©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regions Outside of the CAAX Motif Influence the Specificity of Prenylation of G Protein Subunits (*)

Vivian K. Kalman , Robert A. Erdman , William A. Maltese , Janet D. Robishaw (§)

From the (1)Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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 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.

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 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.


EXPERIMENTAL PROCEDURES

Infection of Sf9 Cells with Recombinant Baculoviruses Encoding the Subunits

For the 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 Cversus 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 Cversus C were comparable in each of the four labeling conditions (data not shown). Furthermore, the ratios of Cversus 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.

To determine the type of prenyl group added to these proteins, lysates were loaded onto SDS-polyacrylamide gels. Segments of gels containing the [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% MeSO. About 2 h after addition of inhibitor, cells were incubated with [H]MVA for an additional 4 h.


RESULTS

Prenylation of Subunits in the Baculovirus Expression System

The prenylation of proteins that terminate in CAAX sequences is carried out by distinct prenyltransferases in mammalian cells(9, 8) . FTase adds a C 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.

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 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 Cversus 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 Subunits in Insect Cells

One possible explanation for the existence of two types of prenyl modification on 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 Subunits by Prenyltransferases

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 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.




DISCUSSION

With the growing awareness that the G protein 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 Subunits

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 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) .

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 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.

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 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.

Finally, it is generally assumed that other members of the 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

Sixty-five h after infection with the designated 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

Sf9 cell cultures were infected with the designated 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.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM39867 (to J. D. R.) and CA34569 (to W. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, Danville, PA 17822-2614. Tel.: 717-271-6682; Fax: 717-271-6701.

With the cloning of three new subunits (K. Ray, C. Kunsch, and J. D. Robishaw, submitted for publication), this brings the total number of known subunits to 10.

The abbreviations used are: FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; MVA, mevalonolactone; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

The recombinant virus encoding the 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.


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