Anti-Insulin Receptor Antibodies Improve Hyperglycemia in a Mouse Model of Human Insulin Receptoropathy.

Loss-of-function mutations in both alleles of the human insulin receptor gene (INSR) cause extreme insulin resistance (IR) and usually death in childhood, with few effective therapeutic options. Bivalent antireceptor antibodies can elicit insulin-like signaling by mutant INSR in cultured cells, but whether this translates into meaningful metabolic benefits in vivo, wherein the dynamics of insulin signaling and receptor recycling are more complex, is unknown. To address this, we adopted a strategy to model human insulin receptoropathy in mice, using Cre recombinase delivered by adeno-associated virus to knockout endogenous hepatic Insr acutely in floxed Insr mice (liver insulin receptor knockout [L-IRKO] + GFP), before adenovirus-mediated add back of wild-type (WT) or mutant human INSR Two murine anti-INSR monoclonal antibodies, previously shown to be surrogate agonists for mutant INSR, were then tested by intraperitoneal injections. As expected, L-IRKO + GFP mice showed glucose intolerance and severe hyperinsulinemia. This was fully corrected by add back of WT but not with either D734A or S350L mutant INSR. Antibody injection improved glucose tolerance in D734A INSR-expressing mice and reduced hyperinsulinemia in both S350L and D734A INSR-expressing animals. It did not cause hypoglycemia in WT INSR-expressing mice. Antibody treatment also downregulated both WT and mutant INSR protein, attenuating its beneficial metabolic effects. Anti-INSR antibodies thus improve IR in an acute model of insulin receptoropathy, but these findings imply a narrow therapeutic window determined by competing effects of antibodies to stimulate receptors and induce their downregulation.

Evaluation of anti-insulin receptor antibodies as potential novel therapies for human insulin receptoropathy using cell culture models.

AIMS/HYPOTHESIS: Bi-allelic loss-of-function mutations in the INSR gene (encoding the insulin receptor [INSR]) commonly cause extreme insulin resistance and early mortality. Therapeutic options are limited, but anti-INSR antibodies have been shown to activate two mutant receptors, S323L and F382V. This study evaluates four well-characterised murine anti-INSR monoclonal antibodies recognising distinct epitopes (83-7, 83-14, 18-44, 18-146) as surrogate agonists for potential targeted treatment of severe insulin resistance arising from insulin receptoropathies. METHODS: Ten naturally occurring mutant human INSRs with defects affecting different aspects of receptor function were modelled and assessed for response to insulin and anti-INSR antibodies. A novel 3T3-L1 adipocyte model of insulin receptoropathy was generated, permitting conditional knockdown of endogenous mouse Insr by lentiviral expression of species-specific short hairpin (sh)RNAs with simultaneous expression of human mutant INSR transgenes. RESULTS: All expressed mutant INSR bound to all antibodies tested. Eight mutants showed antibody-induced autophosphorylation, while co-treatment with antibody and insulin increased maximal phosphorylation compared with insulin alone. After knockdown of mouse Insr and expression of mutant INSR in 3T3-L1 adipocytes, two antibodies (83-7 and 83-14) activated signalling via protein kinase B (Akt) preferentially over signalling via extracellular signal-regulated kinase 1/2 (ERK1/2) for seven mutants. These antibodies stimulated glucose uptake via P193L, S323L, F382V and D707A mutant INSRs, with antibody response greater than insulin response for D707A. CONCLUSIONS/INTERPRETATION: Anti-INSR monoclonal antibodies can activate selected naturally occurring mutant human insulin receptors, bringing closer the prospect of novel therapy for severe insulin resistance caused by recessive mutations.

Cell-autonomous programming of rat adipose tissue insulin signalling proteins by maternal nutrition.

