The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site.

Abstract

To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor a-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain ofhigh affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.The polypeptide hormone insulin plays an essential role in metabolic regulation (1-3). It exerts its physiological effects through its interaction with a specific high-affinity receptor, which is an integral cell surface membrane glycoprotein with a relative molecular mass of about 400 kDa (4-6). The receptor is a disulfide-linked heterotetramer, made up of two a-and two B-subunits (7-12). Its ligand binding domains are formed by the extracellular a-subunits, whereas the /3-subunits consist of a short extracellular domain, a single transmembrane segment, and an intrinsic intracellular tyrosine protein kinase domain involved in signal transduction (13-15).The insulin-like growth factor I (IGF-I) receptor shows extensive similarity to the insulin receptor in amino acid sequence, domain structure, and signaling mechanism (11,16). Although insulin and IGF-I are very similar in amino acid sequence, they bind only weakly to the receptor for the other hormone (17,18).Ligand binding affinity and specificity are central to receptor activation, regulation, and function. Chimeric molecules comprising parts of related receptors have been useful for elucidating relationships between structure and specificity in several systems (e.g., refs. 19-21). In a previous study we showed that it is possible to convert the insulin receptor into a receptor with high affinity for IGF-I and low affinity for insulin by substituting an insulin receptor cDNA fragment (exons 1, 2, and 3) with that for the corresponding . This suggested to us that the ligand specificity of both receptors resides in the same region. Here, we extend this approach to a more detailed analysis of the ligand specificity of this receptor family. The first 68 N-terminal amino acids of the insulin receptor are found to determine insulin specificity. The corresponding 62 N-t...

Insulin Stimulates the Clonogenic Potential of Angiogenic Endothelial Progenitor Cells by IGF-1 Receptor-Dependent Signaling

“…The concentration of insulin needed to observe this effect corresponds to high physiologic serum levels around 0.15 nM (31) and increased at concentrations of ≥1.5 nM, which are present in patients treated with high doses of insulin or analogs (31)(32)(33). It seems reasonable that the high concentrations of human insulin studied are needed to elicit this growth effect, because affinity of insulin for its receptor is two to three orders of magnitude higher than for the cognate IGF-1 receptor (30). The efficient silencing of the IR [which has a half life of ~6-12 h (34,35)] through blocking its synthesis by a 3-day antisense knockdown, as well as the lack of an effect of IR-neutralizing antibodies, make involvement of the IR in this novel effect of insulin unlikely.…”

“…However, only stimulation with 30 nM glargine led to EPC outgrowth (186% ± 41%, P < 0.001) similar to that of human insulin (180% ± 33%, P < 0.001) (Figure 2c). The dosedependent effect of insulin on EPC outgrowth was observed between 0.15 and 15 nM, reflecting concentrations at which the IR is saturated (22,30). Therefore, we speculated that the effects are not mediated by the IR, and examined alternative receptors and signaling cascades involved in the increased outgrowth of EPCs upon stimulation with human insulin.…”

Modifications in the B10 and B26-30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor

“…Interestingly, substitution of Lys 65 and Lys 68 in the D region with Ala also reduced IGF-I receptor affinity approximately 10-fold, suggesting that multiple basic residues (Lys 27 , Arg Rutter [35] demonstrated that 56 and 52 amino acid stretches in the cysteine-rich regions of the insulin and IGF-I receptors, respectively, contained major determinants for ligand binding specificity. Others have confirmed the importance of the cysteine-rich region of the IGF-I receptor in ligand specificity [36][37][38], but have suggested that the N-terminal 68 amino acid portion of the insulin receptor a -subunit contains the key residues for insulin selectivity [38]. It is interesting to note that the C-terminal 20 amino acids of the region of the IGF-I receptor described by Gustafson and Rutter contains one basic residue and five acidic residues, which might interact with the essential basic residues on IGF-I.…”

The adipose conversion process: regulation by extracellular and intracellular factors

“…Supraphysiological concentrations of insulin enhance adipose conversion of preadipose cell lines by acting through the IGF-1 receptor [215]. Cross-reactions with this receptor is indeed possible when insulin is added at high concentrations [45,140]. Because no or poor effects are observed with physiological concentrations, similar interpretation concerns human and rabbit preadipocytes [105,182].…”