Carboxyl-terminal Tetrapeptide Research Articles

Cholecystokinin receptors in GtoPdb v.2023.1

Cholecystokinin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CCK receptors [90]) are activated by the endogenous peptides cholecystokinin-8 (CCK-8), CCK-33, CCK-58 and gastrin (gastrin-17). There are only two distinct subtypes of CCK receptors, CCK1 and CCK2 receptors [64, 124], with some alternatively spliced forms most often identified in neoplastic cells. The CCK receptor subtypes are distinguished by their peptide selectivity, with the CCK1 receptor requiring the carboxyl-terminal heptapeptide-amide that includes a sulfated tyrosine for high affinity and potency, while the CCK2 receptor requires only the carboxyl-terminal tetrapeptide shared by each CCK and gastrin peptides. These receptors have characteristic and distinct distributions, with both present in both the central nervous system and peripheral tissues.

Cholecystokinin receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Cholecystokinin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CCK receptors [89]) are activated by the endogenous peptides cholecystokinin-8 (CCK-8), CCK-33, CCK-58 and gastrin (gastrin-17). There are only two distinct subtypes of CCK receptors, CCK1 and CCK2 receptors [63, 123], with some alternatively spliced forms most often identified in neoplastic cells. The CCK receptor subtypes are distinguished by their peptide selectivity, with the CCK1 receptor requiring the carboxyl-terminal heptapeptide-amide that includes a sulfated tyrosine for high affinity and potency, while the CCK2 receptor requires only the carboxyl-terminal tetrapeptide shared by each CCK and gastrin peptides. These receptors have characteristic and distinct distributions, with both present in both the central nervous system and peripheral tissues.

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.

Effects of mutations on the subcellular distribution and localization of the HIV-1 VPU

Vpu is a small type 1 integral membrane phosphoprotein of the human immunodeficiency virus type 1 (HIV-1), predominantly localized in the endoplasmic reticulum (ER)/Golgi compartments of infected or vpu-transfected cells. Although localized in the membrane of the ER, there is no obvious sequence element identified for its retention in the ER or its distribution in the Golgi apparatus. In the present study, we assess the role of the carboxyl-terminal tetrapeptide sequence, VDDL, and the consensus glycosylation signal sequence, NES, on the subcellular distribution and localization of the Vpu protein of HIV-1LAI (subtype B) in HeLa cells. We fused the wild-type Vpu gene and Vpu genes that carry either the VDDL deletion (Vpu) and/or a glycosylation site mutation, to the enhanced green fluorescent protein (EGFP) gene, and monitored the subcellular distribution of the proteins in co-localization and fluorescence confocal microscopy studies. We found that both the EGFP-wtVpu and EGFP- Vpu proteins co-localized with an ER marker, while a species of the EGFP- Vpu protein also co-localized with a Golgi marker. However, alteration of the putative glycosylation signal in the EGFP- Vpu background resulted in the accumulation of Vpu in the Golgi. Our results suggest that the enhanced Golgi accumulation of Vpu proteins harboring both the VDDL deletion and a point mutation in NES sequence may have resulted from the removal of a restriction that would otherwise retain Vpu to the ER compartment of the cell. (Supported by RCMI Grant #5G12RR017581-05 from NCRR-NIH).

Differential Docking of High-Affinity Peptide Ligands to Type A and B Cholecystokinin Receptors Demonstrated by Photoaffinity Labeling

Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-l-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) probe, 0.58 +/- 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa(24) probe labeled Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.

Conserved Cysteine and Tryptophan Residues of the Endothelin-converting Enzyme-1 CXAW Motif Are Critical for Protein Maturation and Enzyme Activity

The neprilysin (NEP)/endothelin-converting enzyme (ECE) family of metalloproteases contains a highly conserved carboxyl-terminal tetrapeptide sequence, CXAW, where "C" is cysteine, "X" is a polar amino acid, "A" is an aliphatic residue, and "W" is tryptophan. Although this sequence strongly resembles a prenylation motif, human ECE-1 did not appear to be prenylated when labeled in vivo using various isoprenoid precursors in cell lines expressing ECE-1. We used site-directed mutagenesis to investigate the role of the CXAW motif and determined that the conserved cysteine residue of the CXAW motif in ECE-1, Cys(755), is critical for proper folding of the enzyme, its export from the endoplasmic reticulum, and its maturation in the secretory pathway. In addition, site-directed mutagenesis revealed that the conserved tryptophan residue of the sequence CEVW appears to be important for endoplasmic reticulum export and is essential for enzyme activity. Deletion of Trp(758) or substitution with alanine greatly slowed maturation of the enzyme, and resulted in more than a 90% loss of enzyme activity relative to the wild type. Conservative substitution of the tryptophan with phenylalanine did not reduce activity, whereas replacement with tyrosine, methionine, or leucine reduced enzyme activity by 50%, 75%, and 85%, respectively. Together, these data indicate that the conserved CEVW sequence does not serve as a prenylation signal and that both the conserved cysteine and tryptophan residues are necessary for proper folding and maturation of the enzyme. Furthermore, the conserved tryptophan appears to be critical for enzyme activity.

