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 (it) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

There is convincing evidence that the cyto.plasmnic domains of multisning 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 amphiphilici at all. Mastoparan enhances the GTPase activity of recombinant G~a 5-fold in phospholipid vesicles. Like mastoparan, four of the synthetic variants can form amphiphilic a-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 ofthe positively charged residues as well as that of the hydrophobic side chains appear to be of fundamental importance. The other five peptides are not amphiphlc 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.The guanine nucleotide-binding proteins (G proteins) that link cell surface receptors to cytosolic effectors in signal transduction comprise a family of apy heterotrimers associated with the plasma membrane. Upon activation by a liganded receptor the a-subunit binds GTP, dissociates from by, and interacts with one or more effectors (for recent reviews see refs. 1-4). Most known G protein-coupled receptors are characterized by seven hydrophobic transmembrane domains (multispanning receptors), but there is increasing evidence that indicates that certain receptors for growth factors, with a single transmembrane domain, also interact with G proteins (5).The G protein coupling sites ofthe multispanning receptors presumably are located in the second and third cytoplasmic loops (6-8). Synthetic peptides with sequences corresponding to segments of the cytoplasmic loops of the f2-adrenergic receptor have been tested for their effect on adenylate cyclase in erythrocyte membranes. Results suggest that parts of the second, third, and fourth intracellular loops interact with the G protein (9). In contrast, two peptides, comprising the N-and C-terminal 15 amino acids ofthe third intracellular loop of the P2-adrenergic receptor, stimulate the GTPase activity of a recombinant a-subunit of G, ( Because of its amphiphilic nature, MP assumes an a-helical structure when bound to phospholipids, with the positively charged residues exposed to the aqueous medium (13). Presumably, the multispanning receptor regions that interact with G proteins also form cationic amphiphilic a-helices (14).Are there interactions that do not cause stimulation of G protein activity and, ins...

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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.

The effect of peptides homologous to segments of a G protein-coupled receptor on the GTPase activity of recombinant Goa (rGoa) and G~a (rG~a) 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 oz2 adrenergic receptor. Interestingly, two peptides (comprising residues 212-227 and 214-227) strongly inhibit the basal GTPase activity of both rGo~ and rG~. Instead, a C-terminally extended peptide (residues 216-229) stimulates rGo~ but slightly inhibits rG~a. 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 a-subunits.