(Received for publication, December 13, 1995; and in revised form, January 29, 1996)
From the
Previously we showed that interleukin 1 stimulates the
conversion of sphingomyelin to ceramide in the caveolae fraction of
normal human fibroblasts. The ceramide, in turn, blocked
platelet-derived growth factor (PDGF) stimulated DNA synthesis. We now
present evidence that the PDGF receptor initiates signal transduction
from caveolae. Cell fractionation and immunocytochemistry show caveolae
to be the principal location of PDGF receptors at the cell surface.
Multiple caveolae proteins acquire phosphotyrosine when PDGF binds to
its receptor, but the hormone appears to have little effect on the
tyrosine phosphorylation of non-caveolae membrane proteins. Five
proteins known to interact with the phosphorylated receptor were found
to be highly enriched in caveolae membrane. PDGF caused the
concentration of three of these proteins to significantly increase in
the caveolae fraction. Finally, PDGF stimulated the association of a
190-kDa phosphoprotein with the caveolae marker protein, caveolin.
Therefore, ceramide may modulate PDGF receptor function directly in
caveolae.
The PDGF (
)receptor belongs to a family of
growth factor receptors that transmit information across the membrane
by stimulating the phosphorylation of specific effectors such as PI
3-kinase, non-receptor tyrosine kinases, protein-tyrosine phosphatases,
and various adapter molecules(1) . Activation of the receptor
requires the binding of PDGF, the dimerization of the receptor, and
receptor tyrosine phosphorylation(2, 3) . The
phosphorylated receptor then attracts effector molecules containing
both SH2 and SH3 domains where they become active in relaying
information to various cellular compartments. Shortly after ligand
binding, the ligand-receptor complex is internalized and
degraded(4) . Internalization appears to be mediated by
clathrin-coated pits(5, 6) .
The exact location on
the cell surface where the PDGF receptor initiates its signaling
cascade has not been determined. The receptor may do this either from a
random position on the plasma membrane or from a specific membrane
domain. Recently, plasmalemmal caveolae have been identified as a site
where several PDGF receptor effector molecules are
concentrated(7, 8, 9) . This raises the
possibility that the receptor is also located in this compartment.
Caveolae were first identified as distinctive membrane invaginations on the cell surface. The shape of the membrane suggested they were involved in endocytosis. Indeed, caveolae appear to be involved in the uptake of bulk proteins from the blood(10) , the internalization of small molecules such as folate by potocytosis(11) , and the internalization of glycolipid binding toxins(12) . During potocytosis, caveolae internalize molecules in a cyclic process involving the progressive invagination of the plasma membrane, the formation of a closed, vesicle-like compartment, and the return of the vesicle to the plasma membrane. This cycle is hormonally regulated (13) as well as sensitive to pharmacological agents that block either the initial invagination of the membrane (14) or the return of the vesicle from the cell interior(15) .
Cells may also use caveolae to
compartmentalize signal transduction at the cell
surface(11, 16) . Recently we reported that IL-1
stimulates the conversion of sphingomyelin to ceramide only in the
caveolae membrane of normal human fibroblasts(17) . The
ceramide, in turn, blocks PDGF-stimulated DNA synthesis in these cells.
The modulation of PDGF signaling could occur directly in caveolae or at
a remote location in the cell. To distinguish between these
possibilities, we first needed to know where the PDGF-stimulated
phosphorylation cascade originates. We now present evidence this occurs
in caveolae.
Figure 1:
PDGF receptors are concentrated in
caveolae. A, confluent, normal human fibroblasts were grown in
the absence of serum for 12 h. Caveolae were isolated and 5 µg of
protein from either the post-nuclear supernatant (PNS), the
plasma membrane (PM), the non-caveolae membrane (NCM), or the caveolae membrane (CM) fractions were
separated by polyacrylamide electrophoresis, transferred to PVDF
membrane (Millipore), and then blotted with either polyclonal
anti-PDGF receptor IgG (PDGFR) or mAb anti-caveolin IgG (Caveolin). B-E, indirect immunofluorescence
co-localization of PDGF receptors and caveolin. Normal human
fibroblasts grown in the absence of serum for 12 h were processed for
immunofluorescence. Two different cells were processed using either a
nonimmune rabbit IgG (B) or an irrelevant mAb (C). A
separate set of cells was processed to localize the PDGF receptor (D) and caveolin (E) in the same cell. F,
immunogold co-localization of PDGF receptor (5 nm gold) and caveolin
(10 nm gold) in isolated fibroblast plasma membranes. Normal human
fibroblasts were grown in the absence of serum for 12 h. Upper plasma
membranes were attached to Formvar-coated grids and incubated in the
presence of a mixture containing polyclonal anti-PDGF
receptor IgG
and mAb anti-caveolin IgG. G, immunogold localization on
isolated plasma membrane using a nonimmune rabbit IgG as the primary
antibody.
