Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-mediated signaling.

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein alpha subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Gialpha. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Galpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Galpha subunits on cellular membranes.

The stoichiometry of G alpha(s) palmitoylation in its basal and activated states.

Palmitoylation is the dynamic modification of proteins by the addition of palmitate to cysteine residues. The alpha subunits of heterotrimeric G proteins undergo palmitoylation on their amino terminus, and activation of alpha(s) accelerates its palmitate turnover. In previous studies, palmitoylation was assessed by incorporation or turnover of [3H]palmitate. These studies did not determine the fraction of alpha(s) that is palmitoylated because the specific activity of [3H]palmitoyl-CoA within cells is indeterminate. We developed an HPLC method to determine the fraction of alpha(s) that was palmitoylated in the basal and activated states. COS and S49 cells were radiolabeled with [35S]methionine, and alpha(s) was immunoprecipitated from the particulate fraction. The immunoprecipitated proteins were separated by reverse phase HPLC into two peaks that were determined to contain the modified and unmodified forms of alpha(s). Approximately 77% of the endogenous alpha(s) in COS cells and 70% in S49 lymphoma cells were palmitoylated. The fraction of alpha(s) that was modified did not change after treatment with isoproterenol, a beta-adrenergic receptor agonist that causes turnover of palmitate on alpha(s). These results suggest that receptor activation of alpha(s) caused a rapid turnover of palmitate to maintain most of alpha(s) in its palmitoylated form.

Post-translational processing of RhoA. Carboxyl methylation of the carboxyl-terminal prenylcysteine increases the half-life of Rhoa.

RhoA and related GTP-binding proteins are modified post-translationally at their carboxyl terminus to form a prenylcysteine methyl ester. The synthesis and post-translational modification of RhoA and Cdc42 were examined in the RAW264 macrophage cell line, and the effect of carboxyl methylation on protein turnover was determined. Cells were labeled with [35S]cysteine, and RhoA or Cdc42 was immunoprecipitated with specific antibodies. Both RhoA and Cdc42 were methylated rapidly in control cells, with little accumulation of unmethylated protein. Carboxyl methylation of RhoA was inhibited by incubation of cells with a carbocyclic adenosine analog, 3-deazaaristeromycin, resulting in the accumulation of unmethylated RhoA. Under these conditions, Cdc42 methylation was inhibited only partially. When methylation was inhibited, the RhoA half-life decreased from 31 to 12 h, and the Cdc42 half-life decreased from 15 to 11 h. The increased degradation of unmethylated RhoA demonstrates a novel function for carboxyl-terminal prenylcysteine carboxyl methylation in protecting RhoA and related proteins from degradation.

Reciprocal regulation of Gs alpha by palmitate and the beta gamma subunit.

Hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase, promotes dissociation of alpha s from G beta gamma, accelerates removal of covalently attached palmitate from the G alpha subunit, and triggers release of a fraction of alpha s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant alpha s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated alpha s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound beta gamma with 5-fold higher affinity. beta gamma protected GDP-bound alpha s, but not alpha s-GTP[gamma S], from depalmitoylation by a recombinant esterase. We conclude that beta gamma binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of alpha s and that dissociation of alpha s.GTP from beta gamma is likely to mediate receptor-induced alpha s depalmitoylation and translocation of the protein to cytosol in intact cells.

Fractionation and characterization of protein C-terminal prenyl-cysteine methylesterase activities from rabbit brain.

Reversible carboxyl methylation of the C-terminal geranylgeranylcysteine of G25K may regulate its activity and cellular localization. Brain homogenates were examined for enzyme activities which hydrolyze the methyl ester of [3H]methyl-G25K to produce [3H]methanol. Methylesterase activity was detected in both soluble and membrane fractions. The soluble activity was fractionated into at least two distinct activities. One soluble activity appears to be due to the lysosomal protease, cathepsin B, based on sensitivity to certain protease inhibitors, acidic pH optimum, size, and ability to cleave the peptide substrate N alpha-CBZ-Arg-Arg-7-amido-4-methylcoumarin. A second soluble activity, associated with a protein of approximately 25 kDa, exhibits a neutral pH optimum, insensitivity to protease inhibitors, and inhibition by the esterase inhibitor, ebelactone B. The membrane fraction contains larger amounts of a similar methylesterase that may represent the physiologically relevant form of the enzyme.

