Proc. Natl. Acad. Sci. USA Vol. 88, pp. 732-736, February 1991 Biochemistry Sequence requirement for peptide recognition by rat brain p2lras protein farnesyltransferase (covalent modification/prenylation/mevalonate/tetrapeptides/enzyme inhibition) YUVAL REISS*, SARAH J. STRADLEYt, LILA M. GIERASCHt, MICHAEL S. BROWN*, AND JOSEPH L. GOLDSTEIN* Departments of *Molecular Genetics and tPharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Contributed by Michael S. Brown, October 30, 1990

ABSTRACT We tested 42 tetrapeptides for their ability to a heterodimer (10, 11). The enzyme recognizes sequences as bind to the rat brain p2l' protein farnesyltransferase as short as four amino acids provided that cysteine is at the estimated by their ability to compete with p2lH"- in a farnesyl- fourth position from the COOH terminus. Recognition was transfer assay. Peptides with the highest affinity had the struc- demonstrated by the ability ofthese peptides to compete with ture Cys-Al-A2-X, where positions Al and A2 are occupied by p21Ha-ras in the protein farnesyltransferase assay. In addition, aliphatic amino acids and position X is-occupied by a COOH- the enzyme adhered to an affinity column containing a terminal methionine, serine, or phenylalanine. Charged resi- hexapeptide corresponding to the COOH-terminal sequence dues reduced affinity slightly at the Al position and much more of p2lKi-msB. A similar enzymatic activity was identified by drastically at the A2 and X positions. Effective inhibitors in- Schaber et al. (12) in crude extracts of bovine brain. cluded tetrapeptides corresponding to the COOH termini of all The above findings suggest that the recognition site for the animal cell proteins known to be farnesylated. In contrast, the protein farnesyltransferase consists only of cysteine and the tetrapeptide Cys-Ala-Ile-Leu (CARL), which corresponds to the three COOH-terminal residues. This conclusion is consistent COOH termini ofseveral neural guanine nucleotide binding (G) with the observation that the various classes of farnesylated protein y subunits, did not compete in the farnesyl-transfer proteins do not share any sequences except the Cys-A1-A2-X assay. Inasmuch as several of these proteins are geranylgera- motif. It is also in line with the experiment of Hancock et al. nylated, the data suggest that the two isoprenes (farnesyl and (8), who attached a DNA sequence encoding the COOH- geranylgeranyl) are transferred by different enzymes. A biotin- terminal four amino acids ofp21Haras to the 3' end ofa cDNA ylated heptapeptide corresponding to the COOH terminus of encoding bacterial protein A. The chimeric protein was p21K'-1B was farnesylated, suggesting that at least some of the prenylated when it was expressed in COS cells, as judged by peptides serve as substrates for the transferase. The data are studies of [3H]mevalonate labeling. Subsequent enzymes in consistent with a model in which a hydrophobic pocket in the the protein farnesylation pathway may also recognize limited protein farnesyltranferase recognizes tetrapeptides through COOH-terminal sequences. Thus, Stephenson and Clarke interactions with the cysteine and the last two amino acids. (13) recently showed that the last enzyme in the pathway, the carboxylmethyltransferase, recognizes short peptides that A farnesyl residue is attached in thioether linkage to the contain a COOH-terminal farnesylcysteine. COOH-terminal cysteine of a variety of intracellular mem- In addition to the farnesyl group, which contains 15 carbon brane-associated proteins. The list includes cellular p21lS atoms, a 20-carbon polyisoprenoid, geranylgeranyl, can also proteins (1), nuclear lamin B (2), and the 'y subunit of bovine be attached covalently to proteins (14, 15). This modification transducin (3). This modification is also found on mating in animal factors secreted by fungi (4, 5). appears to be more common than farnesylation In each case the farnesylated cysteine is initially the fourth cells, but the nature of the recognition sequence is not yet residue from the COOH terminus (for review, see ref. 6). understood. The only known examples ofgeranylgeranylated Farnesylation is followed by proteolytic removal ofthe three proteins are the y subunits of guanine nucleotide binding terminal residues and carboxylmethylation of the cysteine. proteins (G proteins) from bovine brain (16) and rat PC12 These reactions render the COOH termini hydrophobic, cells (17), some of which have the COOH-terminal sequence presumably facilitating the initial attachment of the proteins Cys-Ala-Ile-Leu (CAIL) (18, 19). Whether these proteins are to cell membranes. In some instances the hydrophobicity is modified by the same enzyme that transfers farnesyl groups increased by palmitoylation of nearby cysteines. Inspection to other proteins is unknown. of the sequences of the known farnesylated proteins has To learn more about the peptide recognition site on the defined a weak consensus sequence for farnesylation that protein farnesyltransferase, we have carried out a study consists of Cys-A1-A2-X, where positions Al and A2 are of the ability of various tetrapeptides to compete with occupied by aliphatic amino acids and position X is occupied p21Ha-,s for acceptance ofa farnesyl residue. As a framework by an undefined amino acid (7, 8). for these peptides, we have used the COOH terminus of The likely donor offarnesyl residues is farnesyl pyrophos- p2lKi-sB, Cys-Val-Ile-Met (CVIM), which was the highest phate (FPP), an intermediate in the synthesis of sterols and affinity peptide among those studied (10). The results indicate polyisoprenes in eukaryotic cells (9). Recently, we have that the Al position of the Cys-Al-A2-X motif will accept a isolated from rat brain an enzyme that transfers a farnesyl variety of amino acids, but the A2 position is much more group from [3H]FPP to p2lHa-as protein (10, 11). The purified strict in its requirement for an uncharged residue. At the X enzyme preparation contains two proteins, each with an position, methionine, phenylalanine, and serine are strongly apparent molecular mass ofabout 50 kDa, that appear to form preferred.

The publication costs of this article were defrayed in part by page charge Abbreviations: DTT, dithiothreitol; FPP, famesyl pyrophosphate; G payment. This article must therefore be hereby marked "advertisement" protein, guanine nucleotide binding protein; CAIL, Cys-Ala-Ile-Leu; in accordance with 18 U.S.C. §1734 solely to indicate this fact. CVIM, Cys-Val-Ile-Met; CVFM, Cys-Val-Phe-Met. 732 Downloaded by guest on September 26, 2021 Biochemistry: Reiss et A Proc. Natl. Acad. Sci. USA 88 (1991) 733 MATERIALS AND METHODS RESULTS Peptides. Peptides were prepared by established procedures To screen peptides for their affinity for protein farnesyltrans- of solid-phase synthesis (20). Tetrapeptides were synthesized ferase, we measured the ability of the peptides to compete on the Milligen 9050 synthesizer using Fmoc (9-fluorenylme- with p21Ha-ras for acceptance of [3H]farnesyl from [3H]FPP as thyloxycarbonyl) chemistry. After deprotection of the last catalyzed by a partially purified rat brain protein farnesyl- residue, a portion of the resin was used to make the N-acetyl- transferase. As a reference point for the peptides, we used modified version ofCVIM. This was done off-line in a solution CVIM, which corresponds to the COOH-terminal sequence of acetic anhydride and dimethylformamide at pH 8 (adjusted ofp21Ki-rasB. Fig. 1 A-C shows a series oftypical experiments with diisopropylethylamine). The