Alanine Scanning of a Putative Receptor Binding Surface of Insulin-like Growth Factor-I

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 ␣ 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. 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. IGF-I2 is a single chain polypeptide that plays an important role in regulating growth and development (1, 2). IGF-I consists of 70 amino acid residues arranged in four domains. The A and B domains are homologous to the A and B chains of insulin (Fig. 1), whereas the C-domain connecting the A-and B-domain and the D-domain extending from the C-terminal end of the A-domain are only present in IGF-I (3-5). The three-dimensional structures of insulin and IGF-I are also very similar. The B-domain of the two molecules is arranged in a central ␣-helix including residues 8 -17 (B9 -B19) (insulin residues in parentheses), and the A-domain contains two anti-parallel ␣-helices including residues 43-48 (A1-A8) and residues 54 -60 (A13-A20) (4, 6, 7).The insulin receptor family consists of the insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR) and the insulin-receptor-related receptor, all of which are receptortyrosine kinases. The members of the insulin receptor family consist of two receptor halves, each comprising an extracellular ␣-subunit and a transmembrane ␤-subunit, linked by a disulfide bridge. The receptor exists as a dimer when no ligand is bound, making an ␣2␤2 receptor held together by disulfide bridges between the two ␣-subunits. The IR and the IGF-IR have sequence similarities varying from 41 to 84% depending on which regions are being compared. The overall structure of the L1-Cys-rich-L2 domains of the two receptors have been shown by x-ray crystallography to be similar (8 -11).Numerous studies indicate that the IR and IGF-IR contains two separate binding sites for the ligand; however, only one ligand binds and activates the ␣2␤2 receptor dimer. Insulin is believed to interact with the receptor using two binding surfaces, which cross-link the two receptor halves. This cross-

A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II

“…This observation was affirmed in the present study where receptor binding by the Glu 12 3 Ala analogue, for instance, was more affected when binding whole cells than immunocaptured receptor, an observation also made for the equivalent analogue of IGF-I (Glu 9 3 Ala IGF-I) (22). The opposite was true for Phe 19 3 Ala on the IGF-1R and Leu 53 3 Ala on the IR-A where a greater effect was seen in immunocapture assays than whole cell binding assays.…”

“…Given evidence in the literature that some analogues may behave differently in the two assay formats (22,35), the affinities of all IGF-II analogues were measured using both immunocaptured receptors (IGF-1R, IR-A, or IR-B) and whole cell receptor binding assays with cells expressing IGF-1R or IR-A. This observation was affirmed in the present study where receptor binding by the Glu 12 3 Ala analogue, for instance, was more affected when binding whole cells than immunocaptured receptor, an observation also made for the equivalent analogue of IGF-I (Glu 9 3 Ala IGF-I) (22).…”

Separation of Fast from Slow Anabolism by Site-specific PEGylation of Insulin-like Growth Factor I (IGF-I)

“…As evident from the three-dimensional model of IGF-I computationally docked through its site 1 on the L1-CR-L2 domains of the IGF-IR ( Fig. 2A), the only area of IGF-IR for which a crystallographic structure has been determined (36,39), Lys-68 is not located in this interaction site 1. PEGylation at this site is likely to sterically hinder both receptor association and closing of the receptor dimer through site 2.…”

“…Such a profile is definitely therapeutically undesirable for an insulin analog, but to the contrary beneficial for an IGF-I analog like ours. Given the similarity of the InsR and IGF-IR binding mechanisms (32,36), it is not unreasonable to extrapolate the consequences of impaired site 2 interaction to this analog.…”

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

“…1, A and B). Mutagenesis of insulin and IGF-I suggests that in each case this ␣-helix makes a key contribution to respective receptor recognition (21,22). The sequences of the A1-A8 ␣-helices are homologous (Fig.…”