From the
A family of GTP-binding regulatory proteins (G proteins)
transduces signals across the plasma membrane from a large number of
receptors to a smaller number of effectors. Recent studies indicate
that a series of post-translational modifications are required for
their association with the plasma membrane and for their function. In
the case of the G protein
Heterotrimeric G proteins are positioned on the inner face of
the plasma membrane, where they are responsible for linking numerous
receptors with various enzymes and ion channels (for review, see Refs.
1 and 2). A growing body of evidence indicates that several
post-translational modifications are required for their localization on
the inner surface of the membrane and for their function. In the case
of the G protein
Although the amino acid
composition of the CAAX motif appears to play the major role
in determining whether certain proteins of the Ras superfamily are
recognized by FTase or GGTase I(10, 11) , it is not
entirely clear whether the same is true for members of the G protein
To determine the type of prenyl group added to
these proteins, lysates were loaded onto SDS-polyacrylamide gels.
Segments of gels containing the [
These results differ significantly from those obtained on
prenylation of other types of proteins with the farnesylation target
sequence CAAS in the baculovirus expression system. As shown
previously by Buss et al.(30) , the H-Ras protein
containing the farnesylation target sequence CVLS was modified
appropriately with a C
With the growing awareness that the G protein
The possibility that sequences other than
the CAAX motif may play a role in determining the specificity
of prenylation of some proteins has been raised by recent studies of
Rho proteins (40) and yeast mating factor(41) . In this
regard, it is noteworthy that in the present study, overexpression of
mammalian FTase in Sf9 cells increased the proportion of the
Finally, it is interesting to note that the general region
upstream of the CAAX sequence that appears to influence the
specificity of prenylation of the
Finally, it is generally assumed that other members of the
Sixty-five h after infection
with the designated
Sf9 cell
cultures were infected with the designated
The recombinant virus encoding the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunits, the post-translational
modifications include the prenylation of a cysteine residue within a
carboxyl-terminal CAAX motif. Although prenylation has been
shown to involve the addition of either a C
farnesyl or a
C
geranylgeranyl group to proteins, the structural
requirements and functional consequences of adding different types of
prenyl groups to various members of the
subunit family have not
been examined. In the present study, we have employed the baculovirus
expression system to study the structural requirements for attaching
different types of prenyl groups to various members of the
subunit family. We show that the
subunit is modified
by a C
geranylgeranyl group, consistent with the presence
of a geranylgeranylation target sequence in this protein. However, we
found that the
and mutant
subunits are modified by both C
farnesyl and
C
geranylgeranyl groups, despite the presence of an
accepted farnesylation target sequence in both of these proteins. Using
chimeras of the
and
subunits, we
provide evidence indicating that structural elements upstream of the
carboxyl-terminal CAAX motif play a role in the recognition of
members of the
subunit family by the appropriate insect and
mammalian prenyltransferases.
subunits, the post-translational modifications
involve a cysteine residue within a carboxyl-terminal CAAX motif (where C = cysteine, A = aliphatic
amino acid, and X = any amino acid). Three sequential
modifications have been identified, beginning with the addition of a
prenyl group to the Cys residue, the proteolytic cleavage of the final
three residues, and the methylation of the newly exposed Cys residue at
the carboxyl terminus (for review, see Refs. 3 and 4). Two types of
prenyl groups have been shown to be added to proteins with the CAAX motif. A C
farnesyl group is added to the
subunit of transducin(5) , the major G protein in retina,
whereas a longer C
geranylgeranyl moiety is added to the
subunit of G
, G
, and
G
(6, 7) , the major G proteins in brain. The
addition of these prenyl groups has been shown to be catalyzed by
either farnesyltransferase (FTase)
(
)(
)or geranylgeranyltransferase (GGTase I),
whose specificity is determined in part by the residue in the
``X'' position of the substrate
protein(8, 9) . In this regard, it has been reported
that proteins with a CAAX motif ending in Ser or Met are
modified with a C
moiety in a reaction catalyzed by FTase,
whereas those ending in Leu are modified with a C
moiety
in a reaction catalyzed by GGTase I.