The role of cysteine 78 in fluorosulfonylbenzoyladenosine inactivation of rat liver S-adenosylhomocysteine hydrolase.

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site-directed reagent 5'-p-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi, T., Ogawa, H., and Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425). To characterize the inactivation mechanism more precisely, the properties of four hydrolase proteins mutated at Cys-78 or Cys-112 were compared to those of the wild-type enzyme. When Cys-78 was mutated to either a serine or an alanine, proteins with greatly reduced enzymatic activity were obtained, large effects on kinetic constants were observed, and enzymatic activity was not affected by incubation with FSBA. When Cys-112 was mutated to either a serine or an alanine, the activity was similar to the wild-type protein, only small changes in the kinetic constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant protein was accompanied by the formation of a disulfide between Cys-78 and Cys-52. The data indicate that FSBA initially reacts with Cys-78 and that Cys-78 has an important role in the structure of the enzyme.

Carboxyl methylation of the low molecular weight GTP-binding protein G25K: regulation of carboxyl methylation by rhoGDI.

A soluble form of G25K from brain co-purified with a 28 kDa protein, which was identified as guanine nucleotide dissociation inhibitor (GDI) protein, similar or identical to rhoGDI. The GDI protein inhibited the dissociation of GDP from G25K. G25K and the GDI protein form a heterodimer and remain associated with either GDP or GTP gamma S bound. The GDI protein inhibited carboxyl methylation of G25K in the presence of magnesium and GDP. The GDI protein appears to be an important regulator of G25K methylation by blocking methylation of G25K in the inactive GDP-bound conformation.

GTP-stimulated carboxyl methylation of a soluble form of the GTP-binding protein G25K in brain.

The GTP-stimulated carboxyl methylation of an M(r) 23,000 protein was investigated in brain homogenates. An M(r) 23,000 methylation substrate was purified from brain homogenates, using an assay for protein methyl-acceptor activity in the presence of a membrane-bound methyltransferase. The M(r) 23,000 methyl-acceptor protein was identified as a soluble form of the GTP-binding protein G25K, based on antibody reactivity and amino acid sequences of tryptic peptides. Two forms of methylated G25K, differing in isoelectric points, were isolated. The soluble G25K could be methylated with a stoichiometry approaching 1 mol of methyl group per mol of G25K, and guanosine 5'-O-3-(thio)triphosphate stimulated the methylation by decreasing the Km for G25K from 0.79 to 0.17 microM. After methylation, the G25K was associated with the membrane fraction. The soluble G25K was isolated as a heterodimer of G25K and an M(r) 28,000 protein. The G25K and M(r) 28,000 protein complex was dissociated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate detergent, and the subunits were separated by Mono-Q chromatography. The association of the M(r) 28,000 protein with G25K decreased the methylation of G25K and altered the guanine nucleotide specificity, indicating that the M(r) 28,000 protein may regulate the methylation of G25K.

Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.

To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.

The G protein connection: molecular basis of membrane association.

Two distinct types of lipid modification, myristoylation and isoprenylation, are critical for membrane association of heterotrimeric G proteins. Elucidation of the molecular basis for G protein membrane association has important implications for understanding G protein structure and function, and is relevant to potential therapeutic approaches to AIDS and cancer.

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding.

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).

Carboxyl methylation and COOH-terminal processing of the brain G-protein gamma-subunit.

The enzymatic methylation of the guanine nucleotide-binding proteins (G-proteins) gamma-subunit was investigated in brain membranes. Brain membranes were methylated in vitro using [3H-methyl]S-adenosylmethionine, and the G-protein beta gamma-complex was purified using an anti-beta antibody to assay for the protein during purification. The isolated G-protein beta gamma-complex was found to be carboxyl methylated on the gamma-subunit. The methyl group was localized by tryptic digestion to the carboxyl-terminal of the protein. The methylated tryptic peptides contained a modified cysteine and were very hydrophobic, suggesting additional modification by lipidation. The evidence suggests that the COOH-terminal of G-gamma is modified in a manner similar to the processing that occurs with the ras proteins.

Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site.