acetylated and unacetylated in which we systematically substituted alanine, lysine, or peptides were cleaved with 50 ml of trifluoroacetic acid/ leucine, respectively, at each of the three positions after phenol, 95:5 (vol/vol), plus approximately 1 ml of ethane- cysteine in CVIM. In each experiment the results were dithiol added as a scavenger. The N-octyl-modified version of compared with those obtained with CVIM. Alanine and an model 430A lysine were tolerated only at the Al position. Insertion of CVIM was synthesized on Applied Biosystems these amino acids at the A2 or X positions decreased the synthesizer using tBoc (t-butoxycarbonyl) chemistry. The affinity for the enzyme by more than 30 times, as estimated octyl group was added in an amino acid cycle using octanoic by the concentration required for 50% inhibition. Leucine acid. The peptide was cleaved from the resin at 0C with a 10:1:1 ratio of HF (ml)/resin (g)/anisole (ml). The peptides were purified by high pressure liquid chromatography (HPLC) on a Beckman C18 reverse-phase column (21.1 cm x 15 cm), eluted with a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Identity was confirmed for all peptides by quantitative amino acid analysis and for the NH2-terminally modified peptides by fast atom bombardment mass spectrom- etry. Just prior to use, each peptide was dissolved at a concentration of0.8 mM in 10mM dithiothreitol (DTT) except for several of the most hydrophobic peptides, which were dissolved at a concentration of 1 mM in dimethyl sulfoxide/10 mM DTT. All dilutions were made in 10 mM DTT in the absence of dimethyl sulfoxide. Biotinylated Lys-Thr-Ser-Cys-Val-Ile-Met (KTSCVIM) was synthesized on an Applied Biosystems model 430A synthesizer. The biotin group was added after removal of the NH2-terminal protecting group before cleavage ofthe peptide from the resin. Specifically, a 4-fold molar excess of biotin 4-nitrophenyl ester was added to 0.5 g of resin in 75 ml of dimethylformamide at pH 8 and incubated for 5 hr at room temperature. Cleavage, identification, and purification were carried out as described above. To synthesize S-acetoamido-CVIM, purified CVIM was dissolved at a final concentration of 1 mM in 0.1 ml of 0.5 M Tris-Cl, pH 8.0/15 mM DTT. The tube was flushed with nitrogen for 2 min, sealed, and incubated for 2.5 hr at 37°C to reduce the cysteine residue, after which iodoacetamide was added to achieve a final concentration of 35 mM. After incubation for 15 min at 37°C, the reaction was stopped by addition of 10 mM DTT. Complete alkylation of CVIM was confirmed by fast atom bombardment mass spectrometry and HPLC. The molecular mass of the product corresponded to the expected molecular mass of S-acetoamido-CVIM. Assay for Protein Farnesyltransferase. The standard assay 0.1 0.3 1 3 involved measuring the amount of [3H]farnesyl transferred Tetrapeptide (pM) from all-trans-[3H]FPP to recombinant human p2lHa-ras as described (10). Each reaction mixture contained the follow- FIG. 1. Inhilition of protein famesyltransferase by tetrapeptide ing concentrations of components in a final volume of 25 ,ul: analogues of CVIM containing alanine (A), lysine (B), or leucine (C) 50 mM Tris Cl (pH 7.5), 50,M ZnCI2, 20 mM KCl, 1 mM in position X. The standard assay mixture contained 15 pmol of DTT, 30 or 40 ,uM p21Ha-,s, 15 pmol of [3H]FPP (12,000- [3H]FPP, 4-7.5 ,ug of partially purified farnesyltransferase, 30 or 40 ,IM p2lHa,-as, and the indicated concentration of competitor tet- 23,000 dpm/pmol), 4-7.5 /kg of partially purified protein rapeptide. After 30 or 60 min, the amount of [3H]farnesyl attached to farnesyltransferase (Mono Q fraction, ref. 10), and the indi- p2lHa-ras was measured by trichloroacetic acid precipitation. Each cated concentration of competitor peptide