Characterization of insulin-like growth factor-free interaction between insulin-like growth factor binding protein 3 and acid labile subunit expressed from Xenopus oocytes.
The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (Delta11-60) demonstrated a moderate reduction. D88 (Delta11-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.
Choi
Kyung-Yi
KY
Department of Life Science, University of Seoul, 90 Jeonnong-dong, Seoul 130-743, Korea.
Kyung
Yoon-Joo
YJ
Lee
Chul Young
CY
Lee
Dong-Hee
DH
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005260:Female; D017353:Gene Deletion; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D007700:Kinetics; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013002:Somatomedins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013002:Somatomedins; C075944:insulin-like growth factor binding protein, acid labile subunit