From the Department of Biochemistry and Molecular Biology East
Tennessee State University, James H. Quillen College of Medicine,
Box 70581, Johnson City, Tennessee 37614-0581
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INTRODUCTION |
Post-translational modification with isoprenoids results in
considerable structural diversity at the carboxyl terminus of many
proteins. Four such carboxyl-terminal structural motifs have been
identified (for review see Ref. 1). These are
farnesylated-methylated cysteine, geranylgeranylated-methylated
cysteine, digeranylgeranylated vicinal cysteines of the -CC rab
proteins, and digeranylgeranylated residue-interrupted cysteines of the
-CXC rab proteins. The carboxyl-terminal cysteine of the
-CXC rabs is methylated, whereas the carboxyl-terminal cysteine of the -CC rabs is not. Since these modifications always increase the hydrophobic character of the substituted proteins, it is
generally assumed that they are involved in the association of proteins
so modified with lipid bilayer membranes. This assumption is supported
by the observation that most prenylated proteins are bound to cellular
membranes, at least under some physiological conditions.
A more recent hypothesis for the function of protein prenylation
that is more in keeping with the observed structural diversity and
membrane specificity is that these lipid modifications also serve to
mediate protein-protein interactions (2). A well studied example of
this function for protein prenylation is the heterodimeric association
of rab proteins with GDP dissociation inhibitor molecules to form
soluble complexes that appear to be dependent on the
digeranylgeranylation of the rab proteins (3). Another recent example
from our laboratory is the mechanism of endoproteolytic cleavage of the
farnesylated prelamin A molecule, which is mediated by an enzyme that
possesses a specific farnesyl binding site (4).
It has been noted that it is possible that even membrane-associated
prenylated proteins may utilize a polyisoprenoid-dependent protein-protein interaction for membrane binding (2). Recognition of
the polyisoprenoid and other structural elements of the protein by
membrane receptors could confer the necessary localization of
particular prenylated proteins to particular subcellular compartments. Sequestration of the polyisoprenoid would be important in preventing nonspecific association of the protein with inappropriate
membranes.
Farnesylated proteins are found in a number of cellular compartments
(1) including plasma membrane (p21ras,
-transducin),
peroxisomes (PxF) and nuclei (lamin B, prelamin A). For the naturally
occurring forms of N, H, and K p21ras, it is clear that plasma
membrane localization shows an absolute requirement for farnesylation
(5), yet the occurrence of farnesylated proteins in other compartments
suggests the existence of some high affinity plasma membrane receptor
that can recognize not only the isoprenoid but other structural
features of the protein as well. Studies on site-directed mutagenic
alterations of p21ras proteins are consistent with this concept
and have clearly identified the hypervariable region of p21ras
as a second domain that specifies its plasma membrane localization (6).
In this report we present quantitative in vitro binding studies that confirm the existence of a high affinity plasma membrane receptor for p21ras.
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MATERIALS AND METHODS |
NIH3T3 Mouse Fibroblast Culture and Plasma Membrane
Isolation--
NIH3T3 mouse fibroblast cultures were grown in
Dulbecco's modified Eagle's medium (high glucose) (Nova-Tech, Inc.)
supplemented with 10% fetal bovine serum (v/v) (Life Technologies,
Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml
amphotericin B. Twenty-four h before harvesting the cells for plasma
membrane isolation, culture media was supplemented with 8 µg/ml
lovastatin to deplete the association of endogenous p21ras with
the plasma membrane.
A highly enriched plasma membrane fraction from NIH3T3 cells was
isolated as described (7). A protease inhibitor mix (1 mM
each of antipain, aprotinin, bestatin, chymostatin, pepstatin A,
leupeptin, and phenylmethylsulfonyl fluoride) was added to the plasma
membrane fractions that were stored at
70 °C until used. The
purity of plasma membrane fraction was assessed via marker enzyme
activities of 5
-nucleotidase (8) and glucose 6-phosphatase (9).