added in 3 ,ul of 10 value is the average ofduplicate or triplicate incubations (no peptide) mM DTT. After incubation for 30-60 min at 37°C, the amount or a single incubation (with peptide). Each tetrapeptide was tested in of [3H]farnesyl present in trichloroacetic acid-precipitable a separate experiment together with equimolar concentrations of p21Ha-,s was measured by a filter assay as described (10). A CVIM. The data for inhibition by CVIM (i) are derived from mean value (<0.6% of input was determined in values from 21 experiments in which the variation of individual blank [3H]FPP) values at each concentration was -15%. The mean "100% of parallel incubation mixtures containing no enzyme. This control" value was 13 pmol per min per mg of protein. Ki, concen- blank value was subtracted before calculating percent of tration of tetrapeptide giving 50%o inhibition. The single-letter amino control values. acid code is used. Downloaded by guest on September 26, 2021 734 Biochemistry: Reiss et al. Proc. Natl. Acad. Sci. USA 88 (1991) was tolerated at the A2 position, but it decreased the affinity when inserted at the X position. The substitution of phenylalanine for isoleucine at the A2 position [Cys-Val-Phe-Met (CVFM)] increased the affinity for the partially purified enzyme by 6- to 20-fold, with half-maximal inhibition occurring at 10-25 nM (Fig. 2A; also see Fig. 4). No such effect was observed when phenylalanine was inserted at either of the other two positions. In addition to performing assays with p21Ha-s as a sub- strate, we also performed assays in which the substrate was the biotinylated heptapeptide KTSCVIM, which contains the 4 COOH-terminal amino acids of p21Ki-(IB (21). The biotin was attached to the NH2 terminus by coupling to the resin- Tetrapeptide (pM) attached peptide. The [3H]farnesyl-labeled product was iso- lated by allowing it to bind to beads coated with streptavidin FIG. 3. Inhibition of protein farnesyltransferase by modified 2 A and B shows that the CVFM was 10- to tetrapeptides. Enzyme activity was measured in the presence of (11). Fig. peptide various concentrations of the indicated tetrapeptide as described in 20-fold more potent than CVIM when either p2lHaas or the Fig. 1. The 100lo of control values were 9.3 and 9.2 pmol per min per biotinylated heptapeptide was used as acceptor, respectively. mg of protein in A and B, respectively. In contrast to the experiments in Fig. 1, which were conducted with a partially purified enzyme, experiments shown in Fig. 2 were carried out with a homogeneous preparation of affinity- acids of the Cys-A1-A2-X type. As a reference sequence for purified protein farnesyltransferase. these studies, we used the peptide CVIM, which inhibited the The free sulfhydryl group of the cysteine is likely required protein farnesyltransferase by 50%o at a concentration of0.15 for tetrapeptide inhibition, as indicated by the finding that ,M. Substitution of various amino acids into this framework derivitization with iodoacetamide abolished inhibitory activ- yielded peptides that gave 50% inhibitions at a spectrum of ity (Fig. 3A). A blocked NH2 terminus is not required, as concentrations ranging from 0.025 MM (CVFM) to greater indicated by similar inhibitory activity ofN-acetyl-CVIM and than 50 MM (Fig. 4). N-octyl-CVIM (Fig. 3B) compared to that ofCVIM (Fig. 3A). In general, the highest inhibitory activities were achieved Fig. 4 summarizes the results of all competition assays in when the Al and A2 positions were occupied with nonpolar which we made substitutions in the CVIM sequence. The aliphatic or aromatic amino acids. This stringency was more results are presented in terms of the peptide concentration severe at the A2 than at the Al position. Thus, peptides required for 50% inhibition. Table 