subunit family. Moreover, the functional consequences of adding
different types of prenyl groups to this family of proteins remains to
be elucidated. This is of particular interest in view of the importance
of prenylation not only for membrane
association(12, 13, 14) but also for the
biologic activities and protein-protein interactions of heterotrimeric
G proteins(15, 16) , Ras-related
GTPases(11, 17) , and rhodopsin kinase(18) . One
way to explore the structural requirements of prenylation is to express
the G protein
subunits in the baculovirus system. In previous
papers, the baculovirus system has been shown to permit the individual
expression of the
subunits and to possess the enzymes needed to
carry out their prenylation and carboxyl
methylation(19, 15, 20) . In the present paper,
we have employed this system to study the structural requirements for
prenylation of the
,
, and
subunits. We show that the
subunit is modified exclusively by a geranylgeranyl group,
whereas the
and
subunits
are modified by varying proportions of farnesyl and geranylgeranyl
groups in this system. This has important ramifications for use of
subunits expressed in this system for functional studies.
Furthermore, using chimeric
subunits, we provide evidence
indicating that prenyltransferases of both insect and mammalian origin
recognize structural elements of the
subunits other than the
CAAX sequence.
Infection of Sf9 Cells with Recombinant Baculoviruses
Encoding the
For the Subunits
subunit(21) , a PstI/XbaI fragment
containing the entire coding region was generated by polymerase chain
reaction amplification of reverse-transcribed mRNA and was subcloned
into the pVL1392 transfer vector. The
subunit (22, 23) was subcloned into the pVL1393 transfer vector,
as described previously(20) . For the
subunit, a BglII/XbaI fragment containing the
entire coding region of the
subunit, except for the
substitution of Leu-71 with Ser, was prepared by polymerase chain
reaction amplification and subcloned into the pVL1392 transfer vector.
The
,
, and
chimeras were prepared and subcloned into pVL1392 and 1393
transfer vectors, as described previously(24) . All constructs
were sequenced to ensure that no changes were introduced by polymerase
chain reaction amplification. Recombinant viruses were generated by
co-transfection of Spodoptera frugiperda (Sf9) insect cells
with the recombinant pVL1392 and 1393 transfer vectors along with
mutant A. californica nuclear polyhedrosis virus, as described
by the supplier (Pharmigen Corp.). All recombinant viruses were
plaque-purified and were verified by their ability to direct the
expression of the appropriate proteins, as detected by immunoblotting.
The recombinant virus encoding mammalian FTase (25) was
generously provided by Dr. Thomas Kost, Glaxo Corp. Sf9 insect cells
were infected with these recombinant baculoviruses at a multiplicity of
infection of 2 for the
subunits or O.2 for the mammalian FTase.
Sf9 insect cells were grown in monolayers or spinners in TNM-FH medium
containing 10% fetal calf serum at 27 °C. Prior to infection of
cells, the medium was supplemented with gentamicin (50 µg/ml) and
amphotericin B (2.5 µg/ml). Prenyl Group Determination of
Subunits Expressed in Sf9
Cells-To assess the specificity of prenylation of the
subunits, Sf9 cells were infected with viruses encoding the
,
, or
subunits or chimeras with or without the virus encoding the
mammalian FTase. After infection, the cells were maintained in TNM-FH
medium containing 10% calf serum. Between 67 and 72 h after infection,
the cells were labeled in medium containing 200 µCi
[
H]mevalonolactone (MVA), the precursor for the
prenylation reaction (DuPont NEN). This labeling period was selected
after careful study showing this period of labeling to be
representative. In this regard, we compared the ratio of C
versus C
on the
subunits in cells
labeled with [
H]MVA for 5 or 24 h at either 40 or
67 h after infection. The ratios of C
versus C
were comparable in each of the four labeling
conditions (data not shown). Furthermore, the ratios of C
versus C
on the
subunits were not
affected by their co-expression with
subunit (data
not shown). At 72 h after infection, cells were lysed in HME buffer
containing 20 mM HEPES, pH 8.0, 2 mM MgCl
, 1 mM EDTA, 2 mM dithiothreitol, and protease inhibitors by passage through a
25-gauge needle.