Evolution of the Cholecystokinin and Gastrin Peptides and Receptors1

The intestinal hormone, cholecystokinin (CCK), and the stomach hormone, gastrin, form a simple two member family of peptides with much to offer students of hormone and receptor evolution. They share a common carboxyl-terminal tetrapeptide sequence, which is the bioactive site of each peptide and is also antigenic, making heterologous biological and immunological assays feasible. Current evidence indicates that CCK evolved in chordate ancestors and that gastrin-like peptides that separately regulate stomach functions evolved from an ancestral CCK at the level of the divergence of tetrapods from fish. This tentative conclusion may require modification when the two separate CCK- and gastrin-like peptides recently identified in the dogfish shark are characterized further. The CCK-X receptor appears to be ancestral to the CCK-A and CCK-B receptors identified in amniotes. The evolution of gastrin and of CCK-A and -B receptors may have played roles in the evolution of the stomach and the evolution of endothermy in vertebrate phylogeny.

Evolution of the Cholecystokinin and Gastrin Peptides and Receptors

The intestinal hormone, cholecystokinin (CCK), and the stomach hormone, gastrin, form a simple two member family of peptides with much to offer students of hormone and receptor evolution. They share a common carboxyl-terminal tetrapeptide sequence, which is the bioactive site of each peptide and is also antigenic, making heterologous biological and immunological assays feasible. Current evidence indicates that CCK evolved in chordate ancestors and that gastrin-like peptides that separately regulate stomach functions evolved from an ancestral CCK at the level of the divergence of tetrapods from fish. This tentative conclusion may require modification when the two separate CCK- and gastrin-like peptides recently identified in the dogfish shark are characterized further. The CCK-X receptor appears to be ancestral to the CCK-A and CCK-B receptors identified in amniotes. The evolution of gastrin and of CCK-A and -B receptors may have played roles in the evolution of the stomach and the evolution of endothermy in vertebrate phylogeny.

Importance of the Carboxyl-terminal FTSL Motif of Membrane Cofactor Protein for Basolateral Sorting and Endocytosis: POSITIVE AND NEGATIVE MODULATION BY SIGNALS INSIDE AND OUTSIDE THE CYTOPLASMIC TAIL

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.

Molecular Cloning, Characterization, and Chromosomal Localization of FKBP23, a Novel FK506-Binding Protein with Ca2+-Binding Ability

We have identified and characterized a cDNA encoding a novel FK506-binding protein (FKBP), named FKBP23, from mouse heart by the signal sequence trap method. The deduced amino acid sequence has significant homology to other FKBP family members around the peptidylprolylcis-trans-isomerase motifs. FKBP23 also has two Ca2+-binding (EF-hand) motifs, and purified FKBP23 protein was shown to have Ca2+-binding ability. This is the first report of a Ca2+-binding FKBP. FKBP23 is a glycoprotein retained in the endoplasmic reticulum by its carboxyl-terminal tetrapeptide His-Asp-Glu-Leu, as demonstrated by immunostaining, retention, and deglycosylation assays. FKBP23 mRNA is expressed most strongly in heart, lung, and testis, beginning at day 8.5 of embryonic development. The FKBP23 gene was mapped to mouse chromosome 2.

RF-amide peptides isolated from the midgut of the corn earworm, Helicoverpa zea, resemble pancreatic polypeptide

The midgut of the corn earworm, Helicoverpa zea, contains endocrine cells which exhibit immunoreactivity resembling Phe-Met-Arg-Phe-NH 2 (FMRFa), a molluscan cardioactive peptide and a member of a recognized family of molecules termed RF-amide peptides. An extract of 10 000 midguts was fractionated by HPLC and FMRFa immunoreactivity was determined by radioimmunoassay (RIA). Two peptides were isolated and their sequences determined by tandem mass spectroscopy were: Gln-Ala-Ala-Arg-Pro-Arg-Phe-NH 2 and Ala-Ala-Arg-Pro-Arg-Phe-NH 2. These new peptides are termed Hez-MP-I and Hez-MP-II, respectively. Their sequences resemble the carboxyl terminal tetrapeptide of (a) the pancreatic polypeptides of lower vertebrates and (b) a related neuropeptide from squid. An antiserum was raised against Hez-MP-I to develop a specific RIA, which was used with HPLC to demonstrate that each of these peptides occur in hemolymph, as well as in midgut and brain. Hez-MPs thus qualify as putative hormones which may play a role in coordination of digestion in this insect.

