A highly conserved insulin-like growth factor-binding protein (IGFBP-5) is expressed during myoblast differentiation.
Insulin-like growth factor-binding proteins (IGFBPs) are a family of secreted proteins that bind insulin-like growth factors I and II (IGFs I and II) and are capable of modulating IGF actions on target cells. We have shown previously that C2 myoblasts secrete a single approximately 29-kDa IGFBP during their terminal differentiation (Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989) J. Biol. Chem. 264, 13810-13817). In this study, we have purified the protein from C2 cell-conditioned media by conventional and IGF-affinity chromatography, cloned its cDNA by PCR-based and traditional library screening, and identified it as mouse IGFBP-5. The resultant 5561 nucleotide cDNA encodes a 252-amino acid mature protein (predicted M(r) approximately 28,400) that is 97% identical to rat and human IGFBP-5. In differentiating C2 myoblasts and in F3 azamyoblasts the > 6-kilobase IGFBP-5 mRNA accumulates concomitantly with induction of myogen mRNA, an early marker of muscle differentiation. Ligand blot analysis shows that IGFBP-5 protein is secreted within 12 h of the onset of differentiation in these cells and that it is the only IGFBP produced in several fusing skeletal muscle cell lines. In vivo, IGFBP-5 transcripts are expressed in a variety of mouse tissues including striated muscle, but, unlike other IGFBPs, it is barely detectable in liver. IGFBP-5 is more conserved than other IGFBPs in mammals; its conserved structure and sequence also extends to non-mammalian vertebrates. Hybridization of a mouse BP5 coding region probe to RNA from several chicken and Xenopus tissues demonstrated similarly sized transcripts in these species. A partial Xenopus cDNA is identical in 38/45 deduced amino acids to the mammalian proteins. Identification of an IGF-binding protein that is produced during myoblast differentiation provides a model system in which to study the potential modulatory role of IGFBPs in development.
James
P L
PL
Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Jones
S B
SB
Busby
W H
WH
Clemmons
D R
DR
Rotwein
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002454:Cell Differentiation; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014981:Xenopus
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II