We used immunocytochemistry to verify the location of the receptor. Normal human fibroblasts grown on coverslips were processed to localize the receptor by indirect immunofluorescence using the same antibody as the one used for the immunoblotting experiments (Fig. 1, B-E). Nonimmune rabbit IgG (Fig. 1B) and an irrelevant mAb (Fig. 1C) gave only background staining. By contrast, the anti-PDGF receptor IgG fluorescence staining was concentrated in patches on the cell surface (Fig. 1D). These patches either stretched along the margin of the cell (arrows) or appeared as irregularly shaped regions over the cell body (arrowheads). The same cell processed using an anti-caveolin IgG (Fig. 1E) had nearly an identical staining pattern. A comparison of both images showed extensive co-localization of the two antigens. This suggests PDGF receptors are concentrated in caveolae. Immunogold labeling of fibroblast plasma membranes attached to Formvar-coated EM grids confirmed the immunofluorescence images (Fig. 1F). The anti-caveolin IgG (10 nm gold) and the anti-PDGF receptor IgG (5 nm gold) were located together in membrane structures with the characteristic morphology of caveolae (arrows). Anti-PDGF receptor IgG was absent from clathrin-coated pits (arrowhead) as well as other regions of membrane. Only background labeling was seen on membranes exposed to the nonimmune rabbit IgG (Fig. 1G).
Figure 2:
Tyrosine phosphorylation of the PDGF
receptor in caveolae. Normal human fibroblasts were grown in the
absence of serum for 12 h. A, cells were incubated in the
presence (lanes 5-8) or absence (lanes
1-4) of 30 ng/ml PDGF for 15 min. Caveolae were isolated and
immunoblotted with either anti-PDGF receptor IgG (PDGFR)
or mAb anti-phosphotyrosine IgG (PY-PDGFR). B, cells
were incubated in the presence of PDGF for the indicated time before
caveolae were isolated and immunoblotted with either anti-PDGF
receptor IgG (PDGFR) of anti-phosphotyrosine IgG (PY-PDGFR).
The kinetics of PDGF-dependent receptor phosphorylation are shown in Fig. 2B. Caveolae were isolated at various times after PDGF was added to the culture media. Before the addition of PDGF, the receptor band did not react with the anti-Tyr(P) IgG (0 min, PY-PDGFR). After 5 min of incubation in the presence of PDGF, strong reactivity appeared (5 min, PY-PDGFR). The phosphorylated receptor remained in caveolae until, after 30 min of incubation, the concentration began to decline (30 min). Coincident with the nearly complete loss of the receptor by 120 min (PDGFR), little phosphoprotein was detected in the caveolae fraction at this time (PY-PDGFR).
PDGF-stimulated
phosphorylation of the PDGF receptor was accompanied by the tyrosine
phosphorylation of a number of proteins in the caveolae fraction (Fig. 3A). Caveolae (CM) and non-caveolae (NCM) fractions were isolated from cells after exposure to
either media alone (0 min) or PDGF for various times (5-120 min).
Fractions were immunoblotted with anti-Tyr(P) IgG and exposed for
either 30 s (Exp. 1) or 5 min (Exp. 2). In the
absence of PDGF, only one band of 63 kDa reacted with the
anti-Tyr(P) IgG (0 min, CM). The addition of PDGF, however,
stimulated a complex pattern of phosphorylation in the caveolae
fraction. At least 7 bands could be resolved (189, 97, 78, 73, 43, 38,
23 kDa) after just 5 min of incubation. An additional three bands
appeared after 15-30 min (126, 86, and 73 kDa). Between 60 and
120 min, many of these bands disappeared, commensurate with the loss of
the PDGF receptor from the caveolae fraction (see Fig. 2B). Nevertheless, there were five bands that
persisted. We could only detect a single reactive band in the
non-caveolae fractions (NCM) during this interval, even though
5 times more protein was loaded in each lane.
Figure 3: Binding of PDGF stimulates phosphorylation of caveolae proteins. Normal human fibroblasts were grown in the absence of serum for 12 h. A, cells were incubated for the indicated time in the presence of 30 ng/ml PDGF. Five µg/lane caveolae membrane (CM) and 25 µg/lane non-caveolae membrane (NCM) protein were separated by electrophoresis and immunoblotted with anti-Tyr(P) IgG. In one experiment (Exp. 1), blots were exposed for 30 s using the ECL method while for the second experiment (Exp. 2) a 5-min exposure was used. B, cells were incubated in the presence (+, lanes 5-8) or absence (-, lanes 1-4) of 30 ng of PDGF for 15 min. Standard fractions were isolated, and 5 µg of protein per lane was separated by electrophoresis and immunoblotted with the indicated antibodies.