Natural inhibitors for protein prenyltransferase.

Farnesyl protein transferase (FPT) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell transforming activities of Ras. Inhibitors of FPT have been demonstrated to inhibit Ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers (1). In the present study, the inhibitory principles for protein prenyltransferases were isolated and identified from Ganoderma lucidum and garlic. The inhibitors from Ganoderma lucidum were identified as ganoderic acid A and ganoderic acid C by comparison with the reported spectral data. Ganoderic acid A has an IC50 value of 100 microM against FPT and its methyl ester (methyl ganoderate A) has an IC50 value of 38 microM for the same enzyme. These inhibitors appear to be competitive with farnesyl pyrophosphate (FPP), and Ki values of ganoderic acid A and methyl ganoderate A are 54 microM and 20 microM, respectively. The inhibitors from garlic were identified as diallyl thiosulfinate (allicin), methyl allyl thiosulfinate, and allyl methyl thiosulfinate. These inhibitors are more effective against geranylgeranyl protein transferase (GGPT) than FPT and IC50 values of allicin, methyl allyl thiosulfinate, and allyl methyl thiosulfinate for GGPT were 43 microM, 57 microM, and 53 microM, respectively. Methyl allyl thiosulfinate appears to be competitive with geranylgeranyl pyrophosphate (GGPP) and its Ki was determined to be 15 microM. The molecular structures of triterpenes and thiosulfinates are expected to be useful in designing lead compounds for new potent antitumour agents.

Expression Cloning of a Novel Farnesylated Protein, RDJ2, Encoding a DnaJ Protein Homologue

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in cellular proteins which terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is most often methionine or serine. Substrates include the p21rasproteins, nuclear lamins, and a series of retinal proteins. To date, a limited number of substrates for the farnesyltransferase have been identified, predominantly by demonstration of the attachment of a farnesyl group to previously identified cDNA clones which encode proteins containing an appropriate carboxyl-terminal tetrapeptide. We describe here the use of a cDNA fusion protein expression library, together with enzymaticin vitro[3H]farnesyl radiolabeling, as a means of identifying novel farnesylated proteins. One candidate cDNA was fully cloned and found to be a homologue of theEscherichia coliheat shock genednaJ.The predicted amino acid sequence of this protein was found to terminate with the tetrapeptide Cys-Ala-His-Gln, which conforms to the consensus sequence for recognition by farnesyltransferase, and was shown to undergoin vivofarnesylation. This farnesylated protein, designated RDJ2 (rat DnaJ homologue 2), is a novel and ubiquitously expressed DnaJ homologue and is the newest member of the subfamily of DnaJ-related proteins which are posttranslationally modified by protein farnesylation.

Calumenin, a Ca2+-binding protein retained in the endoplasmic reticulum with a novel carboxyl-terminal sequence, HDEF.

We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.

Functional dissection of the Drosophila enhancer of split protein, a suppressor of neurogenesis.

The Enhancer of split [E(spl)] gene complex of Drosophila comprises seven related genes encoding a special type of basic helix-loop-helix proteins, the function of which is to suppress the neural developmental fate. One of these proteins is E(spl) itself. To gain insight into the structural requirements for E(spl) function, we have expressed a large number of deletion variants in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. Lack of the basic helix-loop-helix domain, helix III or IV, only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as groucho and the proneural proteins.

