STRUCTURAL CHARACTERIZATION AND ALTERNATE SPLICING OF THE GENE ENCODING THE PREADIPOCYTE EGF-LIKE PROTEIN PREF-1

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein famil y, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished durin g the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C. M., and Sul, H. S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic cl ones for pref-1 and have identified multiple pref-1 transcripts genera ted by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analys is, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional p romoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1 -luciferase fusion gene construct gave 20-fold higher promoter activit y as compared to the promoterless vector. Analysis of exon-intron junc tions reveals that unlike the majority of the mammalian EGF-like genes , EGF-like repeats of pref-1 are not encoded by discrete exons. Throug h RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones , we have identified, in addition to full-length pref-1, five alternat ely 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 al l of the multiple forms of pref-1 transcript have in-frame deletions g enerated by alternate splicing within exon 5.