Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages.

Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.

Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes.

The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of 22Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.

The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids.

The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet-activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses.

Regulation of a liver plasma membrane phosphoinositide phosphodiesterase by guanine nucleotides and calcium.

Rat liver plasma membranes are enriched in a Ca2+-dependent phosphodiesterase active on phosphatidylinositol 4,5-P2 and phosphatidylinositol 4-P, but not phosphatidylinositol. Inositol-P3 is the first product of the reaction, but is rapidly degraded. Micromolar concentrations of GTP and its nonhydrolyzable analogues stimulate the reaction, whereas GDP, GMP and other nucleoside triphosphates are inactive. GTP and its analogues decrease the requirement of the reaction for Ca2+ and also increase its activity at saturating Ca2+. These results support the hypothesis that guanine nucleotides and a guanine nucleotide binding regulatory protein are involved in coupling the receptors for Ca2+-mediated agonists to the breakdown of plasma membrane phosphatidylinositol 4,5-P2.

Functional high efficiency expression of cloned leukocyte chemoattractant receptor cDNAs.

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 +/- 34,000 receptors per cell with two affinity states (Kds of ca. 0.43 nM and 39 nM), which in the presence of 100 microM GTP gamma S decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5-1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.

Metal: ATP characteristics of insulin- and epidermal growth factor-stimulated phosphorylation in detergent extracts of rat liver plasma membranes.

The metal: ATP characteristics of insulin- and epidermal growth factor-(EGF)-stimulated protein kinase activities were examined in Nonidet P40 extracts of rat liver plasma membranes. The two kinase activities were capable of utilizing either manganese or magnesium, although differences were observed. Insulin-stimulated 32P incorporation into an Mr 95 000 protein exhibited a higher affinity for ATP in the presence of manganese compared to magnesium. At 200 microM ATP, insulin stimulated 32P incorporation into the Mr 95 000 protein 3- to 5-fold after 5 min in the presence of either metal. At 1 mM ATP, insulin-stimulated 32P incorporation was significantly greater in the presence of magnesium. In contrast, EGF-stimulated 32P incorporation into an Mr 170 000 protein exhibited similar ATP dependencies in the presence of magnesium or manganese. Basal phosphorylation of the Mr 170 000 protein was 2- to 3-fold higher in the presence of manganese, however. Since the higher basal phosphorylation persisted after chromatography on wheat germ lectin-Sepharose, it may represent an inherent activity of the receptor kinase. In the presence of magnesium: ATP, low concentrations of manganese enhanced both insulin- and EGF-stimulated phosphorylation of angiotensin II suggesting involvement of a second metal binding site which regulates the kinase activity. The results presented show major differences in the metal: ATP properties of the two major hormonally regulated protein kinase activities observed in detergent-extracted liver membranes.

Role of guanine nucleotide regulatory proteins and inositol phosphates in the hormone induced mobilization of hepatocyte calcium.

Treatment of isolated hepatocytes with F- produced a concentration-dependent activation of phosphorylase, efflux of Ca2+, rise in [Ca2+]i, increase in Ins 1,4,5-P3 levels, decrease in PI-4,5-P2 levels, and increase in DAG levels. The levels of intracellular cAMP were decreased by NaF. The effects of NaF were potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of PI-4,5-P2 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Gi, Gs, and transducin). Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation was increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release was evident in the presence of GTP or GTP gamma S. The guanine nucleotide and hormonal stimulation was evident on both inositide production and PI 4,5-P2 degradation. Treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein did not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. The results suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. The GTPase activity of rat liver plasma membranes was stimulated 20% by 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, most of the protein-bound [3H]vasopressin migrated as a single band, also, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased 90% when the sucrose gradient centrifugation was run in the presence of 10 M GTP gamma S. Direct evidence that a GTP-binding protein was present in the [3H]vasopressin peak was obtained by the immuno-detection of a 35 kDa beta subunit of a GTP-binding protein and a 40 kDa alpha subunit. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular events in the activation of murine macrophages.

Recent studies have identified some of the early molecular transductional events, which occur during the activation of murine macrophages. Our current evidence indicate a central role for protein kinase C for the priming effect of interferon-gamma (IFN gamma). IFN gamma also initiates Na+/H+ exchange and 45Ca efflux from murine macrophages (cascade I). Our data further indicate the involvement of multiple transductional pathways in the actions of bacterial lipopolysaccharide (LPS). Specifically, molecular events involved in the action of LPS include production of inositol phosphates and calcium mobilization as well as IFN gamma-regulated alterations in intracellular pH (cascade II). Our data further indicate that additional transductional events (e.g., synthesis of early or competence proteins) in response to LPS (cascade III) are also necessary for macrophage activation. Finally, regulation of important surface (e.g., Ia) and secreted molecules (TNF or IL-1) is exerted at the levels of both transcription and stabilization of specific mRNA in response to transductional cascades I, II and III. Taken together, the data indicate macrophage activation is complexly regulated at multiple levels.