Generation of Recombinant Baculovirus-expressing His-tagged H, N,
and K p21ras, CVLL-p21Ha-ras and Lamin B; Purification
of Recombinant Proteins and Iodination of
p21Ha-ras--
Recombinant baculovirus-expressing
p21Ha-ras with a histidine tag affixed to its N terminus was a
gift from Dr. Sandra L. Hoffmann (University of Texas Southwestern
Medical Center, Dallas, TX). A BamHI/SalI
fragment containing full-length coding sequence of p21Ki-ras4B
and a BamHI/HindIII fragment containing
full-length sequence of p21N-raswere cloned into compatible
cloning sites of pBlueBacHis2 vector (Invitrogen, San Diego, CA). The
constructs were cotransfected with Bac-N-Blue baculovirus DNA into sf-9
cells (Invitrogen) with the aid of a cationic liposome-mediated
transfection kit (Invitrogen) employed according to the manufacturer's
instructions. The recombinant baculoviruses were screened for and
isolated in sf-9 cells as described (10). A single virus clone for each
of the proteins was chosen for all further experimentation.
Approximately, 5 × 106 sf-9 cells
(Invitrogen)/75-cm2 flask were infected (~1
plaque-forming unit/cell) with recombinant baculoviruses derived from
p21Ha-ras, p21Ki-ras, and p21N-ras constructs,
respectively. The infected cells were grown in complete TNM-FH media
(Invitrogen) supplemented with 10 µg/ml gentamicin at 27 °C for
48 h. The infected sf-9 cells were collected by centrifugation and
after several washings with
PBS,1 were processed for
membrane and cytoplasmic fractions isolation (11, 12). Both
cytoplasmic- and membrane-associated forms of recombinant
p21Ha-ras and membrane-associated forms of p21Ki-ras
and p21N-ras were purified via metal affinity chromatography
Xpress System (Invitrogen) and imidazole elution (50-75
mM). Recombinant baculovirus-expressing CVLL-p21Ha-ras was a gift from (Dr. Paul Kirschmeier,
Schering-Plow Research Institute, Kenilworth, NJ). sf-9 cells were
infected with recombinant baculovirus-expressing CVLL-p21Ha-ras
as above, and the resultant protein was purified as described (10). A
highly purified preparation of lamin B was obtained from rat liver
using published procedures (13).
Recombinant p21Ha-ras derived from the membrane fraction of
infected sf-9 cells was tagged with 125I (NEN Life Science
Products/Dupont) in the presence of Iodo-Beads (Pierce) according to
the manufacturer's instructions. Unbound 125I was removed
via dialysis.
Binding of 125I-labeled p21Ha-ras to
NIH3T3 Plasma Membranes and Competition Assays--
Aliquots of plasma
membranes (100 µg of protein) were incubated with varying
concentrations of 125I-p21Ha-ras (5-150
nM) with or without a 100-fold excess of unlabeled
p21Ha-ras in a total volume of 100 µl. The incubation medium
consisted of phosphate-buffered saline containing 5 mM
MgCl2, 30 µM guanosine 5
-diphosphate, 0.1%
bovine serum albumin, 0.5 M NaCl, and 0.8% Triton X-100.
The binding of the radioactive ligand was allowed to proceed for 1 h at 25 °C. Incubations were terminated by the addition of ice-cold
PBS containing 0.8% Triton X-100 and 0.1% bovine serum albumin. The
unbound radioactivity was removed via filtration of the reaction
mixture through Whatman glass microfiber filters. The filters were
washed >6 times with PBS containing 0.8% Triton X-100 and 0.1%
bovine serum albumin and air-dried, and radioactivity was counted in a
gamma counter.
A time course experiment for binding was performed for 15, 30, 45, 60, and 75 min, utilizing the optimal conditions outlined above. Saturation
of the binding was observed after 45 min, and therefore all experiments
were conducted for 1 h. To determine reversibility of binding,
matched samples of plasma membranes (100 µg of protein) were
incubated with 125I-p21Ha-ras (125 nM),
and the binding of the radioactive ligand was allowed to proceed at
25 °C. After a 1-h incubation, a 100-fold excess of unlabeled
p21Ha-ras was added to one set of samples, and incubation was
continued for an additional 1 h. Incubations were then terminated
by the addition of ice-cold PBS containing 0.8% Triton X-100 and 0.1% bovine serum albumin. The reaction mixture was then centrifuged at
13,000 × g for 15 min, and the difference between the
membrane-bound counts in the presence and absence of excess unlabeled
p21Ha-ras was determined.