1 summarizes the results containing lysine or glutamic acid at the Al position gave 50% of other experiments in which we studied tetrapeptides inhibition at 0.7 and 1.5 AM, respectively. When these two corresponding to the COOH termini of 19 proteins, many of residues were inserted at the A2 position, the affinity for the which are known to be farnesylated. The implications of enzyme declined by more than 50-fold. Glycine and proline these studies are discussed below. lowered inhibitory activity moderately at the Al position (50% inhibition at 3 and 7 MM) and somewhat more severely DISCUSSION at the A2 position (8 and 20 MM). The X position showed the The current data extend previous observations on the p2lS highest stringency. When the first three amino acids were protein farnesyltransferase (10, 11) and further indicate that CVI, methionine was the preferred residue at the X position the recognition site for this enzyme is restricted to four amino but phenylalanine and seine were tolerated with only modest losses in activity (0.5 and 1 MM, respectively). Aliphatic _; 1 X A. p21 H; B. Biotinylated KTSCVIM residues and proline were disruptive at position X, with 50%o .~100 CIM ~VMM inhibitions in the range of 5-11 MM. Glutamic acid, lysine, co 820 KK0..01K 0.35 pM coo0~ 60- Leu Ala Lys CCo040( Cxl Mle Val Ser Pe1 Glu Gly Pro F- CVFM CF II I I co.20 K=0 pM Ki =0.035pM I__II_Val _I E 0,'0.1 0. 1 1 Leu Ser CZ 0.001 0.01 0.1 1 ILe Pro 0.01 0.1 1 10 CVxM Phe Tyr Thr Ala Gly GluLys Tetrapeptide (pM) I, 1 I I I II Val I FIG. 2. Inhibition of farnesylation of p2lHa-ras (A) and biotiny- cvix{_____ .L ~ I Pro ILe Lys lated KTSCVIM (B) by CVFM. (A) Each reaction mixture contained CvI x Met Phe Ser Ala Leu Glu Gly 15 pmol of [3H]FPP, 4.5 or 6 ng of affinity-purified protein farnesyl- ___1___I 1 1 I 1 transferase (10), 40,uM p2lHaras, and the indicated concentration of competitor tetrapeptide. After incubation for 30 min at 370C, the 0.01 0.1 1 10 100 amount of [3H]farnesyl transferred to p21Ha, was measured by the Concentration for 50% Inhibition (pM) standard filter assay. Values shown are the average of two experi- ments. The "100%o of control" values were 16 and 19 nmol per min FIG. 4. Inhibition of protein famesyltransferase by tetrapeptides per mg of protein. (B) Each reaction mixture contained 15 pmol of with single amino acid substitutions in CVIM. Enzyme activity was [3H]FPP, 4.5 or 6 ng of affinity-purified protein farnesyltransferase, measured in the presence ofthe indicated competitor tetrapeptide as 3.4,uM biotinylated KTSCVIM, and the indicated concentration of described in Fig. 1. Each tetrapeptide was tested at seven or eight competitor tetrapeptide. After incubation for 30 min at 37TC, the concentrations ranging from 0.01 to 100 ,uM. The concentration of [3H]farnesyl-labeled peptide was trapped on streptavidin-agarose, tetrapeptide giving 50M inhibition was calculated from the inhibition washed, and separated from the unincorporated [3H]FPP, and ra- curve. The single and double underlines denote tetrapeptides cor- dioactivity was measured by scintillation counting as described (11). responding to the COOH-terminal sequence of mammalian and Values shown are the mean of three experiments. The 100%6 of fungal proteins, respectively, that are candidates for farnesylation control values were 10, 17, and 21 nmol per min per mg of protein. (see Table 1). Downloaded by guest on September 26, 2021 Biochemistry: Reiss et al. Proc. Natl. Acad. Sci. USA 88 (1991) 735 Table 1. Inhibition of rat protein farnesyltransferase by COOH-terminal tetrapeptides corresponding to known proteins Concentration COOH-terminal for 50%o Protein Species tetrapeptide inhibition, ,uM p21Ki-rasB* Human, mouse CVIM 0.15 p21Ki-rasA Human CIIM 0.15 p2lN-ms Human