A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells

We have purified a novel GTP-binding protein, designated as the smg-25A protein (smg p25A), from bovine brain membranes and determined its primary structure. In the present studies, the smg-25A mRNA levels in various tissues have been studied. The 1.6-kilobase smg-25A mRNA is detected in rat brain by Northern blot analysis. This mRNA is not detected in other rat tissues including thymus, lung, heart, liver, small intestine, kidney, and skeletal muscle. The 1.6-kilobase smg-25A mRNA is also detected in bovine adrenal medulla but not in the cortex. Moreover, this mRNA is detected in rat pheochromocytoma PC-12 cells and its level increases after differentiation of the cells into sympathetic neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. This mRNA level does not increase in response to 12-O-tetradecanoylphorbol-13-acetate incapable of inducing differentiation. These results suggest that the smg-25A gene is specifically expressed in nerve tissues and that smg p25A plays a role in some neuronal functions.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.