Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-α mRNA, encoding precursors lacking carboxyl-terminal valine residues

Abstract

The human transforming growth factor-alpha (TGF-a) gene is thought to contain ®ve introns and six exons, encoding a transmembrane precursor (proTGF-a) from which the mature polypeptide is released by proteolytic cleavage. We identi®ed a novel 32-nucleotide exon (exon a) within intron 5 and an alternative splice acceptor site in exon 6, splitting exon 6 into two segments: 6A and 6B. Therefore, in addition to wild type (wt) proTGF-a mRNA, which skips exon a, two novel proTGF-a variants are produced: Variant I (VaI), skipping exons a and 6A, and Variant II (VaII) which includes exon a and skips exon 6A. The only signi®cant di erence between variant and wt proTGF-a proteins is that the two wt carboxyl-terminal valines are replaced in the variants by ®ve or four other amino acids, respectively. Both variant TGF-a mRNAs were readily detected in human keratinocytes and tumor-derived cell lines. Their protein products were cleaved as e ciently as wt TGF-a in response to the calcium ionophore A23187. However, both variants (but not wt) reduced serum requirements for proliferation in CHO cells. In addition, VaIIexpressing CHO cells (not VaI or wt) formed foci in monolayer cultures. These results suggest that variant TGF-a precursors induce autonomous growth.

Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-α precursors mediate specific interactions with ErbB4 and ErbB2

“…We detected abundant surface staining in non-permeabilized CHO cells expressing WT or variant TGF-a precursors, but not in CHO expressing the VVDEL mutant. As expected based on our previous processing data (Xu et al, 1999) treatment with calcium ionophore dramatically decreased cell surface (and also total) staining in WT, VaI and VaII constructs (data not shown). CHO expressing the`Hook' moiety alone, or the chimeric TACT-WT and TMCT-WT constructs also exhibited abundant cell surface staining with the anti-HA antibody indicating that the TACT-WT and TMCT-WT trac normally to the cell surface (data not shown).…”

“…When the starved cells were re-stimulated by medium containing 10% serum for 1 h, ErbB2 phosphorylation was induced in mock transfectants as well (Figure 1b). Surprisingly, ErbB2 phosphorylation levels were not signi®cantly dierent between CHO cells expressing WT and variant TGF-a precursors, despite the fact that VaI and VaII reduced serum requirements, and VaII (but not VaI or WT) exhibited transforming abilities (Xu et al, 1999). Nevertheless, this observation suggested a possible association between the TGF-a precursors and the ErbB2 receptor.…”

“…We reasoned that the growth advantage provided to CHO cells by TGF-a VaI and VaII (Xu et al, 1999) might be mediated through ErbB2 activation. However, it has been demonstrated that mature TGF-a does not bind to and activate ErbB2, and no high-anity ligand so far has been identi®ed for ErbB2.…”

“…We have previously isolated two variant TGF-a precursors derived from alternative splicing, designated as VaI and VaII (Xu et al, 1999). The two carboxylterminal valine residues in WT are replaced by ®ve (GCRLY) or four (ATLG) amino acids in VaI and VaII, respectively.…”

“…The carboxyl-terminal peptide GCRLY in VaI contains a consensus prenylation site (Xu et al, 1999) where the cysteine residue could be the prenylation acceptor site (Zhang and Casey, 1996). Therefore, we generated a VaI point mutant in which 160 Cys was mutated to 160 Ala, and a series of deletion mutants removing a single amino acid at a time from the extreme carboxyl-terminus of VaI: VaIC?A, VaID1, VaD2, VaID3, and VaID4 (Figure 6).…”