cAMP signal within
the cell (19), cAMP
accumulation assays were conducted in the presence of nicotinic stimulation.
First, we confirmed the effect of nicotine to elevate
[Ca2+]i in PC-12 cells
(Fig. 3A). The
treatment of PC-12 cells with 100 µM nicotine elicited a rise in
[Ca2+]i at two peaks of the elevation as
reported previously (14). This
rise of [Ca2+]i was blocked in the presence
of -BTX, a specific nAChR
7 antagonist, indicating
that the effect of nicotine was due to the stimulation of
nAChR
7.
|
Stimulation of PC-12 with forskolin (1 µM), a direct AC stimulator, for
10 min increased cAMP accumulation by 10-fold over the basal level. In the
presence of nicotine, this accumulation was inhibited by 35%
(Fig. 3B). The
subcellular distribution of nAChR
7 and AC was not shifted by
nicotine stimulation (data not shown). In the presence of
-BTX, the
effect of nicotine on cAMP accumulation was negated
(Fig. 3B), suggesting
that nAChR
7 contributed to the inhibition of AC6.
Effect of cholesterol depletion from plasma membrane. It is known
that MCD depletes plasma membrane cholesterol, leading to the
attenuation of the function of several molecules within lipid rafts
(11). PC-12 cells were
incubated with 10 mM M
CD, followed by fractionation and immunoblotting.
Figure 4A shows the
subcellular distribution of nAChR
7, AC6, and flotillin in
M
CD-treated cells. In contrast to the finding in the absence of
M
CD pretreatment (Fig.
1C), these molecules were found diffusely in
fractions 410, indicating that the localization of
nAChR
7, AC6, and flotillin was altered by cholesterol
depletion. When cholesterol was added to M
CD to negate its
cholesterol-depleting effect
(12),
nAChR
7, AC6, and flotillin were relocalized to lipid rafts,
suggesting that the localization of these molecules to lipid rafts requires
the presence of cholesterol in the plasma membrane. Furthermore, the
association between nAChR
7 and AC6 was mostly lost by
M
CD treatment, as demonstrated by reciprocal immunoprecipitation assays
with nAChR
7 and AC6 antibodies
(Fig. 4B,
center). This ablation of the association between
nAChR
7 and AC6 was also restored by adding cholesterol to
M
CD (Fig. 4B,
right).
|
Figure 5A
demonstrates that MCD or cholesterol treatment did not alter
nicotine-induced rise in [Ca2+]i relative to
that in nontreated cells shown in Fig.
3A. The peaks of [Ca2+]i
showed no significant differences among the control, M
CD, and M
CD
+ cholesterol groups (282.3 ± 4.1, 273.5 ± 3.9, and 280.8
± 5.1 nM, respectively). The second elevation of
[Ca2+]i also showed no differences among
these conditions (127.3 ± 2.9, 124.5 ± 3.4, and 126.3 ±
2.8 nM, respectively). Similarly, M
CD treatment did not alter
forskolin-stimulated cAMP accumulation but negated the nicotine-induced
inhibition of cAMP accumulation in PC-12 cells
(Fig. 5B). We also
examined the specificity of the effect of M
CD. When cholesterol was
added to M
CD in PC-12 cells, the nicotine-induced inhibition of cAMP
production was restored (Fig.
5B). These data suggest that the presence of intact lipid
rafts is necessary to localize nAChR
7 to the lipid rafts as
well as to regulate cAMP signal through the nicotinic activation of
nAChR
7.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that AC3 was also in lipid rafts but was not readily
coimmunoprecipitated with nAChR7. This finding suggests that
AC3 may not be bound to nAChR
7 as tightly as AC6, and
thereby the regulation of AC3 by nAChRs might not be so potent. Unfortunately,
the effect of Ca2+ on this isoform is controversial
(8,
31), and we cannot predict the
effect of nAChRs specifically on AC3. Alternatively, the sensitivity and/or
affinity of the AC3 antibody may not be sufficiently high, although we have
successfully used this antibody in past studies
(17). Nevertheless,
nAChR
7 may play a distinct role from the other nAChR
subunits in regulating intracellular signals because of its unique subcellular
localization in lipid rafts. In support of this hypothesis, it was recently
demonstrated that nAChR
7 associates with
phosphatidylinositol 3-kinase (PI3K) and a tyrosine kinase (Fyn) but
nAChR
4 did not
(21). PI3K and the Src family
of tyrosine kinases are reported to exist within caveolae
(32,
33). These findings suggested
that nAChR
7, PI3K, and Fyn are closely located and interact
with each other. We do not deny, however, that the other nAChR subunits also
play important roles in regulating other signaling pathways.