H]MVA-labeled
subunits were excised and subjected to electroelution. The
possibility that the eluted [
H]MVA-labeled
subunits might be contaminated with endogenous
subunits derived
from Sf9 cells was excluded in a previous paper (19) which
showed that there was no incorporation of [
H]MVA
into proteins in this region of the gel when lysates from noninfected
or wild-type virus-infected Sf9 cells were examined. The
[
H]MVA-labeled proteins were precipitated with
acetone and solubilized in 8 M guanidine HCl, 0.2 M sodium phosphate, pH 7.0. Prenyl thioether bonds were then
disrupted by incubating the proteins with Raney Nickel catalyst, and
the released [
H]MVA-labeled prenyl chains were
extracted into pentane and reduced over platinum oxide(26) . The
radiolabeled hydrocarbons contained in 50 µl (out of approximately
150 µl) of the pentane phase were subjected to high performance gel
permeation chromatography as described previously(26) .
Retention times of the [
H]MVA-labeled groups were
determined in relation to standards of defined chain length; i.e. 2,6,10-trimethyldodecane (15-carbon farnesane) and
2,6,10,14-tetramethylhexadecane (20-carbon phytane), which were mixed
with the samples prior to injection on the column. For the studies
described in Fig. 1& 5 and , the yields of
H-labeled hydrocarbons in the pentane phase averaged 62
± 6% (mean ± S.E.), based on the acetone-precipitable
counts added to the Raney Nickel reaction. Recoveries of injected
H-labeled hydrocarbons from the column averaged 88 ±
4% (mean ± S.E.).
Figure 1:
Prenyl modifications of G protein
subunits expressed in insect cells. Sf9 insect cell cultures were
infected with baculovirus encoding either the
,
, or
subunit. Sixty-seven h
after infection, each culture was labeled for 5 h with 200 µCi of
[
H]MVA. The expressed
subunits derived from
total cell lysates were subjected to SDS-PAGE and electroeluted. The
[
H]MVA-derived prenyl groups were then released
by Raney Nickel cleavage and analyzed by high performance gel
permeation chromatography, as described under ``Experimental
Procedures.'' The retention times of the farnesane (C15)
and phytane (C20) hydrocarbon standards are shown above each
chromatogram, with each chromatogram being representative of results
obtained from three separate experiments.
FTase and GGTase Assays
Sf9 cell lysates prepared
from either noninfected cells or cells infected with the mammalian
FTase virus were assayed for FTase or GGTase activity by quantitating
the amount of H label transferred from either
[
H]farnesyl pyrophosphate (FPP) or
[
H]geranylgeranyl pyrophosphate (GGPP) to
H-ras or G25K proteins, respectively. Both the H-ras and G25K proteins were obtained as fusion proteins with maltose
binding protein following expression in Escherichia
coli(27) . The assays were conducted for 1 h at 37 °C
in a solution consisting of 20 mM HEPES, pH 7.4, 10 mM MgCl
, 2 mM dithiothreitol, 1 µCi of
[
H]FPP or [
H]GGPP, 1 µl
of H-ras or G25K fusion protein, and 10 µg of Sf9 cell
extract. The assays were stopped by acetone precipitation and the
proteins were resolved on a SDS-polyacrylamide gel prior to
fluorography.
Use of FTase Inhibitor
The selective FTase
inhibitor, BZA-5B, was generously provided by Dr. James Marsters,
Genetech Corp. (28). BZA-5B was dissolved in MeSO, yielding
a 100 mM stock solution. Immediately before use, the stock
solution was diluted 1:200 by addition of 10 mM dithiothreitol
prepared in phosphate-buffered saline, which was then diluted 1:20 by
addition of 1
Grace's medium. Approximately 65 h after
infection with virus, Sf9 cells were treated with BZA-5B at a final
concentration of 25 µM BZA-5B, 0.5 mM
dithiothreitol, and 0.025% Me
SO. About 2 h after addition
of inhibitor, cells were incubated with [
H]MVA
for an additional 4 h.