To study the effect of different competitors (non-farnesylated
p21Ha-ras, bovine serum albumin, p21N-ras
p21Ki-ras, CVLL-p21Ha-ras, rat liver lamin B,
LLGNSSPRTQSPQNCfarnesyl, and N-acetyl farnesyl
methyl cysteine) on binding of iodinated p21Ha-ras to plasma
membranes, varying concentrations (5 nM to 5 µM) of competitors were added with
125I-p21Ha-ras (50 nM) under binding
conditions outlined above.
Nonlinear regression analysis of saturation binding and competitive
inhibition data were performed with SigmaPlot (Jandel Scientific, San
Rafael, CA). Scatchard plot analysis and calculations of
IC50 values were performed as described (14) with the
linear regression performed with Sigma Plot.
To evaluate whether the binding of p21Ha-ras to plasma membrane
is dependent on p21Ha-ras being bound to GTP or GDP, the
binding studies were carried out in the presence of 30 µM
GDP or 25 µM GTP
S (Biomol) under the conditions
outlined for binding of 125I-labeled p21Ha-ras to
NIH3T3 plasma membranes above. These concentrations of unbound ligand
are approximately 1,000-fold excess to the concentration of GDP-loaded
p21Ha-ras, which gives half-maximal binding to plasma
membranes. Binding reaction mixes were preincubated at 37 °C for 100 min in the absence of membranes to allow nucleotide exchange to occur
(15) and transferred to a 25 °C water bath, and the binding reaction
was initiated by the addition of membranes.
Ligand Blotting Assays--
Ligand blots were performed by a
previously described method (16) with some modifications. Approximately
25 µg of NIH3T3 plasma membrane proteins were separated via
SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes. The blots were incubated in PBS containing
0.3% Tween, 0.5% Triton X-100, 5 mM MgCl2, 30 µM guanosine 5
-diphosphate, 0.1% bovine serum albumin,
and 125I-p21Ha-ras (50 nM) with or
without a 100-fold excess of unlabeled p21Ha-ras with constant
shaking at 25 °C. After an 18-h incubation, blots were washed (>5
times) with PBS containing 0.3% Tween and 0.5% Triton X-100,
air-dried, and exposed to Kodak X-Omat AR film at
70 °C.
Binding of Iodinated p21Ha-ras with
Trypsin-treated Plasma Membranes--
To determine if the binding of
iodinated p21Ha-ras is to the protein component(s) of the
plasma membrane, NIH3T3 plasma membranes were pretreated with trypsin
(10 µg/ml) for 30 min at 25 °C followed by the addition of
protease inhibitor mix. For control experiments, trypsin was
inactivated with protease inhibitor mix and then added to the binding
reaction mixture. For other series of experiments, plasma membrane
proteins were also inactivated by incubation at 100 °C for 5 min.
The binding studies were carried out exactly as above.
Other Assays--
Protein concentrations were determined by BCA
method (Pierce). N-Acetyl farnesyl methyl cysteine was
prepared by acid-catalyzed methylation of commercial
N-acetyl farnesyl cysteine (Calbiochem) as described
previously (17). Gas-liquid chromatographic analysis of the
radiolabeled isoprenoid group of baculovirus-generated p21Ha-ras was done after Raney nickel cleavage of
immunoprecipitated [3H]mevalonate-labeled protein, as
described previously (18).
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RESULTS |
Saturation Binding of Farnesylated p21Ha-ras to 3T3
Cell Plasma Membranes--
Farnesylated, geranylgeranylated, and
nonprenylated p21Ha-ras proteins were synthesized in the
baculovirus insect cell expression system, and the proteins were
purified to homogeneity as described under "Materials and Methods"
(Fig. 1). All of the
baculovirus-generated p21Ha-ras proteins showed
immunoreactivity with Y13-259 antibody in Western blots (data not
shown). Synthesis of protein in the presence of [3H]mevalonate followed by immunoprecipitation gave rise
to labeled material from membranes. This material was analyzed for
farnesylation by Raney nickel cleavage followed by radiolabeled
gas-liquid chromatography and radiodetection (Fig.
