Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1

CM Smas, D Green, HS Sul - Biochemistry, 1994 - ACS Publications
CM Smas, D Green, HS Sul
Biochemistry, 1994ACS Publications
Revised Manuscript Received May 5, 1994® abstract: Preadipocyte factor 1 (pref-1), a
member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like
repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in
vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of
pref-1 in preadipocytes inhibits their differentiation [Smas, CM, & Sul, HS(1993) Cell 73,725-
734]. In the present studies, we have isolated and characterized genomic clones for pref-1 …
Revised Manuscript Received May 5, 1994® abstract: Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C. M., & Sul, H. S.(1993) Cell 73,725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bpupstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-15'flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxtamembrane, and predicted transmembrane domains. We conclude, by comparing cDNA and genomic sequences, that all of the multiple forms of pref-1 transcript have in-frame deletions generated by alternate splicing within exon 5.
Preadipocyte factor 1 (pref-1) 1 is a newly described member of the epidermal growth factor-like familyof proteins that we have cloned from 3T3-L1 preadipocytes (Smas & Sul, 1993). Analysis of pref-1 cDNA sequence predicts a transmembrane protein containing six tandem epidermal growth factor-like (EGF) repeats in the putativeextracellular region. This 35-40 amino acid motif, first identified in EGF and characterized by 6 cysteines that form 3 disulfide loops (Carpenter & Cohen, 1990), is present in a variety of proteins that function incell growth, cell adhesion, and differentiation. These include the diffusible growth factors EGF, TGF-a (Massague, 1990), amphiregulin (Plowman et al., 1990), and HB-EGF (Higashiyamaetal., 1991). Each of these molecules exhibits high-affinity binding to the EGF receptor, produces mitogenic responses in EGF-sensitive cells, and in addition to the characteristic cysteine spacing in the EGF-like domain has other conserved amino acid residues involved in receptor interaction. In addition to those proteins that act through the EGF receptor, EGF-like domains are also present in a number of molecules that function in protein-protein interaction including extracellular matrix proteins and cell adhesion molecules (Siegelman et al., 1990; Krusius et al., 1987), and the Drosophila proteins notch and delta that interact via their EGF-like domains to determine cell fate choice in the neurogenic ectoderm (Rebay etal., 1991; Fehonet al., 1990).
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