CVVM 0.15 p21N-ras Mouse CVLM 0.15 Lamin B* Human, Xenopus laevis CAIM 0.15 Lamin A Human, X. laevis CSIM 0.20 Retinal cGMP phospho- Bovine CCVQ 0.35 diesterase, a subunit rasl* Saccharomyces cerevisiae CIIC 0.35 ras2* S. cerevisiae CIIS 0.35 Transducin, y subunit* Bovine CVIS 1.0 p2lHa-ras Chicken CVIS 1.0 p21Ha-ras* Human, rat CVLS 3.0 Mating factor a* S. cerevisiae CVIA 5.0 rap2b Human CVIL 11 Dd-ras Dictyostelium CLIL 17 rapla/krevl Human CLLL 22 Mating factor* Rhodosporidium toruloides CTVA 30 G proteins, y subunits Bovine CAIL 100 HMG CoA reductase 1 S. cerevisiae CIKS >100 Enzyme activity was measured in the presence of the indicated tetrapeptide as described in Fig. 1. Each tetrapeptide was tested at seven concentrations ranging from 0.03 to 100 ,utM. The concentration giving 50%o inhibition was calculated from the inhibition curve. COOH-terminal tetrapeptide sequences are from refs. 3, 18, 19, and 21-25. HMG CoA, hydroxymethylglutaryl coenzyme A. The single-letter amino acid code is used. *Shown to be farnesylated in vivo. and glycine were not tolerated at all; 50% inhibition required sigmoidal. The inhibition curves with the various peptides concentrations above 40 AuM. differ from classic competitive inhibition curves (data not A study of tetrapeptides corresponding to the COOH shown). Finally, as mentioned in an earlier paper (10), the termini of known proteins (Table 1) gave results that were maximal velocity of the purified enzyme is relatively low. generally in keeping with those obtained with the substituted These findings suggest that the binding of the peptides to the CVIM peptides. They provided the additional information enzyme is not a simple equilibrium reaction. Rather, there that glutamine and cysteine are well tolerated at the X may be slow binding that requires conformational change. position [Cys-Cys-Val-Gln (CCVQ) and Cys-Ile-Ile-Cys The observation that the Al position shows a relaxed (CIIC), respectively]. All ofthe proteins that are known to be amino acid specificity suggests that the residue at this posi- farnesylated in intact cells (indicated by the asterisks in Table tion may not contact the famesyltransferase directly. Rather, 1) followed the rules outlined above, and all inhibited farne- the contacts may involve only the cysteine and the residues sylation at relatively low concentrations (5 AM or below) with at the A2 and X positions. A working model for the active site the exception of the Cys-Thr-Val-Ala (CTVA) sequence, ofthe protein farnesyltransferase places the peptide substrate which is found in the mating factor ofR. toruloides (22). This in an extended conformation such that the side chains of the peptide inhibited the rat brain protein farnesyltransferase by cysteine and the A2 residues face the enzyme and the side 50% only at the high concentration of 30 ,M. It is likely that chain of the Al residue faces the solvent. A largely hydro- the protein farnesyltransferase in this fungal species has a phobic pocket of the enzyme apparently interacts with the X different specificity than that of the rat brain. group of the Cys-Al-A2-X-containing substrate. The peptide CAIL, which corresponds to the COOH terminus of some of the y subunits ofbovine brain G proteins Debra Noble, Richard Gibson, Candace Millhouse, and Khuan Ng (18, 19), did not compete efficiently with p21Ha-,s for farne- provided excellent technical assistance. Elizabeth Brown and Jen- sylation (Table 1). We observed 50%o inhibition only at the nifer Maxwell provided excellent help with the synthesis and puri- highest concentration tested (100 ,uM). The inhibitory activ- fication of tetrapeptides. We thank our colleagues Pat Casey and J. R. Falck for helpful discussions. This research was supported by ity was lower than that of