Modulation of human IGF binding protein-3 activity by structural modification.
To delinate regions of IGFBP-3 involved in ligand and cell-surface binding. DNAs encoding human IGFBP-3 [1-264] and several variants were transfected into CHO cells. Of three deletion (delta) mutants. IGFBP-3 [1-88], [1-184], and [delta 89-184], none bound IGF-I tracer by ligand blotting, although all were detectable by immunoblotting. No ALS binding was detectable, as predicted by the lack of IGF binding. Normal-sequence IGFBP-3 associated with the CHO cells and was partly displaceable by IGF-I. Whereas IGFBP-3 [1-88] and [1-184] failed to cell-associate, the non-IGF-binding central deletion variant [delta 89-184] did associate with CHO cells but was not displaced by IGF-I. To further examine the role of the carboxy-terminal domain in cell-association, the basic sequence IGFBP-3 [228-232] (KGRKR) was altered to the corresponding IGFBP-1 residues MDGEA, a major charge reversal. This variant showed reduced IGF-I binding, and bound ALS with decreased affinity as determined by Scatchard analysis. It showed no cell binding, implicating the basic domain in cell-association. We conclude that, whereas the central and carboxy-terminal domain deletions fail to bind IGF-I, the ability to cell associate requires the carboxy-terminal but not the central domain. Specifically, the basic region [228-232] is essential for cell binding, and also affects IGF-I binding, and independently, ALS affinity.
Baxter
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Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, NSW, Australia.
Firth
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SM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D014162:Transfection
D002352:Carrier Proteins; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; C075944:insulin-like growth factor binding protein, acid labile subunit