Farnesyltransferase as a target for anticancer drug design

The currently understood function for Ras in signal transduction is in mediating the transmission of signals from external growth factors to the cell nucleus. Mutated forms of this GTP-binding protein are found in 30% of human cancers with particularly high prevalence in colon and pancreatic carcinomas. These mutations destroy the GTPase activity of Ras and cause the protein to be locked in its active, GTP bound form. As a result, the signaling pathways are activated, leading to uncontrolled tumor growth. Ras function in signaling requires its association with the plasma membrane. This is achieved by posttranslational farnesylation of a cysteine residue present as part of the CA1A2X carboxyl terminal tetrapeptide of all Ras proteins. The enzyme that recognizes and farnesylates the CA1A2X sequence, Ras farnesyltransferase (FTase), has become an important target for the design of inhibitors that might be interesting as antitumor agents. Several approaches have been taken in the search for in vivo active inhibitors of farnesyltransferase. These include the identification of natural products such as the chaetomellic and zaragozic acids that mimic farnesylpyrophosphate, bisubstrate transition state analogs combining elements of the farnesyl and tetrapeptide substrates and peptidomimetics that reproduce features of the carboxyl terminal tetrapeptide CA1A2X sequence. This last group of compounds has been most successful in showing highly potent inhibition of FTase and selective blocking of Ras processing in a range of Ras transformed tumor cell lines at concentrations as low as 10 nM. Certain peptidomimetics will also block tumor growth in various mouse models, with apparently few toxic side effects. These results suggest that farnesyltransferase inhibitors hold considerable promise as anticancer drugs in the clinic.

Design and synthesis of non-peptide Ras CAAX mimetics as potent farnesyltransferase inhibitors.

Cysteine farnesylation of the ras oncogene product Ras is required for its transforming activity and is catalyzed by farnesyltransferase (FTase). The Ras carboxyl terminal tetrapeptide CAAX (C is cysteine, A is any aliphatic amino acid, X is methionine or serine) is the minimum sequence for FTase recognition. We report here the design, synthesis, and biological characterization of Ras CAAX non-peptide mimetics in which the cysteine is linked through a reduced pseudopeptide bond to 4-amino-3'-carboxybiphenyl. These non-peptide mimetics are potent inhibitors of FTase (IC50 = 40 nM for the most potent inhibitor) and are highly selective for FTase over GGTase I (geranylgeranyltransferase I). They are not substrates for farnesylation, do not have peptidic features, and have no hydrolyzable bonds. Structure-activity studies reveal the importance of the position of the carboxylic acid on the aryl ring as well as the reduction of the cysteine amide bond. Substitution at the 2-position of 4-amino-3'-carboxybiphenyl increases inhibitory potency, while the removal of the carboxylic acid results in a 10-fold loss of inhibitory activity.

CAAX Peptidomimetic FTI-244 Decreases Platelet-Derived Growth Factor Receptor Tyrosine Phosphorylation Levels and Inhibits Stimulation of Phosphatidylinositol 3-Kinase but Not Mitogen-Activated Protein Kinase

Cysteine farnesylation of the Ras carboxyl terminal tetrapeptide CAAX motif (where C=cysteine, A=leucine, isoleucine, or valine, and X=methionine or serine) is required for Ras biological activity. In this report, we describe the effects of inhibitors of farnesyltransferase (FTase), the enzyme responsible for this lipid modification, on platelet-derived growth factor (PDGF) signaling in NIH-3T3 cells. In vitro, the CAAX peptidomimetic FTI-232 exhibits potent inhibition of FTase activity (IC 50=150 nM) and its carboxyl-methylated counterpart, FTI-244, inhibits Ras processing in vivo. Treatment of NIH-3T3 cells with FTI-244 inhibits PDGF-induced DNA synthesis but not stimulation of mitogen-activated protein kinase (MAPK). However, FTI-244 significantly reduces PDGF-induced tyrosine phosphorylation levels of PDGF receptor (PDGFR) as well as its association with, and activation of, phosphatidylinositol-3-kinase (PI-3-K), a key enzyme in PDGF-induced mitogenesis.

NMR studies of novel inhibitors bound to farnesyl‐protein transferase

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.

A novel cellular source for endothelin

Cysteine farnesylation of the Ras carboxyl terminal tetrapeptide CAAX motif (where C=cysteine, A=leucine, isoleucine, or valine, and X=methionine or serine) is required for Ras biological activity. In this report, we describe the effects of inhibitors of farnesyltransferase (FTase), the enzyme responsible for this lipid modification, on platelet-derived growth factor (PDGF) signaling in NIH-3T3 cells. In vitro, the CAAX peptidomimetic FTI-232 exhibits potent inhibition of FTase activity (IC50=150 nM) and its carboxyl-methylated counterpart, FTI-244, inhibits Ras processing in vivo. Treatment of NIH-3T3 cells with FTI-244 inhibits PDGF-induced DNA synthesis but not stimulation of mitogen-activated protein kinase (MAPK). However, FTI-244 significantly reduces PDGF-induced tyrosine phosphorylation levels of PDGF receptor (PDGFR) as well as its association with, and activation of, phosphatidylinositol-3-kinase (PI-3-K), a key enzyme in PDGF-induced mitogenesis.