Prenylation of
The prenylation of proteins that terminate in
CAAX sequences is carried out by distinct prenyltransferases
in mammalian cells(9, 8) . FTase adds a C Subunits in the Baculovirus
Expression System
group to proteins in which X is Ser, whereas GGTase I
attaches a C
moiety to proteins in which X is
Leu. To investigate whether the prenyltransferases in insect cells have
similar structural requirements, we infected Sf9 insect cells with
baculoviruses encoding various
subunits of the G proteins.
Approximately 67 h after infection, cells were labeled with
[
H]MVA for 5 h. After treatment of the proteins
with Raney Nickel to release the prenyl groups, the
H-labeled hydrocarbons were analyzed by gel filtration (Fig. 1). The
subunit in which X is
Leu was found to be modified by a C
group following
expression in insect cells (upper panel). This result confirms
earlier studies showing the
subunit was modified
exclusively by a geranylgeranyl moiety in mammalian cells (7) and following expression in insect cells(20) . To
determine whether the nature of the CAAX motif influences the
specificity of prenylation in insect cells, we constructed a virus
encoding a mutant
subunit (
subunit) in which the Leu in the X position was replaced
with Ser. In contrast to the
subunit, the mutant
subunit was found to be modified by a mixture
of C
and C
groups following expression in
insect cells (middle panel). This result suggests that, unlike
the
subunit, the mutant
subunit can serve as a substrate for the insect FTase. Thus, the
insect FTase recognizes substrates with a CAAX sequence in
which X is Ser in a manner analagous to the mammalian
FTase(29) . However, it is not clear from this result why much
of the mutant
subunit was still modified by a
C
group. One possible explanation is that structural
elements outside the CAAX sequence might be important for
recognition, thereby rendering the
subunit a
substrate for both insect FTase and GGTase. To examine this
possibility, we infected Sf9 insect cells with a virus encoding the
subunit, which was shown previously to be modified
exclusively by a farnesyl moiety in mammalian cells(5) .
Surprisingly, the
subunit in which X is Ser
was also found to be modified by a mixture of C
and
C
groups in insect cells (lower panel). However,
it is noteworthy that the
subunit contained a
lower proportion of the C15 group than the
subunit
(compare middle and lower panels in Fig. 1).
Thus, those
subunits with the geranylgeranylation target sequence
CAAL were modified appropriately with a C
group,
whereas those
subunits with farnesylation target sequence
CAAS were modified inappropriately with both C
and C
groups in the baculovirus expression system.
moiety in Sf9 insect cells in a
manner analagous to mammalian cells. This result was confirmed in the
present study by monitoring the ability of Sf9 cell extracts possessing
both FTase and GGTase activities to transfer
H label from
either [
H]FPP or [
H]GGPP to
the H-Ras fusion protein. As shown in Fig. 2, incorporation of
H label into the H-Ras protein was detected only in the
presence of [
H]FPP. Thus, despite the fact that
the
and
subunits and the
H-Ras protein both contain a CAAS motif, they appear to behave
differently as substrates for the insect prenyltransferases. Taken
together, these results raise a number of questions, particularly with
regard to the identities and structural requirements of the
prenyltransferases responsible for adding the C
versus C
group to the mutant
and
subunits.
Figure 2:
Prenyl modifications of H-Ras protein in
insect cells. The soluble extracts from non-infected Sf9 cells were
assayed for FTase and GGTase activity by visualizing the amount of H label transferred from [
H]FPP or
[
H]GGPP to H-Ras fusion protein by fluorography
(overnight exposure) of the amplified SDS-polyacrylamide
gel.
Effect of FTase Inhibitor on Specificity of
Prenylation
From the results described above, it is not clear
whether the insect FTase or GGTase was responsible for the addition of
the C moiety to the
and
subunits. To examine this question, we treated Sf9
cells with the compound BZA-5B, which has been shown to be a highly
selective inhibitor of the mammalian FTase(28) . To compare the
inhibitory activity of BZA-5B on the insect FTase and GGTase, Sf9 cells
were cultured in the presence of 0, 25, or 50 µM BZA-5B
for 2 h. Subsequently, cell extracts prepared from these cultures were
assayed for FTase or GGTase activity by monitoring the transfer of
H label from [
H]FPP or
[
H]GGPP to H-Ras protein containing the
farnesylation target sequence CVLS or G25K protein containing the
geranylgeranylation target sequence CVLL, respectively. As shown in Fig. 3, BZA-5B completely inhibited the transfer of
[
H]FPP to H-Ras protein at a concentration of 25
or 50 µM (compare lanes 1-3), without
affecting the transfer of [
H]GGPP to G25K protein
(compare lanes 4-6). Thus, BZA-5B was able to
selectively inhibit the insect FTase in a manner analogous to the
mammalian FTase.
