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.
Department of Life Science, University of Seoul, 90 Jeonnong-dong, Seoul 130-743, Korea.
Kyung-Yi
KY
Choi
Yoon-Joo
YJ
Kyung
Chul Young
CY
Lee
Dong-Hee
DH
Lee
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