We have not examined the mechanism that localizes nAChR7
and AC to the same lipid rafts. In caveolae of peripheral cells, caveolin, a
structural component of caveolae, directly binds to and anchors the molecules
within caveolae (24). However,
we did not find caveolin expression in lipid rafts of our PC-12 cells. A few
studies in which the same antibodies were used as in our study reported that
caveolin was readily detected in PC-12 cells
(13,
23). This difference may be
due to the difference in PC-12 cell type or experimental conditions, such as
the state of cellular differentiation. We instead demonstrated that flotillin,
a marker for lipid rafts (3),
was in the same fractions as nAChR
7 and AC6. Nevertheless,
our results suggest that the cholesterol depletion that is known to disrupt
both caveolae and lipid rafts
(11) destroyed the interaction
between nAChR
7 and AC. The robust finding of our study is
the suggestion that subcellular microdomains such as lipid rafts provide a
site of regulation within neuronal cells between a specific
Ca2+-handling molecule subtype and a
Ca2+-regulated enzyme subspecies.
![]() |
DISCLOSURES |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Berger F, Gage
FH, and Vijayaraghavan S. Nicotinic receptor-induced apoptotic cell death
of hippocampal progenitor cells. J Neurosci
18: 68716881,
1998.
3. Bickel PE,
Scherer PE, Schnitzer JE, Oh P, Lisanti MP, and Lodish HF. Flotillin and
epidermal surface antigen define a new family of caveolae-associated integral
membrane proteins. J Biol Chem
272: 1379313802,
1997.
4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254, 1976.[ISI][Medline]
5. Brusés JL, Chauvet N, and Rutishauser U. Membrane lipid
rafts are necessary for the maintenance of the 7 nicotinic
acetylcholine receptor in somatic spines of ciliary neurons. J
Neurosci 21:
504512, 2001.
6. Chen D and
Patrick JW. The -bungarotoxin-binding nicotinic acetylcholine
receptor from rat brain contains only the
7 subunit. J Biol
Chem 272:
2402424029, 1997.
7. Chiono M, Mahey
R, Tate G, and Cooper DMF. Capacitative Ca2+ entry
exclusively inhibits cAMP synthesis in C62B glioma cells. J
Biol Chem 270:
11491155, 1995.
8. Choi EJ, Xia Z, and Storm DR. Stimulation of the type 3 olfactory adenylyl cyclase by calcium and calmodulin. Biochemistry 31: 64926498, 1992.[ISI][Medline]
9. Clementi F, Fornasari D, and Gotti C. Neuronal nicotinic receptors, important new players in brain function. Eur J Pharmacol 393: 310, 2000.[ISI][Medline]
10. Drisdel RC and
Green WN. Neuronal -bungarotoxin receptors are
7 subunit
homomers. J Neurosci 20:
133139, 2000.
11. Fagan KA, Smith
KE, and Cooper DMF. Regulation of the
Ca2+-inhibitable adenylyl cyclase type 6 by capacitative
Ca2+ entry requires localization in cholesterol-rich
domains. J Biol Chem 275:
2653026537, 2000.
12. Furuchi T and
Anderson RGW. Cholesterol depletion of caveolae causes hyperactivation of
extracellular signal-related kinase (ERK). J Biol Chem
273: 2109921104,
1998.
13. Galbiati F,
Volonte D, Gil O, Zanazzi G, Salzer JL, Sargiacomo M, Scherer PE, Engelman JA,
Schlegel A, Parenti M, Okamoto T, and Lisanti MP. Expression of caveolin-1
and -2 in differentiating PC12 cells and dorsal root ganglion neurons:
caveolin-2 is up-regulated in response to cell injury. Proc Natl
Acad Sci USA 95:
1025710262, 1998.
14. Gueorguiev VD,
Zeman RJ, Hiremagalur B, Menezes A, and Sabban EL. Differing temporal
roles of Ca2+ and cAMP in nicotine-elicited elevation of
tyrosine hydroxylase mRNA. Am J Physiol Cell Physiol
276: C54C65,
1999.