Figure 3:
Selective inhibition of insect FTase
activity by BZA-5B. Sixty-five h after plating, cultures of Sf9 insect
cells were treated with BZA-5B at a final concentration of 0, 25, or 50
µM BZA-5B. Six h later, the lysates from these cultures
were assayed for FTase or GGTase I activity by visualizing the amount
of H label transferred from [
H]FPP or
[
H]GGPP to H-Ras fusion protein (lanes
1-3) or G25K fusion protein (lanes 4-6),
respectively, by fluorography of the amplified SDS-polyacrylamide gel
for 2 days. Lanes 1 and 4, represent lysates from
untreated cultures; lanes 2 and 5, represent lysates
from cultures treated with 25 µM BZA-5B; lanes 3 and 6, represent lysates from cultures treated with 50
µM BZA-5B. The data shown have been reproduced in two
separate experiments.
To study the effect of this compound on prenylation in vivo, we infected Sf9 cells with virus encoding the
subunit. Approximately 65 h after infection, cells
were treated with 0 or 25 µM BZA-5B for 2 h and then
labeled with [
H]MVA for 4 h. Equal amounts of
protein from untreated and BZA-5B-treated cells were resolved by
SDS-PAGE and the
H-labeled protein(s) were visualized by
fluorography. Prenylation of the
subunit was
visualized by fluorography and quantitated by scintillation counting of
the excised gel slice containing the labeled
subunit.
As shown in , BZA-5B markedly decreased prenylation of the
subunit by 42 or 61% in two separate experiments. To
determine whether the inhibitory effect of BZA-5B represented loss of
the C
or the C
moiety from the
subunit, equal amounts of protein from untreated and
BZA-5B-treated cells were incubated with Raney Nickel, and the
H-labeled hydrocarbons were analyzed by gel filtration. As
shown in , BZA-5B drastically reduced the proportion of C15 versus C20 chains in the prenyl mixture extracted from the
subunit in two separate experiments. Moreover, from
the distribution of [
H]MVA between C
and C
, it is apparent that the overall decline in
radiolabeling of the
subunit was due mainly to the
loss of the C
modification with little or no decrease in
the C20 modification. Taken together, these results indicate that
GGTase I, rather than FTase, is responsible for the addition of the
C
group to the
subunit following its
expression in insect cells. Thus, despite the presence of a
farnesylation target sequence CVIS, the
subunit is a
substrate for both the FTase and GGTase I in insect cells. Furthermore,
since the fraction of the
subunit modified with the
C
group was drastically decreased, but the fraction
modified with the C
group was not significantly increased,
this suggests that the insect GGTase I is operating at its maximal rate
on this substrate.