The effect of solvents on nucleotide regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase is controlled by two nucleotide sites. We have found that organic solvents affect the regulatory properties of phosphorylase by altering the binding at these two sites. At the activator site, the Ka for AMP is lowered 10-fold in the presence of 10% 1,2-dimethoxyethane while, at the inhibitor site, the Ki for caffeine is increased 6-fold. The stimulation of activity by organic solvents is highly dependent on the enzyme's activity state. Phosphorylase b, which has a requirement for a nucleotide activator, loses this requirement in the presence of organic solvents, while the active form of the enzyme, phosphorylase a, is only slightly stimulated by organic solvents. The activation profile obtained with rabbit liver phosphorylase suggests that differences in the properties of this enzyme from rabbit muscle phosphorylase might be explained by a change in the relative affinity for AMP at the two nucleotide sites. The results also suggest that 1,2-dimethoxyethane may be useful to determine accurately the activities of different forms of liver phosphorylase.

Effects of 1,2-dimethoxyethane on the catalytic and coenzyme properties of glycogen phosphorylase.

Dimethoxyethane, a good activator of phosphorylase b, has been used to study mechanisms of phosphorylase activation and the catalytic reaction. Activation can be explained best by an alteration of the allosteric equilibrium in favor of the active R conformation. Lesser effects are seen with phosphorylase a, and activation does not alter appreciably the equilibrium between the dimeric and tetrameric forms. With 20% 1,2-dimethoxyethane, the Vm value of phosphorylase b is 74% of that obtained in the presence of adenosine monophosphate. In the presence of 10% 1,2-dimethoxyethane, the Ki value for glucose inhibition is increased 3-fold, but inhibition by 1,5-gluconolactone is increased. The allosteric activation of glycogen phosphorylase results in a change in pK1 for the pH-activity profile. The formation of the dianionic form of the phosphoryl group of the coenzyme, pyridoxal phosphate, may account for this change. By analogy to the effects of anions and a change in dielectric on the acid hydroylsis of glucose 1-phosphate, it is suggested that the dianion of the coenzyme could stabilize the developing positive charge of an oxonium ion intermediate. Dimethoxyethane also affects the interaction of pyridoxal phosphate with phosphorylase. It influences the rates of both resolution and reconstitution. Good preparations of apophosphorylase a can be made by using 1,2-dimethoxyethane in the resolution medium.

Quantification of contributions of phospholipid precursors to diradylglycerols in stimulated mononuclear phagocytes.

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line beta-radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the composition of accumulated DG. The molecular species composition of early DG (up to 1 min stimulation time) was very similar to that of unstimulated DG, whereas the proportions of the species present in later DG were substantially altered. In the same experiments, we extracted native phospholipids from unstimulated macrophages, separated phosphatidylinositol (PI), PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), converted them to the corresponding DGs by using phospholipase C, and determined their molecular species compositions as above. In comparison with the diradyl compositions of stimulated DG, the diradyl composition of PI closely matched that of early DG, the differences between the PC and PI compositions matched the differences between early and late DG very closely, and the compositions of PE and PS were unique and unrelated. We quantified these relationships more precisely by multilinear regression analysis to calculate the theoretical best mix of five molecular species compositions (PI, PC, PE, PS and unstimulated DG) that would most closely replicate the early and late accumulated DG compositions. We found that by both h.p.l.c. and t.l.c. analyses, 15-30% (PAF) or 25-50% (ionomycin and PMA) of the later DG could be accounted for by PC hydrolysis. These results represent quantifications of phospholipid class contributions to stimulated DG formation, and demonstrate the potential importance of PC hydrolysis in phagocytic leucocytes.

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate.

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.

A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction.

Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C.

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters.

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment of the PMNs with pertussis toxin (PT) or 4-beta-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTP gamma[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 microM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTP gamma S binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTP gamma S alone at low concentrations of Ca2+ (0.1-1 microM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTP gamma S, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTP gamma S. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.

Isolation of GTP-binding proteins from myeloid HL-60 cells. Identification of two pertussis toxin substrates.

We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.

Solubilization of the vasopressin receptor from rat liver plasma membranes. Evidence for a receptor X GTP-binding protein complex.