The cDNA for rat liver S-adenosylhomocysteinase has been cloned, and the nucleic acid sequence has been determined. By comparison of the deduced amino acid sequence for S-adenosylhomocysteinase with that of the dinucleotide binding region for other proteins, the sequence from amino acids 213 to 244 in rat liver S-adenosylhomocysteinase was proposed to be part of the NAD binding site (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). A vector has been constructed that expresses S-adenosylhomocysteinase in Escherichia coli in the presence of isopropyl beta-D-thiogalactopyranoside by inserting the coding sequence of rat liver S-adenosylhomocysteinase cDNA downstream from the lac promoter of plasmid pUC118. The enzyme that is produced comprises as much as 10% of the soluble cellular proteins. The purified enzyme is a tetramer, contains 4 mol of tightly bound NAD, and has kinetic properties indistinguishable from those of the liver enzyme. Tryptic peptide mapping and NH2-terminal sequence analysis indicate that the recombinant enzyme is structurally identical to the liver enzyme except for the absence of the NH2-terminal blocking group. The rat liver enzyme has a blocked NH2-terminal alanine residue (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). By oligonucleotide-directed mutagenesis mutant vectors have been generated that express proteins in which each of the glycines in the Gly-Xaa-Gly-Xaa-Xaa-Gly sequence of the putative NAD binding site (residues 219-224) was changed to valine. Immunoblot analysis of extracts of the cells transformed with these vectors reveals the presence of immunoreactive proteins with the subunit molecular weight of S-adenosylhomocysteinase. The mutant proteins have no catalytic activity, contain no bound NAD, and do not form the same quaternary structure as the recombinant S-adenosylhomocysteinase.

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins.

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol. The specific methyl acceptor proteins and methyltransferases varied between tissues and cell types, suggesting that these methylations have cell-specific functions. The guanine nucleotide-dependent carboxyl methylations provide a possible mechanism for regulating the function of GTP-binding membrane proteins in the transduction of receptor-mediated signals of eukaryotic cells.

Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go.

Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of alpha and associated beta/gamma subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the alpha subunits of the purified proteins and cloned cDNAs encoding putative alpha subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoresis of the purified proteins, and of the in vitro translated products of cDNAs encoding alpha subunits. Purified G proteins with alpha subunits of 39, 41, and 40 kDa (G39, G41, and G40 in order of abundance) correspond to the products of Go, Gi1, and Gi2 cDNAs. We purified a novel G protein with an alpha subunit slightly above 39 kDa (G39*). G39* is less abundant than G39, elutes earlier than G39 on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G39. G39 and G39*, however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G39* may represent a novel form of Go, differing in posttranslational modification rather than primary sequence.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence.

S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours. The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase. When the sequences from the two species were aligned, 74% of the amino acids were in identical positions. If conservative changes were taken into account the homology was 84%. Because differences have been reported in the binding characteristics of NAD+ to the D. discoideum and rat AdoHcy hydrolases, changes in the amino acids of the putative NAD+-binding site were of particular interest. Six changes were observed in this region but the changes appeared to be in regions that are not critical to the three dimensional folding of the NAD+-binding site.

Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line.

The pertussis toxin substrate from RAW264 macrophage cell membranes was characterized by two-dimensional gel electrophoresis and by immunoblots using antibodies directed against different guanine nucleotide binding proteins. RAW264 membranes were found to contain one major pertussis toxin substrate, which was recognized by both antibodies AS/6 and LE/3. The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line. The RAW264 pertussis toxin substrate was not recognized by either antibody CW/6 or antibody RV/3, which recognize the 41-kilodalton alpha-subunit of brain Gi (Gi-1-alpha) and Go-alpha, respectively. Pertussis toxin substrates from bovine brain were resolved into four major alpha-subunits by two-dimensional gel electrophoresis, and the LE/3 antibody recognized only one of the four proteins. The brain LE/3 reactive protein also reacted with the AS/6 antibody, migrated with a 40K molecular weight, and had an isoelectric point slightly more basis than the RAW264 pertussis toxin substrate. Therefore, the major pertussis toxin substrate in RAW264 cells appears to be Gi-2, and bovine brain contains a relatively minor amount of a closely related guanine nucleotide binding protein.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino- and carboxyl-terminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD+, which is required for the activity of the enzyme.

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis.

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.