Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Christoffersen, Claus T.; Bornfeldt, Karin E.; Rotella, Carlo Maria; Gonzales, Noe; Vissing, Henrik; Shymko, Ronald M.; Hoeve, Johanna ten; Groffen, John; Heisterkamp, Nora; Meyts, P De

doi:10.1210/endo.135.1.8013387

Cited by 63 publications

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“…Although the functional epitopes of the equivalent ligand binding site of both forms of the receptor are similar, certain amino acids appear to make ]insulin were performed with receptors isolated from detergent lysates of transfected cells. Data were analyzed by curve fitting to a two-site sequential model as described under "Experimental Procedures" (8,30). Results are mean Ϯ S.E.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

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Section: Resultsmentioning

confidence: 99%

“…Washing, blocking, receptor binding, and competitive binding assays with labeled and unlabeled peptides were performed as described. Binding data were analyzed by a two-site sequential model (8,30).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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“…At very high insulin concentrations, more than one molecule can bind to the IR, and this results in a bell-shaped doseresponse curves for dissociation, whereas IGF-IR competition binding assays with IGF-I binding to the IGF-IR do not display this characteristic (53) (Fig. 3).…”

Section: Discussionmentioning

confidence: 99%

“…3). Christoffersen et al (53) concluded that IGF-I also has a bivalent binding mode and crosslinks two ␣-subunits at sites that are distinct from the IR binding sites. In this study, we show for the first time that the same is true for IGF-II binding to the IGF-IR, since we do not see a bell-shaped dose-response curves for dissociation.…”

Section: Discussionmentioning

confidence: 99%

An Investigation of the Ligand Binding Properties and Negative Cooperativity of Soluble Insulin-like Growth Factor Receptors

Surinya

Forbes

Occhiodoro

et al. 2008

Journal of Biological Chemistry

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To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the selfassociating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors. Despite their similarity in sequence and structure IGF-I and IGF-II can stimulate both overlapping and distinct biological functions (reviewed in Ref. 9). This is evident in patients with IGF-I deficiency, which results in severe growth and mental retardation, where IGF-II does not compensate for the loss of IGF-I activity (10 -12). Therefore, in order to understand how both IGF-I and IGF-II stimulate their respective biological outcomes, we first need to understand the mechanism by which both ligands bind and in turn activate the IGF-1R. Insulin-like growth factors (IGFsThe IGF-1R is a member of the tyrosine kinase family of receptors and, together with the insulin receptor (IR) and insulin-related receptor, forms a subfamily with similar structural organization (reviewed in Ref. 9). The IGF-1R and IR are homodimers composed of two ␣ and two ␤ subunits and are synthesized as a single precursor polypeptide, which is then post-translationally processed by dimerization, proteolytic cleavage, and glycosylation.The amino-terminal regions of the ␣ chain of these receptors are composed of three domains, two structurally homologous subdomains designated large homologous domain 1 (L1) and large homologous domain 2 (L2), which are separated by a cysteine-rich (CR) domain of ϳ160 amino acids (3) (see supplemental Fig. S1). The major ligand binding determinants of the structurally related IGF-1R and IR reside within the extracellular ␣ subunits of the receptors (reviewed in Refs. 9, 13, and 14). Studies performed with truncated IRs have demonstrated that dimeriza...

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“…Primers used for PCR were purchased from Genosys and all 5'-deoxynucleotide triphosphates were from Boehringer. Plasmids carrying cloned fulllength cDNAs encoding IR [19,20] and p85 [21 23] were generously provided by A. Ullrich and cloned full-length cDNA for IGF-1R [24,25] was obtained from P. De Meyts. The plasmids used for hybrid gene constructs were pBTMll6 ( [26]; carrying trpl) and pGAD GH ( [27]; carrying leu2), encoding the DNA binding domain of LEXA and GAL4 activation domain (GAD), respectively.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Interaction of p85 subunit of PI 3‐kinase with insulin and IGF‐1 receptors analysed by using the two‐hybrid system

et al. 1995

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Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Cited by 63 publications

References 0 publications

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

An Investigation of the Ligand Binding Properties and Negative Cooperativity of Soluble Insulin-like Growth Factor Receptors

Interaction of p85 subunit of PI 3‐kinase with insulin and IGF‐1 receptors analysed by using the two‐hybrid system

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