Generation of structural and functional diversity in furin-like proteins in Drosophila melanogaster by alternative splicing of the Dfur1 gene.

Abstract

To investigate whether or not alternative splicing might be a mechanism by which in Drosophila melanogaster diversity is generated in endoproteases of the novel eukaryotic family of subtilisin‐like proprotein processing enzymes, we determined structural and functional characteristics of the Dfur1 gene. Northern blot analysis revealed Dfur1 transcripts of 7.6, 6.5, 4.5 and 4.0 kb. By comparative nucleotide sequence analysis of Dfur1 genomic and cDNA clones, 10 coding exons were identified and, together with Northern blot analysis using exon‐specific probes, evidence was obtained that the four transcripts are generated by alternative splicing and polyadenylation. The apparently complete open reading frames of three Dfur1 cDNAs revealed that these coded for three furin‐like proteins, Dfurin1 (892 residues), Dfurin1‐CRR (1101 residues) and Dfurin1‐X (1269 residues), which possessed common but also unique structural domains. These various isoforms of furin in Drosophila were characterized in gene transfer studies using immunoprecipitation analysis. Differential expression of Dfur1 transcripts was found in Northern blot analysis of RNA from various developmental stages of Drosophila. RNA in situ hybridization experiments revealed that the Dfurin1‐X and Dfurin1‐CRR isoforms are expressed in non‐overlapping sets of tissues during Drosophila embryogenesis. In gene transfer experiments in which the Dfurin1, Dfurin1‐CRR and Dfurin1‐X proteins were expressed at high levels together with the precursor of the beta A‐chain of activin‐A, a member of the transforming growth factor beta (TGF beta) superfamily, or the precursor of von Willebrand factor, all three proteins appeared capable of processing these substrates. Our studies indicate that the Dfur1 gene encodes structurally different subtilisin‐like proprotein processing enzymes with distinct physiological functions in Drosophila.

Notch in vertebrates - molecular aspects of the signal

“…The Notch receptor is originally produced as a single path transmembrane protein, but it receives at least three processings. The cleavages are created by different proteases; Furin (Roebroek et al, 1993), Kuzbanian (TACE) (Yavari et al, 1998) and Presenilin (Boulianne et al, 1997). These modifications and Fringe glycosylation are well conserved in the evolution of vertebrates and invertebrates.…”

Identification of Two Amino Acids in Activin A That Are Important for Biological Activity and Binding to the Activin Type II Receptors

“…Like other members of the TGF-␤ family, the activins are synthesized as large precursor proteins consisting of a signal peptide, a glycosylated prodomain and a mature domain. The maturation of activin requires intracellular cleavage by protein convertases, such as furin, at the basic cleavage site which separates the mature chain from the prodomain (13,14). Removal of the prodomains from the precursor dimer is necessary for biological activity of the mature 25-kDa dimer, since unprocessed high molecular weight forms of activin A display no biological activity (13,15,16).…”

The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology.

“…Recently, genes that encode kex2/subtilisin-like endoproteases have been isolated from Drosophila melanogaster and the nematode Caenorhabditis elegans. Two genes isolated from Drosophila, called Dfur-1 (Roebroek et al 1992(Roebroek et al , 1994; also called dKLIP-1; Hayflick et al 1992), and Dfur-2 (Roebroek et al 1992), encode convertases with sequence similarity to hFURIN. Genes isolated from C. elegans include CELPC2, which shows sequence similarity to PC2 (G6-mez-Saladin et al 1994), and an additional gene identified by the genome sequencing group that most closely resembles hFURIN (Waterston et al 1992;C.…”