Abstract
Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.
Highlights
Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor
We have analyzed whether insulin-like growth factor-I (IGF-I) residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor
The results show that introducing alanine in positions Glu9, Asp12, Phe16, Leu54, and Glu58 results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding
Summary
Materials and Cell Lines—Molecular biological procedures, including agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation, and DNA sequencing were performed by standard methods. Oligonucleotides were purchased from Geneworks Pty Ltd (Adelaide, South Australia). Restriction enzymes and ligase were from New England. Recombinant LongTMR3IGF-I and IGF-I were purchased from GroPep Ltd (Adelaide, South Australia). Europium-labeled IGF-I was produced as described by Denley et al [29] according to the manufacturer’s instructions. Human embryonic renal cells (293EBNA) transfected with cDNA encoding full-length human IGF-IR (293EBNA IGF-IR). Were made as previously described [30]. The P6 BALB/c3T3 cell line overexpressing the IGF-IR (P6 IGF-IR) was a kind gift from. The L6 rat muscle myoblast cell line was kindly provided by M.
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