AIMS/HYPOTHESIS: Individuals with a low birthweight have an increased risk of developing type 2 diabetes mellitus in adulthood. This is associated with peripheral insulin resistance. Here, we aimed to determine whether changes in insulin signalling proteins in white adipose tissue (WAT) can be detected prior to the onset of impaired glucose tolerance, determine whether these changes are cell-autonomous and identify the underlying mechanisms involved. METHODS: Fourteen-month-old male rat offspring born to dams fed a standard protein (20%) diet or a low (8%) protein diet throughout gestation and lactation were studied. Fat distribution and adipocyte size were determined. Protein content and mRNA expression of key insulin signalling molecules were analysed in epididymal WAT and in pre-adipocytes that had undergone in vitro differentiation. RESULTS: The offspring of low protein fed dams (LP offspring) had reduced visceral WAT mass, altered fat distribution and a higher percentage of small adipocytes in epididymal WAT. This was associated with reduced levels of IRS1, PI3K p110ß, Akt1 and PKCζ proteins and of phospho-Akt Ser473. Corresponding mRNA transcript levels were unchanged. Similarly, in vitro differentiated adipocytes from LP offspring showed reduced protein levels of IRß, IRS1, PI3K p85α and p110ß subunits, and Akt1. Levels of Akt Ser473 and IRS1 Tyr612 phosphorylation were reduced, while IRS1 Ser307 phosphorylation was increased. CONCLUSIONS/INTERPRETATION: Maternal protein restriction during gestation and lactation changes the distribution and morphology of WAT and reduces the levels of key insulin signalling proteins in the male offspring. This phenotype is retained in in vitro differentiated adipocytes, suggesting that programming occurs via cell-autonomous mechanism(s).

Acute knockdown of the insulin receptor or its substrates Irs1 and 2 in 3T3-L1 adipocytes suppresses adiponectin production.

Loss of function of the insulin receptor (INSR) in humans produces severe insulin resistance. Unlike "common" insulin resistance, this is associated with elevated plasma levels of the insulin-sensitising, adipose-derived protein adiponectin. The underlying mechanism for this paradox is unclear, and it is at odds with the acute stimulation of adiponectin secretion reported on insulin treatment of cultured adipocytes. Given recent evidence for ligand-independent actions of the INSR, we used a lentiviral system to knock down Insr or its substrates Irs1 and Irs2 conditionally in 3T3-L1 murine preadipocytes/adipocytes to assess whether acute loss of their expression has different consequences to withdrawal of insulin. Efficient knockdown of either Insr or Irs1/2 was achieved by conditional shRNA expression, severely attenuating insulin-stimulated AKT phosphorylation and glucose uptake. Dual knockdown of Irs1 and Irs2 but not Insr in preadipocytes impaired differentiation to adipocytes. Acute knockdown of Insr or both Irs1 and Irs2 in adipocytes increased Adipoq mRNA expression but reduced adiponectin secretion, assessed by immunoassay. Knockdown sustained for 14 days also reduced immunoassay-detected adiponectin secretion, and moreover induced delipidation of the cells. These findings argue against a distinct effect of Insr deficiency to promote adiponectin secretion as the explanation for paradoxical insulin receptoropathy-related hyperadiponectinaemia.

Molecular basis of signaling specificity of insulin and IGF receptors: neglected corners and recent advances.

Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of "metabolic" and "mitogenic" responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to "metabolic" and "mitogenic" responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, ß-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in "metabolic" or "mitogenic" signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears to be the major determinant of whether responses to insulin and IGFs are perceived as "metabolic" or "mitogenic."

Two isoforms of the mRNA binding protein IGF2BP2 are generated by alternative translational initiation.

