Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I.
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.
Gauguin
Lisbeth
L
Receptor Systems Biology Laboratory, Hagedorn Research Institute, 2820 Gentofte, Denmark. lgau@hagedorn.dk
Delaine
Carlie
C
Alvino
Clair L
CL
McNeil
Kerrie A
KA
Wallace
John C
JC
Forbes
Briony E
BE
De Meyts
Pierre
P
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D007328:Insulin / Q000737:chemistry; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology; D011485:Protein Binding; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D007328:Insulin; D008024:Ligands; D017526:Receptor, IGF Type 1; D000409:Alanine