15. Hanoune J and Defer N. Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol 41: 145174, 2001.[ISI][Medline]
16. Henderson LP, Gdovin MJ, Liu C, Gardner PD, and Maue RA. Nerve growth factor increases nicotinic Ach receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells. J Neurosci 14: 11531163, 1994.[Abstract]
17. Ishikawa Y, Grant BS, Okumura S, Schwencke C, and Yamamoto M. Immunodetection of adenylyl cyclase protein in tissues. Mol Cell Endocrinol 162: 107112, 2000.[ISI][Medline]
18. Ishikawa Y and
Homcy CJ. The adenylyl cyclases as integrators of transmembrane signal
transduction. Circ Res 80:
297304, 1997.
19. Ishikawa Y,
Katsushika S, Chen SL, Hanlon NJ, Kawabe J, and Homcy CJ. Isolation and
characterization of a novel cardiac adenylyl cyclase cDNA. J Biol
Chem 267:
1355313557, 1992.
20. Kawabe J, Ebina T, Toya Y, Oka N, Schwencke C, Duzic E, and Ishikawa Y. Regulation of type 5 adenylyl cyclase by PMA-sensitive and -insensitive protein C isoenzymes in intact cells. FEBS Lett 384: 273276, 1996.[ISI][Medline]
21. Kihara T,
Shimohama S, Sawada H, Honda K, Nakamizo T, Shibasaki H, Kume T, and Akaike
A. 7 Nicotinic receptor transduces signals to phosphatidylinositol
3-kinase to block a
-amyloid-induced neurotoxicity. J Biol
Chem 276:
1354113546, 2001.
22. Patel TB, Du Z, Pierre S, Cartin L, and Scholich K. Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 269: 1325, 2000.[ISI]
23. Perió S,
Comella JX, Enrich C, Martín-Zanca D, and Rocamora N. PC12 cells
have caveolae that contain TrkA. J Biol Chem
275: 3784637852,
2000.
24. Schwencke C, Okumura S, Yamamoto M, Geng YJ, and Ishikawa Y. Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane. J Cell Biochem 75: 6472, 1999.[ISI][Medline]
25. Schwencke C,
Yamamoto M, Okumura S, Toya Y, Kim SJ, and Ishikawa Y. Compartmentation of
cyclic adenosine 3',5'-monophosphate signaling in caveolae.
Mol Endocrinol 13:
10611070, 1999.
26. Sharma G and
Vijayaraghavan S. Nicotinic cholinergic signaling in hippocampal
astrocytes involves calcium-induced calcium release from intracellular stores.
Proc Natl Acad Sci USA 98:
41484153, 2001.
27. Shaul PW and
Anderson RGW. Role of plasmalemmal caveolae in signal transduction.
Am J Physiol Lung Cell Mol Physiol
275: L843L851,
1998.
28. Smart EJ, Graf
GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, and Lisanti
MP. Caveolins, liquid-ordered domains, and signal transduction.
Mol Cell Biol 19:
72897304, 1999.
29. Song KE, Li S,
Okamoto T, Quilliam LA, Sargiacomo M, and Lisanti MP. Co-purification and
direct interaction of ras with caveolin, an integral membrane protein of
caveolae microdomains. J Biol Chem
271: 96909697,
1996.
30. Vijayaraghavan S, Huang B, Blumenthal EM, and Berg DK. Arachidonic acid as a possible negative feedback inhibitor of nicotinic acetylcholine receptors on neurons. J Neurosci 15: 36793687, 1995.[Abstract]
31. Wayman GA,
Impey S, and Strom DR. Ca2+ inhibition of type 3
adenylyl cyclase in vivo. J Biol Chem
270: 2148021486,
1995.
32. Wu C, Butz S,
Ying Y, and Anderson RGW. Tyrosine kinase receptors concentrated in
caveolae-like domains from neuronal plasma membrane. J Biol
Chem 272:
35543559, 1997.
33. Zundel W,
Swiersz LM, and Giaccia A. Caveolin-1 mediated regulation of receptor
tyrosine kinase-associated phosphatidylinositol 3-kinase activity by ceramide.
Mol Cell Biol 20:
15071514, 2000.