Effect of Overexpression of the Mammalian FTase on the
Type of Prenyl Group Added to the
One possible explanation for the existence of two types of
prenyl modification on Subunits in Insect
Cells
is that attachment of the
C
group to the
subunit could be favored
by a preponderance of GGTase I activity compared to FTase activity in
insect cells. If so, one might expect that increased expression of
FTase might result in a decline in the C
modification of
due to competition between FTase and GGTase I for
available substrate protein. Accordingly, we examined whether
overexpression of the mammalian FTase in insect cells could increase
the proportion of the
subunit that was modified with
the C15 moiety. As shown in Fig. 4, infection of Sf9 insect cells
with virus encoding the
and
subunits of the mammalian FTase
resulted (25) in a large increase in enzyme activity. Comparison
of the enzyme activity in soluble fractions prepared from either
noninfected cells or cells infected with the mammalian FTase virus
revealed more than a 15-fold increase in the ability to farnesylate the
H-Ras fusion protein. Moreover, as shown in , co-infection
of Sf9 insect cells with the mammalian FTase virus along with the
virus resulted in a large increase in the proportion
of the
subunit that was modified with the C
moiety, although some of the C
group was still
transferred to the protein. In contrast, co-infection of Sf9 insect
cells with the mammalian FTase virus along with the
virus showed no significant increase in the proportion of the
subunit that was modified with the C
moiety. Thus, the mutant
subunit was a
much less effective substrate for the mammalian FTase than was the
subunit, despite both subunits having a farnesylation
target sequence CAAS. This result, along with the observation
that a smaller fraction of the
subunit was
modified with the C
moiety compared with the
subunit (Fig. 1), raises the distinct possibility that
elements outside the CAAX motif must be important for
recognition of these proteins by the FTases of both mammalian and
insect origin.
Figure 4:
Augmentation of FTase activity by
overexpression of the mammalian FTase in insect cells. Cultures of Sf9
insect cells were infected with wild-type virus (Control) or
with recombinant baculovirus encoding both subunits of the mammalian
FTase (FTase). Sixty-seven h after infection, the soluble
extracts from each culture were assayed for FTase activity by
visualizing the amount of H label transferred from
[
H]FPP to H-Ras fusion protein as described under
``Experimental Procedures.'' The data shown have been
reproduced in two separate experiments.
Domain(s) Upstream of the CAAX Motif Are Important for
Recognition of
From earlier
studies on the Ras protein family, it was concluded that the CAAX motif was sufficient for recognition of these proteins by the
appropriate prenyltransferases(11, 31) . However, the
results of these studies differ significantly from those obtained on
the Subunits by Prenyltransferases
subunits with a similar farnesylation target sequence
CAAS in the present paper ( Fig. 1and ),
raising the distinct possibility that structural elements other than
the CAAX motif might be important for recognition of the
subunits by the FTases and GGTases of both mammalian and insect origin.
To investigate this possibility, we examined the ability of the
COOH-terminal, middle, and NH
-terminal domains of the
subunit to affect the type of prenyl modification added to these
proteins. As shown in Fig. 5, the
chimera
possessing the COOH-terminal domain of the
subunit
and the NH
-terminal and middle domains of the
subunit was modified exclusively with a C
moiety (upper panel), consistent with this chimera having a predicted
geranylgeranylation target sequence CAAL sequence at its COOH
terminus. The reciprocal
chimera possessing the
NH
-terminal domain of the
subunit and the
middle and COOH-terminal domains of the
subunit was
modified predominantly with a C
moiety (middle
panel), again consistent with this chimera having a predicted
farnesylation target sequence CAAS at its COOH terminus. In
this regard, it is noteworthy that the
chimera
appears to be a better substrate for the FTase than the wild-type
subunit (compare Fig. 1and Fig. 5).
However, of most interest, the
chimera having the
NH
- and COOH-terminal domains of the
subunit and the middle domain of the
subunit
was modified predominantly with a C
moiety (lower
panel), despite this chimera having a predicted farnesylation
target sequence CAAS. This result provides direct evidence
that structural elements other than the CAAX sequence are
important for the recognition of the
subunits by the appropriate
prenyltransferases and points to the importance of a 31-amino acid
region in the middle of these proteins. Within this region of the
subunit (from amino acid 16 to 46), there are only 15
nonconserved amino acid differences between the
and
subunits, further defining the particular residues
involved in recognition. At the present time, it is not clear whether
the greater incorporation of
H counts into the
chimera compared with the
and
the
chimeras is due to higher expression of the
protein or because the
protein
is a better substrate for prenylation.
Figure 5:
Prenyl modifications of G protein
chimeras expressed in insect cells. Sf9 insect cell cultures were
infected with baculovirus encoding either the
,
, or
chimeras. Sixty-seven h
after infection, each culture was labeled for 5 h with 200 µCi of
[
H]MVA and total cell lysates were prepared.