2) as described previously (18). These results further demonstrate that we have prepared bona-fide
p21Ha-ras in sf-9 insect cells, as has also been reported
elsewhere (11, 19-21).

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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of the baculovirus-generated p21ras proteins.
A Coomassie-stained 10% polyacrylamide minigel with
membrane-associated p21Ha-ras (lane 1), cytosolic
p21Ha-ras (lane 2), CVLL-p21Ha-ras
(lane 3), p21Ki-ras (lane 4),
p21N-ras (lane 5), and lamin B from rat liver
(lane 6) is shown.
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Fig. 2.
Farnesylation of membrane-associated
p21Ha-ras expressed in sf-9 cells. Infected cells were
incubated at room temperature overnight with
[3H]mevalonolactone (200 µCi/ml) in the presence of 25 µM lovastatin. Labeled p21Ha-ras derived from the
membrane fraction of infected sf-9 cells was treated with Raney nickel
to release the isoprenoid moiety, which was then analyzed by gas-liquid
chromatography and radiodetection (B). The peaks for
p21Ha-ras were identified by comparison of retention time
values with the ones generated from Raney nickel-treated synthetic
farnesyl (peak 1) and geranylgeranyl cysteine methyl esters
(peak 2) (A). Recombinant p21Ha-ras
released only the farnesyl group (A, peak 1).
FID, flame ionization detector; cps, counts/s;
stds, standards.
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NIH3T3 cells were pretreated with lovastatin for 24 h before
harvest for membrane preparation to decrease any endogenous
p21ras occupancy of potential
farnesylation-dependent binding sites. An enriched plasma
membrane fraction from lovastatin-treated NIH3T3 cells was prepared by
means of discontinuous Ficoll and sucrose density gradients. Marker
enzyme analysis of the plasma membrane fraction showed that the
specific activity of 5
-nucleotidase was 12.5-fold enriched in this
fraction compared with the initial homogenate. Based on
glucose-6-phosphatase activity, the cross-contamination of the plasma
membrane fraction with microsomes was approximately 11%.
We examined the kinetics of binding of the purified,
125I-p21Ha-ras to the NIH3T3 plasma membranes,
subtracting out nonspecific binding estimated from the binding in the
presence of excess unlabeled ligand. The results (Fig.
3) are consistent with a saturable
binding site for p21Ha-ras, presumably a receptor protein. At
saturation, approximately 53% of the total binding was specific
binding of 125I-p21Ha-ras to the NIH3T3 plasma
membranes. Scatchard analysis of the specific ligand binding data (Fig.
3, inset) using a one-site model yielded an estimated
Kd and Bmax values of
24.4 ± 5.3 nM (mean ± S.D.) and 137.3 ± 27.1 pmol/mg of protein, respectively. To determine if the binding is
reversible under these conditions, 125I-p21Ha-ras
was allowed to bind with plasma membranes to saturation, and then a
100-fold excess of unlabeled p21Ha-ras was added to the assay
mixture. After 1 h, nearly half of the counts had been displaced
from the membranes, consistent with reversible association of the
ligand with the binding site.

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Fig. 3.
Saturation binding of
125I-p21Ha-ras (farnesylated) to NIH3T3 plasma
membranes and Scatchard plot (one-site model) of specific binding showing estimates of Kd and
Bmax. Aliquots of plasma membranes were
incubated with varying concentrations of
125I-p21Ha-ras (5-150 nM) with or
without a 100-fold excess of unlabeled p21Ha-ras as described
under "Materials and Methods." Every data point represents an
average (±S.D.) of five replicates of three independent experiments
(n = 15).
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We also examined the effect of the GTP loading on the membrane binding.
To do this, binding was assayed with p21Ha-ras loaded with
GTP
S, a nonhydrolyzable GTP analogue. The GTP
S-loaded p21Ha-ras binding exhibited negative cooperativity, with an
n value of 0.0342 (Fig.
4).

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Fig. 4.
