Role of Insulin Receptor Dimerization Domains in Ligand Binding, Cooperativity, and Modulation by Anti-receptor Antibodies

Abstract

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four aminoterminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the ␣-subunit). The fragments contained one or both cysteine residues (amino acids 524 The insulin receptor (IR)1 contains two ␣-and two ␤-subunits in a disulfide-linked ␤-␣-␣-␤ configuration. Ligand binding determinants reside within the extracellular ␣-subunit, and the cytoplasmic tyrosine kinase activity of the intracellular ␤-subunit is responsible for ligand-induced signal transduction to metabolic and mitogenic responses (1). The structure of the extracellular region of the insulin receptor is predicted to be composed of six distinct structural domains as follows: two homologous domains L1 and L2 flanking a cysteine-rich domain CR followed by three fibronectin type III repeats (Fn0, Fn1, and Fn2) (2). A crystal structure has been determined for the first three domains of the homologous type I IGF receptor (IGFR) (3), and the structure of the Fn domains has been modeled by comparison with similar structures in other proteins (4 -6). The central fibronectin domain Fn1 includes a large inserted loop of 135 amino acids containing the site of proteolytic cleavage that generates ␣-and ␤-subunits from the proreceptor polypeptide. Disulfide links between ␣-subunits have been localized to Cys 524 (Fn0 domain) and Cys 682 (Fn1 insert), whereas the ␣-␤-disulfide link is between Cys 647 (Fn1) and Cys 860 (Fn2) (7, 8). Regions of the IR/IGFR that are involved in ligand binding have been identified by affinity cross-linking, generation of chimeric IR/IGFR, and alanine-scanning mutagenesis (9 -14). Determinants of insulin specificity reside within the L1 domain of the IR and of IGF-I specificity in the CR domain of the IGFR (15-17). The carboxyl-terminal region of the ␣-subunit is critical for ligand binding in both receptors, as revealed by mutagenesis and generation of deletion constructs (12,18,19). We have shown previously that inclusion of a 16-amino acid sequence from the carboxyl terminus of the IR or IGFR ␣-subunit is necessary to confer ligand binding on monomeric receptor fragments containing the first three amino-terminal domains (L1/CR/L2) (20, 21). However, the affinity of such monomeric constructs for insulin is considerably lower than that of the wild type receptor (IRwt), indicating that additional structural elements are necessary to confer binding properties of wild type IR. Full-length half-receptors, generated from IRwt by disulfide reduction, similarly exhibit decreased affinity (22, 23) suggesting that dimerization plays an important part in creating a high affinity binding site.A characteristic feature of ligand binding to wild type IR is negative cooperativity, as revealed by curvilinear Scatchard plots and accelerated dissociation of bound 125 I-insulin in the presence of unlabeled insulin...

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

“…In contrast, the corresponding constructs without the CT domain, IR468 and IR308, did not bind insulin. This supports previous findings that the presence of the CT domain is essential for insulin binding but that it can be effective from different positions in the primary sequence (8,18). Therefore, we investigated whether it was possible to reconstitute insulin binding by mixing the IR468 and IR308 fragments with free synthetic CT peptide.…”

“…Although the CT domain appears to be able to support insulin binding from different locations in the primary sequence, its positioning may be a critical determinant of binding affinity (18). In the present study, this was demonstrated by the IR308.CT and IR255.CT constructs.…”

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

“…The importance of domain organization has been observed when comparing the binding properties of an artificial soluble IR protein (mIR.Fn0/Ex10), which exhibits high affinity ligand binding and negative cooperativity (19,20), whereas the sIR does not show negative cooperativity but contains all of the same domains. Presumably, the arrangement of these domains in the two receptor fragments is different and influences negative cooperativity.…”

“…Full-length disulfide-reduced half-receptors, dimeric receptors truncated at the transmembrane domain (ectodomain), and monomeric IR fragments all exhibit reduced affinity compared with the wild type receptor (17,18). High affinity binding is restored by the inclusion of IR dimerization domains (Fn1, Fn2 insert domain) in soluble IRs (19,20) and following fusion of the IR ectodomain with self-associating domains, such as immunoglobulin Fc (21) or a leucine zipper motif (22). A soluble IGF-1R extracellular-Z domain fusion protein binds IGF-I and IGF-II with high affinity (23), suggesting that all of the determinants required for high affinity ligand binding reside within the extracellular domain of the IGF-1R.…”

Globular Adiponectin Activates Nuclear Factor-κB and Activating Protein-1 and Enhances Angiotensin II–Induced Proliferation in Cardiac Fibroblasts

“…Depending on the epitope recognized, the interaction of antibodies with insulin receptor can be either stimulatory or inhibitory for equilibrium binding of insulin and can result in inhibition or acceleration of dissociation of previously bound ligand (18). In this study, we examined whether gAd activation of two redox-sensitive transcription factors is mediated through AdipoR1 and AdipoR2 (19) using antibodies against AdipoR1 and AdipoR2.…”