After electroelution of the
subunits from SDS-polyacrylamide
gels, the [
H]MVA-derived prenyl groups were
released by Raney Nickel cleavage and analyzed by high performance gel
permeation chromatography, as described under ``Experimental
Procedures.'' The retention times of the farnesane (C15)
and phytane (C20) hydrocarbon standards are shown above each
chromatogram, with each chromatogram being representative of results
obtained from three separate experiments.
subunits play an active role in receptor-effector coupling and receptor
desensitization (for reviews, see Refs. 1, 2, and 32), it is of
particular interest to examine the ability of different types of
subunits to regulate adenylyl cyclase, phospholipase C,
phospholipase A2, ion channels, and receptor kinase activities. Such
studies have not been possible previously because of the difficulty in
resolving the heterogeneous mixtures of
subunits that are
present in tissues and because of the probable existence of as yet
undescribed
and
subunits in these tissues. With the recent
identification of at least five
subunits (33) and ten
subunits(34) ,
studies have been undertaken
recently to produce known combinations of these
subunits in
the baculovirus expression
system(19, 15, 20, 35, 36, 37) .
Of particular interest, this system has been reported to carry out a
number of post-translational modifications(30, 38) ,
including prenylation and carboxyl methylation, that are required for
the function of the
subunits. However, despite the widespread use
of the baculovirus expression system for the production of these
subunits, studies to examine whether the
subunits are
being appropriately processed in this system have been limited to the
subunit(20) .
Structural Requirements for Differential Prenylation of the
It is now well established that two different
types of prenyl moieties (farnesyl and geranylgeranyl) can be added to
proteins terminating in CAAX sequences, with the amino acid
residue in the X position playing a major role in determining
which type of prenyl group is added. In this regard, mutation of the
terminal amino acid in H-Ras from Ser to Leu, or vice versa, has been
shown to reverse the type of prenyl group added to these
proteins(11, 31) . From these earlier studies, it was
concluded that the CAAX motif was the principal determinant
for recognition of Ras and Ras-related proteins by the appropriate
prenyltransferases. The results of the present study show that in many
respects the insect FTase and GGTase I have structural requirements
that are similar to those of their mammalian counterparts. Thus, the
insect FTase catalyzes the addition of a C Subunits
farnesyl moiety
to the
and
subunits ending
in Ser, whereas the insect GGTase I catalyzes the attachment of a
C
geranylgeranyl moiety to the
subunit
ending in Leu. Surprisingly, however, we observed that in Sf9 cells,
C
groups are also attached to the
and
subunits ending in Ser. We speculate that the
alternative modification of the CAAS proteins is catalyzed by
the insect GGTase I, since it is not substantially suppressed by an
inhibitor of FTase (). The possibility that GGTase I can
exhibit cross-specificity under certain circumstances is further
supported by recent data in yeast showing that ram1 null
mutants lacking FTase activity are still able to prenylate some RAS,
presumably by addition of geranylgeranyl group in a reaction catalyzed
by GGTase I(39) .
that underwent C
modification, but did
not similarly increase the proportion of total radioactivity migrating
as C15 in the
mutant. One possible
interpretation of this finding is that the
is
not as good a substrate for mammalian FTase as
,
because it lacks key elements outside the CAAX sequence that
may be important for FTase recognition. Although the precise location
of such upstream recognition domains remains to be defined, the present
study provides two lines of evidence suggesting that both the
NH
-terminal and central region of the
sequence are involved. First, we show that, contrary to
expectations, replacing the NH
-terminal domain of
with the corresponding domain from
(amino acids 1-15) results in an increased proportion of
the
chimera being modified by the C
farnesyl moiety in Sf9 cells. Second, we show that substituting
the central domain of
with the corresponding region
from
(amino acids 16-46) alters the type of
prenyl group added to the
chimera from the C
farnesyl moiety to the C
geranylgeranyl moiety.