Binding of
125I-p21Ha-ras loaded with the GTP analogue
GTP S to NIH3T3 plasma membranes. A Hill plot analysis of the
binding data is also shown. Aliquots of plasma membranes were incubated with varying concentrations of 125I-p21Ha-ras
(5-150 nM) with or without a 100-fold excess of unlabeled
p21Ha-ras in the presence of 30 µM GDP or 25 µM GTP S as described under "Materials and
Methods." Every data point represents an average (±S.D.) of three
replicates of two independent experiments (n = 6).
Y, fractional saturation.
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To verify the protein nature of this putative receptor, we examined the
effect of treatment of the plasma membranes with heat or trypsin on the
binding activity. Treatment of plasma membranes with either heat or
trypsin significantly decreased 125I-p21Ha-ras
binding (Fig. 5). These results clearly
demonstrate that the preferential association of p21Ha-ras with
plasma membranes is due to a nonlipid component, i.e. a protein. Comparison of binding activities of the different membrane fractions from the gradient also confirmed that the putative receptor for p21Ha-ras co-purified with the plasma membrane-enriched
fraction (Fig. 5).

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Fig. 5.
Binding of
125I-p21Ha-ras (farnesylated) to different
subcellular fractions of NIH3T3 cells. NIH3T3 plasma membrane and
microsomal and large particulate fractions were assayed for
p21Ha-ras binding activity with 50 nM
p21Ha-ras. In one set of experiments, the fractions were
pretreated with trypsin or trypsin inactivated with protease inhibitor
mix. In another series of experiments, subcellular fractions were also inactivated by incubation at 100 °C for 5 min. The binding studies were carried out as described under "Materials and Methods." The mean and S.D. of three determinations from two independent experiments are shown.
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Role of the Farnesyl Group in Receptor Binding--
The saturation
binding assay described above provides a method for quantitatively
determining the effect of the farnesyl group on binding of
p21Ha-ras to its receptor in plasma membranes. We performed
competitive inhibition studies with baculovirus-generated
nonfarnesylated cytosolic p21Ha-ras. The cytosolic protein has
been shown to be concentrated in the aqueous layer after Triton X-114
partitioning and, hence, to be nonlipidated. We confirmed this for our
preparations of the cytosolic protein by demonstrating that this
material could be farnesylated by reticulocyte lysates in
vitro (data not shown). We could not obtain any inhibition of
farnesylated p21Ha-ras by the nonfarnesylated protein at
concentrations as much as 1,000-fold higher than the farnesylated
ligand (Fig. 6). On the other hand, unlabeled, fully processed N, H, or K p21ras prepared from sf-9
cells were all efficient competitive inhibitors.

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Fig. 6.
Binding of
125I-p21Ha-ras with NIH3T3 plasma
membranes in the presence of unlabeled competitors.
Nonfarnesylated p21Ha-ras, bovine serum albumin,
p21N-ras, p21Ki-ras, CVLL-p21Ha-ras, rat liver
lamin B, LLGNSSPRTQSPQNCfarnesyl, and N-acetyl
farnesyl methyl cysteine (5 nM to 5 µM) were
added with 125I-p21Ha-ras (50 nM) under
binding conditions outlined under "Materials and Methods."
Calculations of IC50 and Ki values were
performed as described (11). BSA, bovine serum
albumin.
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The farnesylation requirement for receptor binding is indicative of
recognition of the polyisoprenoid group. To examine this point further,
we synthesized N-acetyl-S-farnesyl methyl
cysteine, an analogue of the carboxyl-terminal amino acid residue of
the p21ras proteins. This compound is a weak competitive
inhibitor of p21Ha-ras binding to the receptor (Table
I). Competitive inhibition was also
observed with another farnesylated protein, lamin B, and with an
S-farnesyl cysteine methyl ester peptide corresponding to
the carboxyl-terminal 15 amino acid residues of prelamin A (Table I).
These results indicate that although the receptor recognizes the
polyisoprenoid moiety, binding is also mediated by another domain on
the p21ras protein.