Immunoblotting was
used to determine if any intermediates in the PDGF receptor cascade
were present in the caveolae fractions of either unstimulated or
stimulated cells (Fig. 3B). Normal human fibroblasts
were incubated in the presence (+) or absence(-) of PDGF for
15 min before various fractions were prepared for immunoblotting. In
the molecular weight range of 60,000-75,000, we found the
tyrosine phosphatase Syp (Syp), the adapter molecule Shc 66 (Shc), and
pp60 kinase (Src) to be enriched in caveolae of
unstimulated cells. Nck (47 kDa) and MAPK (42 kDa) were also enriched,
as was the noncatalytic subunit of PI 3-kinase (85 kDa). After the
addition of PDGF, there appeared to be an increase in the amount of
Syp, Shc, and MAPK in the caveolae fraction (compare - with
+; Syp, Shc, MAPK) while the concentration of the other enriched
proteins remained unchanged.
Figure 4: PDGF stimulates the association of a tyrosine phosphoprotein with caveolin. Normal human fibroblasts were grown in the absence of serum for 12 h. A, cells were incubated in the presence (+, lanes 3 and 4) or absence (-, lanes 1 and 2) of 30 ng/ml PDGF for 15 min and immunoprecipitated using either polyclonal anti-caveolin IgG (lanes 2 and 4) or the preimmune IgG from the same rabbit (lanes 1 and 3). B, cells were incubated in the presence of media alone (lane 1) or media that contained either 20 ng/ml PDGF (lane 2), 10 µg/ml insulin (lane 3), 20 ng/ml EGF (lane 4), or 10 ng/ml acidic FGF (lane 5) for 15 min at 37 °C before lysis. One-half of the lysate was immunoprecipitated with polyclonal anti-caveolin IgG and immunoblotted with either anti-Tyr(P) IgG (top panel, PY-190) or anti-caveolin IgG (middle panel, Caveolin). The remaining portion of the lysate was immunoprecipitated with an anti-Tyr(P) IgG (bottom panel) and immunoblotted with anti-MAP kinase (PY-MAPK).
The appearance of the 190-kDa phosphoprotein was specific for PDGF (lane 2, Fig. 4B). We were unable to detect the phosphoprotein in cells exposed to either insulin (compare lane 3 with lane 2), EGF (compare lane 4 with lane 2), or acidic FGF (compare lane 5 with lane 2) even though equal amounts of caveolin were immunoprecipitated (middle panel). On the other hand, all of the peptide hormones stimulated an increase in the amount of tyrosine-phosphorylated MAPK (bottom gel, compare lane 1 with lanes 2-5) immunoprecipitated with an anti-Tyr(P) IgG, indicating they were active in these cells.
The phosphoprotein is approximately the same size as the PDGF receptor (Fig. 5A). Therefore, we separated on polyacrylamide gels samples of anti-caveolin (lanes 4 and 5) and preimmune (lanes 2 and 3) immunoprecipitates from cells incubated either in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of PDGF and immunoblotted with anti-PDGF receptor IgG (Fig. 5A). We could not detect a reactive band in these samples even though the anti-Tyr(P) IgG reacted with the 190-kDa protein specifically in samples from PDGF-stimulated cells (Anti-PY, compare lane 4 with 5). PDGF receptor was detected in isolated caveolae from stimulated cells (Anti-PDGFR, lane 1) together with a strong anti-Tyr(P) positive band (Anti-PY, lane 1). A close comparison of the anti-Tyr(P) IgG reactive bands in lanes 1 and 5 indicates that the two phosphoproteins have slightly different molecular weights.
Figure 5: The 190-kDa phosphoprotein is not the PDGF receptor. Cells were incubated in the presence (+) or absence(-) of PDGF for either 15 min (A-C) or for the indicated time (B). Samples of either whole cell (A and B) or individual fractions collected during caveolae purification (C) were used to immunoprecipitate either caveolin (A, lanes 2-5; B, lanes 5-9), PDGF receptor (B, lanes 1-4) or phosphotyrosine-containing proteins (C). Immunoprecipitates were separated by gel electrophoresis and immunoblotted with either anti-PDGF receptor IgG (A and C), anti-caveolin IgG (B), or anti-Tyr(P) IgG (A and B).