Thus, although the presence of a CAAX motif is sufficient for
prenylation of the
subunit family, it appears that elements other
than the CAAX sequence are influential in determining the specificity of the modification. Whether this means that
distinct prenyltransferases are responsible for modifying the G protein
subunits and proteins such as H-Ras remains unclear. However, to
date, only one FTase and one GGTase I have been cloned from mammalian
cells.
subunits includes a 14-amino
acid region previously shown to be important in conferring the
specificity of association with the
subunits(42) . Since
Higgins and Casey (43) have recently demonstrated that
prenylation of the
subunits is not required for
assembly in vitro, one might speculate that prenylation of the
subunits may actually precede their association with the
subunits in vivo. This will be an important topic for future
investigation.
Functional Consequences of Differential
Prenylation
The present study has important implications for the
use of the baculovirus expression system to produce various
subunits for functional studies. Thus, with the realization that the
baculovirus-expressed
subunit is inappropriately
modified by both farnesyl and geranylgeranyl moieties, it is not clear
whether the observed functional differences between the expressed
and
subunits that have been identified in terms of interaction with G
protein
subunits and effectors (15, 36) are due to
differences in the primary structures of the
subunits, the type
of prenyl group added to these proteins, or some combination of both.
Since studies in yeast have suggested that only the farnesylated, but
not the geranylgeranylated, form of the Ras protein is capable of
stimulating adenylyl cyclase(17) , it will be of particular
interest to examine this question more closely. In this regard, the
ability to express and purify particular
subunits modified by
either a farnesyl or geranylgeranyl group in the baculovirus system
should allow the contribution of the prenyl group to be assessed in
terms of
interaction with G
proteins, receptors,
receptor kinases, and effectors. The feasibility of this approach is
supported by data in the present study showing the use of FTase
inhibitors and/or overexpression of FTase to manipulate the proportion
of protein modified by either a farnesyl or geranylgeranyl group in
this system. Thus, in future studies, it should be possible to design
studies to allow the contribution of the prenyl group to be accurately
assessed.
subunit family with CAAX motifs terminating in Leu will
be modified by a geranylgeranyl group. However, this assumption has yet
to be confirmed in any mammalian or baculovirus expression system. With
the realization in the present study that sequences outside the
CAAX motif are important in determining the type of prenyl
group added to this family of proteins, it will be important to examine
the validity of this assumption. In this regard, it is noteworthy that
Rho proteins terminating in Leu can be modified by both farnesyl and
geranylgeranyl groups, depending on presence or absence of specific
amino acids upstream of the CAAX motif(40) . Thus, it
will be of interest in future studies to determine the type of prenyl
group attached to other members of the
subunit family,
particularly a newly identified
subunit that
contains a predicted farnesylation target sequence.
Table: Effect of FTase inhibitor on prenyl
modifications of the subunits
virus, Sf9 cells were treated with 0 or 25
µM FTase inhibitor BZA-5B. After 2 h, cells were labeled
for additional 2 h with 200 µCi of [
H]MVA.
After rinsing, cell lysates were resolved on a SDS-polyacrylamide gel.
To measure incorporation of [
H]MVA, the excised
band containing the radiolabeled
subunit was analyzed by
scintillation counting. To determine the fraction the fraction of the
incorporated [H]MVA label eluting with the C
and
C
standards by HPLC analysis, the remaining portions of
the cell lysates were resolved by SDS-PAGE and the
[
H]-labeled proteins were subjected to Raney
Nickel cleavage. From these two values, the distribution of
[
H]MVA between C
and C
groups was calculated. The results of two separate experiments
are shown.
Table: Prenyl modification of subunits:
effect of overexpression of mammalian FTase in insect cells
virus with or without
the FTase virus. Sixty-seven h after infection, each culture was
labeled for 5 h with 200 µCi of [
H]MVA. After
electroelution of the
subunits from gels, the
[
H]MVA-derived prenyl groups were released by
Raney Nickel cleavage and analyzed by HPLC. The fraction of total dpm
eluting along with the C
farnesane standard is shown for
each culture.
subunits (K. Ray, C. Kunsch, and J. D.
Robishaw, submitted for publication), this brings the total number of
known
subunits to 10.
and
subunits of farnesyl transferase was generously provided by Dr. Thomas
Kost, Glaxo Corp. The FTase inhibitor, BZA-5B, was kindly supplied by
Dr. James Marsters, Genetech Corp.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.