It has been reported that transfection of 3T3 cells with
geranylgeranylated p21Ha-ras (CAAX = CVLL)
results in a membrane-associated protein that is transforming when
oncogenic but inhibits cellular growth p21Ha-ras when wild type
(22). We considered the possibility that this observation might reflect
some difference in the interaction of this reported (22)
geranylgeranylated protein and the wild-type farnesylated protein with
the receptor. Competitive inhibition studies with this CVLL mutant of
p21Ha-ras, previously shown to be geranylgeranylated and to
produce growth inhibition, were performed. The results (Fig. 6)
indicate that the, presumably, geranylgeranylated p21Ha-ras has
an affinity for the receptor that is comparable to that of the
wild-type p21ras proteins. These results suggest that the
p21ras receptor does not exhibit a strong discrimination
between farnesyl and geranylgeranyl substituents.
Ligand Blot Analysis of p21Ha-ras Binding Sites in the
Plasma Membrane-enriched Fraction--
Another technique that can
visualize a membrane-associated receptor is ligand blotting (16). In
this technique, association of a radioiodinated ligand with its
receptor can be demonstrated by SDS-polyacrylamide gel electrophoresis
separation of the membrane proteins, their transfer to nitrocellulose
filters, and incubation of the filters with ligand. We applied this
method to the p21Ha-ras receptor. The results (Fig.
7) indicate two bands that appear to be
specifically labeled by 125I-p21Ha-ras at 45 and 35 kDa (lane 1); this binding was almost completely eliminated
in the presence of a 100-fold excess of noniodinated farnesylated
p21Ha-ras (lane 2). The binding of
125I-p21Ha-ras with 45 and 35 kDa polypeptides in
ligand blots was not affected in the presence of a 100-fold excess of
nonfarnesylated cytosolic p21Ha-ras (Fig. 7, lane
3). Binding also was seen to be reduced by doing the blots in the
presence of 5 µM N-acetyl farnesyl methyl
cysteine (Fig. 7, lane 4). These results for the proteins
visualized by ligand blot are consistent with the quantitative
competitive inhibition of binding seen in Table I. The binding proteins
(45 and 35 kDa) almost exclusively partitioned into the detergent phase
with Triton X-114 (data not shown), which is consistent with their
being intrinsic membrane proteins.

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Fig. 7.
Ligand blot analysis of
125I-p21Ha-ras binding sites in NIH3T3 plasma
membranes. NIH3T3 plasma membrane proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The blots were incubated with
125I-p21Ha-ras (50 nM) alone
(lane 1) or in the presence of 5 µM unlabeled
farnesylated p21Ha-ras (lane 2), 5 µM
nonfarnesylated cytosolic p21Ha-ras (lane 3), and 5 µM N-acetyl farnesyl methyl cysteine
(lane 4). Proteins capable of binding p21Ha-ras were
visualized by autoradiography. The autoradiograms were processed as
described under "Materials and Methods."
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DISCUSSION |
Plasma membrane localization of p21ras has been shown to
be critical for both normal and oncogenic signaling activities of these proteins (23). Elegant mutant expression studies on recombinant p21ras have demonstrated the structural requirements for N, H,
and K p21ras protein localization to cell plasma membranes (5,
24). Plasma membrane association of p21Ha-ras or
p21N-ras requires post-translational modification of the
protein by two alkyl groups (24). The expression studies indicate that
post-translational modification by acylation (palmitoylation at Cys-181
or Cys-185 or N-terminal myristoylation) and/or prenylation
(farnesylation or geranylgeranylation) produces at least some
association of the dialkylated protein with plasma membranes. Mutants
of p21Ha-ras that only undergo farnesylation do not exhibit
plasma membrane association of p21ras as detected by
immunofluorescence. The carboxyl terminus of p21Ha-ras thus
possesses a dialkyl structure that appears to be required for its
plasma membrane binding. It has been noted (24) that the close
proximity of the N terminus and C terminus of p21ras would
cause myristoylated and carboxyl-terminal-monolipidated p21ras
to present a structure comparable to that of the wild-type protein. The
recognition site for this structure in the plasma membrane has been
hypothesized to be a receptor or docking protein that mediates assembly
of p21ras into the membrane where it is stabilized by
nonspecific lipid-lipid interactions.