We also could not detect caveolin in anti-PDGF receptor IgG immunoprecipitates (Fig. 5B). Strong immunoblot reactivity with an anti-Tyr(P) IgG was present in samples from PDGF-stimulated cells (Anti-PY, lane 4) but caveolin could not be detected (Anti-Caveolin, lane 4). In the same experiment, we detected a time-dependent association of the 190-kDa phosphoprotein with caveolin. Anti-caveolin immunoprecipitates were prepared from cells that had been incubated in the presence of PDGF for various times (lanes 5-9) and blotted with either anti-Tyr(P) IgG (Anti-PY) or anti-caveolin IgG (Anti-Caveolin). Initially there was no phosphoprotein in the immunoprecipitate (Anti-PY, lane 5). After 5 min, a reactive band was present (lane 6) and remained associated with caveolin until 120 min (lane 9). The loss of the 190-kDa protein from anti-caveolin immunoprecipitates coincided with the migration of PDGF receptor out of caveolae (Fig. 2).
The intensity of the anti-Tyr(P) IgG-positive, 190-kDa band in the caveolae fraction was markedly less than the band of similar molecular weight in the anti-caveolin IgG immunoprecipitate (Fig. 5B, compare lanes 4 and 6). The two bands also appeared to have slightly different molecular weights. Nevertheless, we wanted to determine directly that PDGF stimulated receptor phosphorylation in the caveolae fraction (Fig. 5C). Samples (30 µg of protein) of postnuclear supernatant (lanes 1 and 5), plasma membrane (lanes 2 and 6), non-caveolae membrane (lanes 3 and 7), and caveolae membrane (lanes 4 and 8) were prepared from cells that had been incubated in the presence (lanes 5-8) or absence (lanes 1-4) of PDGF. Anti-Tyr(P) IgG immunoprecipitates were prepared from these samples, separated by gel electrophoresis, and immunoblotted with anti-PDGF receptor IgG. PDGF receptor was not detected in any of the fractions from control cells (lanes 1-4). After PDGF treatment, however, the anti-Tyr(P) IgG immunoprecipitated PDGF receptors from the postnuclear supernatant (lane 5) and the plasma membrane (lane 6). Immunoprecipitates of non-caveolae membranes yielded little PDGF receptor (lane 7), but there was a very intense band in the caveolae membrane (lane 8). These results further emphasize the select location of the PDGF receptor on the surface of fibroblasts.
Light and electron microscopic immunocytochemistry, as well as immunoblots of membrane fractions, show PDGF receptors to be concentrated in caveolae. We cannot determine with certainty that this is the exclusive location of these receptors at the cell surface. Nevertheless, we did not detect tyrosine phosphorylation of non-caveolae membrane proteins in stimulated cells, and five molecules known to interact with PDGF receptor during signal transduction were, like the receptor, clearly enriched in caveolae. The receptors began to migrate out of caveolae after 1 h of exposure to PDGF. Coincidentally, the number of phosphotyrosine proteins in this fraction declined. This suggests that the termination of the tyrosine phosphorylation signal is coupled to the loss of the receptor from caveolae. The most parsimonious interpretation of these results is that caveolae are major sites of PDGF-stimulated signal transduction.
We also found that a 190-kDa phosphoprotein interacts with caveolin in cells exposed to PDGF. Neither this protein, nor any other phosphoprotein, was detected in immunoprecipitates from cells exposed to other hormones. This protein is not the PDGF receptor. Nevertheless, it is the third protein found to interact with caveolin. This suggests that caveolin, in addition to functioning in cholesterol transport (21) , plays a role in organizing caveolae proteins during signal transduction(22) .
These results further confirm a role for
caveolae in processing information at the cell surface(16) . So
far we have localized four other signaling events to caveolae:
regulation of caveolae internalization by histamine(13) ,
EGF-dependent activation of Raf-1, ()endothelial
nitric-oxide synthase activity(26) , and IL-1
-stimulated
ceramide production (17) . In addition, a variety of hormone
receptors and signal transducing molecules appear to be enriched in
caveolae(27) . The presence of all these molecules at one
location most likely is required for the proper integration of multiple
signaling events occurring simultaneously in each cell. Integration of
PDGF and IL-1
signaling through ceramide appears to be a good
model system for studying how this occurs. The effect of ceramide on
PDGF-stimulated phosphorylation of effector molecules can now be
determined directly. This will set the stage for understanding how
ceramide function is altered in damaged caveolae from transformed
cells(25) . The conversion of sphingomyelin to ceramide or the
interaction of ceramide with the PDGF receptor might be impaired in
these cells, leading to uncontrolled cell proliferation.
ACKNOWLEDGEMENTS
hspace=3 SRC="/icons/back.GIF">