The GTPase activity of plasma membranes isolated from rat livers was stimulated 20% over basal by vasopressin. A concentration dependency curve showed that maximal stimulation was obtained with 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, the majority of protein-bound [3H]vasopressin migrated as a single band with a sedimentation constant of 16.8 S. Moreover, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10 microM guanosine 5'-O-(thiotriphosphate). When the 16.8 S peak of bound [3H]vasopressin was further purified over a wheat germ lectin-Sepharose column, a GTPase activity co-eluted from the column with the protein-bound [3H]vasopressin. Direct evidence that a GTP-binding protein was present in the 16.8 S peak was obtained by the immunodetection of a 35-kDa beta subunit of a GTP-binding protein. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

Separation and assay of phosphodiesterase isoforms in murine peritoneal macrophages using membrane matrix DEAE chromatography and [32P]cAMP.

Cyclic nucleotide phosphodiesterases (PDE) are a family of exquisitely regulated enzymes which play a central role in regulating the biological half-life of cAMP and cGMP. Hormonal regulation of specific isoforms is the basis for crosstalk and antagonism between several well-described signaling pathways. In the present work improved methods are described which accelerate and simplify the separation and assay of PDE isoforms occurring in mouse peritoneal macrophages. The described method is equally applicable to other cell and tissue types, and is based on the mobilization of DEAE beads on a macroporous support structure which allows high flow rates, high resolution, and reproducible separations. The failure of the resin to undergo compression in this configuration prevents band spreading caused by diffusion into small pores and channeling associated with bed movement. Therefore, the PDE isoforms from a tissue extract can be resolved in a chromatographic run taking only 30-35 min. In addition, the column cartridge can be regenerated and fully reequilibrated in 10-15 min. After each chromatographic run, cAMP PDE activity in the fractions is characterized by incubating each fraction with [32P]cAMP, followed by quantitative conversion of [32P]AMP formed in the initial reaction to 32PO4 and adenosine. The unreacted [32P]-cAMP is then adsorbed onto charcoal and after centrifugation, the 32PO4 remaining in the supernatant is determined by counting an aliquot. The latter step replaces the need to use small columns to separate tritiated cAMP from tritiated adenosine as in previous assays, and provides the expected improvement in sensitivity associated with a high specific activity substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoprenylation of the low molecular mass GTP-binding proteins rac 1 and rac 2: possible role in membrane localization.

Ras proteins can be modified at their COOH-terminal cysteine in the motif Cys-Ali-Ali-Xaa by a farnesyl isoprenoid. This modification is essential for membrane association and biological activity of ras proteins. A similar COOH-terminal amino acid sequence, Cys-Xaa-Ali-Xaa, exists in the ras-related GTP-binding proteins rac 1 and rac 2. To determine whether these proteins were similarly modified, COS cells were transfected with rac 1 and rac 2 cDNA and expressed proteins were labeled with [3H]mevalonic acid. We report here that both rac 1 and rac 2 are post-translationally modified by addition of an isoprenoid group, the likely site of which is the COOH-terminal cysteine. Isoprenylation was found only in racs associated with particulate cell fractions, suggesting that this modification may be associated with membrane localization of the proteins. These data specifically identify mammalian low molecular mass GTP-binding proteins other than ras that undergo post-translational modification and further define the COOH-terminal consensus sequence, Cys-Ali-Ali-Xaa, as an isoprenylation signal. This sequence may identify a larger family of low molecular mass GTP-binding proteins which are isoprenylated.

Insulin and epidermal growth factor do not affect phosphoinositide metabolism in rat liver plasma membranes and hepatocytes.

Recent studies with viral oncogene tyrosine kinases have suggested that these kinases may phosphorylate phosphoinositides and diacylglycerol. Since the receptors for insulin and epidermal growth factor (EGF) also possess tyrosine kinase activity, we have investigated possible effects of insulin and EGF on phosphoinositide metabolism in rat liver plasma membranes and rat hepatocytes. In plasma membranes prepared from rats injected 18 h prior with [3H]myo-inositol or incubated with [gamma-32P]ATP, phosphatidylinositol-4-P and phosphatidylinositol-4,5-P2 were formed, but there were no effects of either insulin or EGF although these agents stimulated protein tyrosine phosphorylation. In hepatocytes incubated with [3H]myo-inositol, label was incorporated into phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2, but there was no effect of insulin. Incubation of hepatocytes with [3H]myo-inositol plus insulin or EGF for 2 h also did not alter the formation of [3H]myo-inositol-1,4,5-P3 from [3H]phosphatidylinositol-4,5-P2 induced by vasopressin. These findings suggest that the tyrosine kinase activity of liver insulin and EGF receptors is not important in phosphoinositide formation.