The studies reported here, confirm and extend these ideas. Based
on the amount of protein per cell (~1 mg/107 cells) and
the fraction of total cellular protein found in the plasma
membrane-enriched fraction (~5%) used in our binding studies, we
estimate that there are approximately 5 × 105
p21ras receptors/cell. This can be compared with the ~5 × 105 sites/cell reported for transmembrane-bridging
mitogenic peptide receptors such as those for platelet-derived growth
factor and epidermal growth factor (25), which are believed to
transduce growth signals through p21ras. If nothing else, this
number suggests that the total number of such receptors is relatively
small and, therefore, inconsistent with being a lipid or a ubiquitous
membrane protein. The Kd (25 nM) we
observe for p21Ha-ras binding can be compared with that seen
for another farnesylation-dependent protein-protein
interaction of p21ras. In vitro binding of
p21Ki-ras(4B) to human SOS1 has been shown to be absolutely
dependent on farnesylation and exhibits 50% saturation at 200 nM (26). In this comparison, the
farnesylation-dependent binding we have observed appears to be at relatively high affinity.
The ligand blot data we report are also consistent with a farnesyl
group-dependent binding site for p21ras in plasma
membranes. Other p21ras binding activities, with the exception
of the cytosolic hSOS1 protein noted above, do not exhibit such a
requirement for farnesylation. For example, it has been reported (27)
that the 21-24-kDa protein caveolin has p21Ha-ras binding
activity. Caveolin was shown not to have an absolute requirement for
farnesylation of p21Ha-ras to bind it, whereas our binding
activity does. We also tested our ligand blots with parallel
immunoblots with anti-caveolin and did not find co-migration of
caveolin with p21Ha-ras binding activity (data not shown).
We have recently reported a kinetic analysis (4) of the prelamin
A endoprotease that demonstrates that this enzyme functions through
specific binding of the farnesyl group of prelamin A as well as another
domain on that protein. The studies reported here are consistent with
an analogous p21ras receptor that recognizes the lipid moiety
but, based on the weak competition for binding of N-acetyl
farnesyl cysteine methyl ester (which is also detectable in the ligand
blots) and the prelamin A peptide, recognizes other structural features
of p21ras as well.
A plasma membrane receptor for p21ras that recognizes the
protein at least in part through its isoprenoid substituent is, as noted above, consistent with a large body of literature regarding the
effect of site-directed mutations on p21ras localization to the
plasma membrane. There are, however, at least two functional
interpretations of such a binding protein. Hancock and co-workers (24),
have suggested that H and N p21ras assemble into plasma
membranes through initial recognition of two alkyl groups by a docking
protein. The bound protein is then unloaded into the membrane bilayer
where it is stabilized by hydrophobic interaction of the alkyl groups
with the lipid bilayer. The binding activity we have identified is
certainly consistent with this hypothesis. A second possibility is that
the p21ras receptor represents a terminal destination for
p21ras. It should be noted in this regard that the
Kd for the p21ras receptor is much lower
than that reported (28) for binding of the alkylated carboxymethylated
model p21Ki-ras4B peptide (Kd = 480 nM) to lipid vesicles, and therefore p21ras
receptors could be occupied even though surrounded by lipid bilayer. It
is also possible that both the p21ras receptor and the lipid
bilayer are terminal destinations for p21ras but mediate
different functions.
The reversibility of binding of the farnesylated p21Ha-ras as
well as the negatively cooperative binding kinetics observed with GDP
and GTP
S-loaded p21Ha-ras is consistent with the possibility
that the association of p21Ha-ras with the plasma membrane may
be regulated. The sigmoidal kinetics observed when the
p21Ha-ras is GTP-loaded suggests that the receptor may exist as
a homodimer in the membrane. In this regard, it is of interest to note
that a pool of farnesylated cytosolic p21ras has been reported
to exist in 3T3-L1 cells (29).
We thank Drs. Paul Kirschmeier and
Adrienne Cox for helpful advice and constructive criticism. We also
thank Robin Burdine for excellent technical assistance. We also thank
Dr. Fusun Kilic for the synthesis of N-acetyl farnesyl
methyl cysteine and for the farnesylation of prelamin A peptide.