Cys-Val-Ile-Leu (CVIL; 11 ,uM) or grants from the National Institutes of Health (HL 20948 and GM Cys-Ala-Ile-Met (CAIM; 0.15 ,uM). Thus, the combination of 37616), the Lucille P. Markey Charitable Trust, the Perot Family alanine at the Al position and leucine at the X position is Foundation, and the Robert A. Welch Foundation. Y.R. is the more detrimental than either single substitution. This finding recipient of a Chaim Weizmann postdoctoral fellowship award. is particularly relevant since the y subunits ofG proteins from bovine brain (16) and rat PC12 cells (17) have been shown to 1. Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. (1989) contain a geranylgeranyl rather than a farnesyl. These find- Proc. Nati. Acad. Sci. USA 86, 8323-8327. ings suggest the existence of a separate geranylgeranyl trans- 2. Farnsworth, C. C., Wolda, S. L., Gelb, M. H. & Glomset, ferase that favors and other related J. A. (1989) J. Biol. Chem. 264, 20422-20429. CAIL perhaps sequences. 3. Fukada, Y., Takao, T., Ohguro, H., Yoshizawa, T., Akino, T. The experiments with the biotinylated heptapeptide (Fig. & Shimonishi, Y. (1990) Nature (London) 346, 658-660. 2B and ref. 11) confirm that at least some ofthe short peptides 4. Kamiya, Y., Sakurai, A., Tamura, S. & Takahashi, N. (1978) act as substrates for the enzyme. The saturation curves Biochem. Biophys. Res. Commun. 83, 1077-1083. relating reaction velocity to the concentration of either 5. Anderegg, R. J., Betz, R., Carr, S. A., Crabb, J. W. & Duntze, p21Ha-,s or the biotinylated heptapeptide are complex and W. (1988) J. Biol. Chem. 263, 18236-18240. Downloaded by guest on September 26, 2021 736 Biochemistry: Reiss et al. Proc. Nati. Acad. Sci. USA 88 (1991)

6. Glomset, J. A., Gelb, M. H. & Farnsworth, C. C. (1990) 16. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Trends Biochem. Sci. 15, 139-142. Fung, B. K.-K., Clarke, S., Gelb, M. H. & Glomset, J. A. 7. Lowy, D. R. & Willumsen, B. M. (1989) Nature (London) 341, (1990) Proc. Natl. Acad. Sci. USA 87, 5868-5872. 384-385. 17. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S. & 8. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. USA 87, 5873- (1989) Cell 57, 1167-1177. 5877. 9. Goldstein, J. L. & Brown, M. S. (1990) Nature (London) 343, 18. Gautam, N., Baetscher, M., Aebersold, R. & Simon, M. I. 425-430. (1989) Science 244, 971-974. 10. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & 19. Robishaw, J. D., Kaiman, V. K., Moomaw, C. R. & Slaughter, Brown, M. S. (1990) Cell 62, 81-88. C. A. (1989) J. Biol. Chem. 264, 15758-15761. 11. Reiss, Y., Seabra, M. C., Goldstein, J. L. & Brown, M. S. 20. Stewart, J. M. & Young, J. D. (1984) Solid Phase Peptide (1991) Methods (Companion Methods Enzymol.), in press. Synthesis (Pierce Chemical, Rockford, IL), 2nd Ed. 12. Schaber, M. D., O'Hara, M. B., Garsky, V. M., Mosser, 21. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827. S. D., Bergstrom, J. D., Moores, S. L., Marshall, M. S., 22. Akada, R., Minomi, K., Kai, J., Yamashita, I., Miyakawa, T. Friedman, P. A., Dixon, R. A. F. & Gibbs, J. B. (1990) J. Biol. & Fukui, S. (1989) Mol. Cell. Biol. 9, 3491-3498. Chem. 265, 14701-14704. 23. Schafer, W. R., Kim, R. Sterne, R., Thorner, J., Kim, S.-H. & 13. Stephenson, R. C. & Clarke, S. (1990) J. Biol. Chem. 265, Rine, J. (1989) Science 245, 379-385. 16248-16254. 24. Ohmstede, C.-A., Farrell, F. X., Reep, B. R., Clemetson, 14. Farnsworth, C. C., Gelb, M. H. & Glomset, J. A. (1990) Sci- K. J. & Lapetina, E. G. (1990) Proc. Natl. Acad. Sci. USA 87, ence 247, 320-322. 6527-6531. 15. Rilling, H. C., Breunger, E., Epstein, W. W. & Crain, P. F. 25. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. & (1990) Science 247, 318-320. Noda, M. (1989) Cell 56, 77-84. Downloaded by guest on September 26, 2021