Differential regulation of G protein alpha-subunit GTPase activity by peptides derived from the third cytoplasmic loop of the alpha 2-adrenergic receptor.

The effect of peptides homologous to segments of a G protein-coupled receptor on the GTPase activity of recombinant Go alpha (rGo alpha) and Gs alpha (rGs alpha) has been tested. These peptides contain overlapping sequences spanning from amino acid 212 of the putative fifth transmembrane domain to amino acid 229 of the third cytoplasmic loop of the alpha 2 adrenergic receptor. Interestingly, two peptides (comprising residues 212-227 and 214-227) strongly inhibit the basal GTPase activity of both rGo alpha and rGs alpha. Instead, a C-terminally extended peptide (residues 216-229) stimulates rGo alpha but slightly inhibits rGs alpha. Circular dichroism spectroscopy of the peptides reveals that an a helical structure is more easily inducible in the inhibitory ones. These findings constitute an example of peptides representing cytoplasmic receptor sequences that differentially modulate the GTPase activity of recombinant G protein alpha-subunits.

Attenuation of GTPase activity of recombinant G(o) alpha by peptides representing sequence permutations of mastoparan.

There is convincing evidence that the cytoplasmic domains of multispanning receptors interact with guanine nucleotide-binding proteins (G proteins). What are the rules governing these interactions? In an attempt to answer this question, we focused our attention on mastoparan, an amphiphilic tetradecapeptide from wasp venom, and on nine of its variants, produced by sequence permutation, which have altered amphiphilicity or no amphiphilicity at all. Mastoparan enhances the GTPase activity of recombinant G(o) alpha 5-fold in phospholipid vesicles. Like mastoparan, four of the synthetic variants can form amphiphilic alpha-helices and two of them indeed stimulate the GTPase activity of the G protein, whereas the other two have no effect. This confirms that the activation of certain G proteins by a number of peptides is mainly due to their cationic amphiphilicity. However, this structural feature is clearly not sufficient. The relative orientation of the positively charged residues as well as that of the hydrophobic side chains appear to be of fundamental importance. The other five peptides are not amphiphilic and do not enhance the rate of GTP hydrolysis. Surprisingly, three of them almost completely inhibit the G protein's intrinsic GTPase activity. This finding is of interest because of the possible role differential regulation of G protein activity can play in cellular functions.

Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation.

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

Reduced maturation of Friend virus in adhesive mutants of Friend leukemia cells.

The replication of Friend Leukemia virus (FLV) has been investigated in adhesive clones (FF) of Friend Leukemia cells which were selected via cultivation on top of human fibroblast monolayers. In these adhesive clones a shut-down of FLV production is observed under conditions of culture confluency; this finding is not due either to a reduced number of cell divisions nor to a defective expression of FLV genome as assessed by Northern blot and immunofluorescence studies. Ultrastructural studies showed that virus budding and release into the medium is not detectable under these conditions. Conversely, in confluent FF cell monolayers abundant imperfect type-A enveloped particles were visible, possibly originating from stacks of granular endoplasmic reticulum with thickened membranes. It is postulated that the reduced virus production in adhesive FF monolayers is due to as yet undetermined events taking place during virus maturation at a time coincident with that of cell-cell adhesion under conditions of culture confluency.

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia.

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.

Friend murine leukemia virus and spleen focus-forming virus expression in highly malignant interferon-sensitive and interferon-resistant Friend leukemia cells.

Analysis of expression of the Friend murine leukemia virus (F-MuLV) and of the spleen focus forming virus (SFFV) has been undertaken in highly malignant interferon (IFN)-sensitive (745) and IFN-resistant (3Cl-8) Friend leukemia cells (FLC), serially passaged intraperitoneally in DBA/2 mice. In vivo passaged 745 cells, as well as the clones derived thereof, did not release Friend virus (FV). Western blot analysis of the plasma membrane fractions of the virus nonproducer 745 cells revealed the lack of gp69/70 glycoprotein expression. At least 10 intraperitoneal passages of virus producer in vitro passaged of virus producer in vitro passaged 745 cells were necessary to obtain the selection of the virus nonproducer phenotype. In contrast in vivo passaged 3Cl-8 cells continued to produce FV even after 100 in vivo passages and expressed gp69/70 antigens to a similar extent as the original in vitro passaged FLC. The expression of F-MuLV and SFFV RNAs in virus producer and virus nonproducer FLC clones has been investigated by means of Northern blot technique using probes specific for either F-MuLV or SFFV. No F-MuLV specific RNA sequences were detected in virus nonproducer 745 clones. SFFV specific RNA transcripts and gp52/55 glycoprotein production could be revealed in all the FLC tested. Southern blot analysis showed the presence of F-MuLV specific sequences in the cellular DNA of virus nonproducer 745 clones. As both in vivo passaged F-MuLV producer 3Cl-8 and F-MuLV nonproducer 745 cells were equally barely immunogenic and highly malignant when injected into syngeneic DBA/2 mice, these results indicate that F-MuLV expression does not result per se in a high immunogenic potential of tumor cells. For the time being, as a specific property of 3Cl-8 versus 745 cells is the interferon-resistant phenotype, it is tempting to speculate that the selection of virus nonproducer cell variants after in vivo passages of interferon-sensitive 745 cells could depend on the presence of low levels of endogenous interferon in normal young mice.

The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival.

It has been recently reported that the pR plasmid enhances the UV survival in E.coli c600. In order to test whether this function may be expressed also in mammalian cells, LTA (tk- aprt-) mouse cells were cotransformed with pR plasmid DNA and ptk1 plasmid as selectable marker. Tk+ transformants were analyzed for their UV survival and for the presence of pR DNA sequences by blot-hybridization. The results show a correlation between the enhanced UV survival and presence of pR DNA sequences in cotransformed LTA mouse cells.