Expression of the TGF alpha integral membrane precursor induces transformation of NRK cells.

TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.

Characterization of the rat transforming growth factor alpha gene and identification of promoter sequences.

We have determined the complete nucleotide sequence of rat transforming growth factor alpha (TGF alpha) mRNA and characterized the six exons that encode this transcript. These six exons span approximately 85 kilobases of genomic DNA, with exons 1 to 3 separated by particularly large introns. What had previously been thought to represent a species-specific difference in the size of the TGF alpha precursor (proTGF alpha) is now shown to be due to microheterogeneity in the splicing of exons 2 and 3. This results from a tandem duplication of the acceptor CAG and gives rise to two alternate forms (159 and 160 amino acids) of the integral membrane precursor. Exon 6, which encodes the 3' untranslated region of TGF alpha mRNA, also encodes, on the opposite strand, a small (approximately 200-nucleotide) transcript whose sequence predicts an open reading frame of 51 amino acids. Expression of this latter transcript does not appear to be coregulated with that of TGF alpha mRNA. Primer extension and S1 nuclease analyses of authentic TGF alpha transcripts revealed two major and multiple minor 5' ends which span more than 200 base pairs of DNA in a G + C-rich region that lacks canonical CCAAT or TATA sequences. The 5' ends of six independently derived cDNAs localized to five different sites in this same region. Restriction fragments that overlap these transcription start sites and extend approximately 300 base pairs in the 5' direction faithfully promote transcription in vitro with HeLa cell nuclear extracts. In addition, they direct the expression of the bacterial chloramphenicol acetyltransferase gene in transient-transfection assays.

Regulation in Escherichia coli of the porin protein gene encoded by lambdoid bacteriophages.

Specialized lambda transducing phages carrying the cloned lc porin gene from the lambdoid bacteriophage PA-2, including various amounts of a sequence 5' to the start of transcription, were used to study the regulation of the porin gene. It was found that a cyclic AMP receptor protein consensus binding site 65 base pairs 5' to the start of transcription was required for catabolite repression of lc but was not sufficient for maximum expression under derepressing conditions. A sequence located more than 209 base pairs 5' to the start of transcription was necessary for maximum expression. By manipulating the copy number of the lc gene and the temperature and by measuring both the rate of synthesis of mRNA and the amount of Lc protein in the outer membrane, it was determined that the expression of lc is regulated primarily at the level of transcription and that expression is not autoregulated. Evidence is also presented that the silent phage porin gene nmpC of Escherichia coli K-12 is transcribed to the same extent as lc even though it does not give rise to a stable pool of mRNA. The structure of the 5' end of lc and nmpC is similar to that of ompF, and a model for transcriptional regulation is presented which may apply to all of these porin genes.

Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage.

The lc gene of the lambdoid bacteriophage PA-2 and the nmpC gene located on a defective lambdoid prophage in the 12-min region of the Escherichia coli K12 chromosome have been sequenced. The porin proteins encoded by these two genes were almost identical, with only 4 of the 365 residues of the precursor forms of the proteins being different. The Lc and NmpC proteins were strongly homologous to the OmpC, ompF, and PhoE proteins, with greater than 56% of the residues identical in each case. Sequencing of the region flanking the lc gene allowed precise positioning of this gene with respect to the rightward cos site of the phage and to sequences which are homologous between PA-2 and lambda. In wild-type strains of E. coli K12, the nmpC gene is not expressed and contains an IS5 insertion near the 3' end of the coding region. This insertion deletes 18 residues from the COOH terminus of NmpC protein and adds 8 residues from an open reading frame extending into IS5 sequence. Expression of this form of the gene in an expression vector plasmid demonstrated that this altered protein is still capable of being translocated to the outer membrane. Plasmid expression experiments using lc-nmpC hybrid genes show that it is the presence of the IS5 insertion which prevents expression of the porin in wild-type E. coli K12. In the nmpC mutant which expresses the protein, there has been a precise excision of the IS5 which regenerates a COOH terminus of NmpC protein which is identical to that of the Lc protein. Blot hybridization detected no mRNA transcripts from the wild-type nmpC gene, although transcripts were readily detected from the lc gene in PA-2 lysogens and from the nmpC mutant which has excised the IS5. This indicates that IS5 affects the production or stability of transcripts from the adjacent nmpC gene.