IGF2BP2 is a member of a family of mRNA binding proteins that, collectively, have been shown to bind to several different mRNAs in mammalian cells, including one of the mRNAs encoding insulin-like growth factor-2. Polymorphisms in the Igf2bp2 gene are associated with risk of developing type 2 diabetes, but detailed functional characterisation of IGF2BP2 protein is lacking. By immunoblotting with C-terminally reactive antibodies we identified a novel IGF2BP2 isoform with a molecular weight of 58 kDa in both human and rodents, that is expressed at somewhat lower levels than the full-length 65 kDa protein. We demonstrated by mutagenesis that this isoform is generated by alternative translation initiation at the internal Met69. It lacks a conserved N-terminal RNA Recognition Motif (RRM) and would be predicted to differ functionally from the canonical full length isoform. We further investigated IGF2BP2 mRNA transcripts by amplification of cDNA using 5'-RACE. We identified multiple transcription start sites of the human, mouse and rat Igf2bp2 genes in a highly conserved region only 50-90 nts upstream of the major translation start site, ruling out the existence of N-terminally extended isoforms. We conclude that structural heterogeneity of IGF2BP2 protein should be taken into account when considering cellular function.

Signalling by insulin and IGF receptors: supporting acts and new players.

The signalling pathways utilised by insulin receptor (IR) and IGF receptor to transduce their diverse effects on cellular metabolism, growth and survival are well established in broad outline, but many details remain to be elucidated. Tyrosine phosphorylation of IR substrates and Shc initiates signalling via canonical phosphoinositide 3-kinase/Akt and Ras/MAP kinase pathways, which together mediate many of the actions of insulin and IGFs. However, a variety of additional substrates and scaffolds have been described that may play roles in modulating the canonical pathways or in specific biological responses. This review will focus on recent studies that have extended our understanding of insulin/IGF signalling pathways, and the elements that may contribute to specificity.

The involvement of microRNAs in Type 2 diabetes.

T2D (Type 2 diabetes mellitus) is a major health issue that has reached epidemic status worldwide. T2D is a progressive metabolic disorder characterized by reduced insulin sensitivity, insulin resistance and pancreatic ß-cell dysfunction. Improper treatment of TD2 can lead to severe complications such as heart disease, stroke, kidney failure, blindness and nerve damage. The aetiology and molecular mechanisms of T2D are not fully understood, but compelling evidence points to a link between T2D, obesity, dyslipidaemia and insulin resistance. Although T2D seems to be strongly linked to environmental factors such as nutrition and lifestyle, studies have shown that genetic factors, such as polymorphisms associated with metabolic genes, imprinting, fetal programming and miRNA (microRNA) expression, could also contribute to the development of this disease. miRNAs are small 22-25-nt-long untranslated RNAs that negatively regulate the translation of mRNAs. miRNAs are involved in a large number of biological functions such as development, metabolism, immunity and diseases such as cancer, cardiovascular diseases and diabetes. The present review examines the various miRNAs that have been identified as being potentially involved in T2D, focusing on the insulin-sensitive organs: white adipose tissue, liver, skeletal muscle and the insulin-producing pancreatic ß-cells.

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.

An investigation of the ligand binding properties and negative cooperativity of soluble insulin-like growth factor receptors.

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 self-associating 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.

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation.

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.

Differential phosphorylation of IRS-1 and IRS-2 by insulin and IGF-I receptors.

The specific contribution of insulin and IGF-I receptors to IRS-protein activation remains elusive. We studied the signalling properties of AspB10-insulin, an analog with enhanced affinity for the IGF-I receptor, in comparison to native insulin using primary human skeletal muscle cells. In myoblasts regular insulin and AspB10-insulin were equipotent in stimulating the IRS cascade, whereas this analog induced a significantly higher Shc phosphorylation. Phosphorylation of IRS-1 in response to insulin was inhibited equally by blocking either the insulin or the IGF-I receptor. IRS-1 activation by AspB10-insulin was only inhibited by blocking the IGF-I receptor. IRS-2 phosphorylation induced by both insulin and AspB10-insulin was nearly insensitive to blocking the insulin receptor, being predominantly mediated by the IGF-I receptor. We conclude that in myoblasts IRS-2, but not IRS-1, functions as preferred substrate for the IGF-I receptor. These data suggest a specific role for IRS-2 in growth and differentiation of human skeletal muscle.

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation.

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.

Grb10 and Grb14: enigmatic regulators of insulin action--and more?

The Grb proteins (growth factor receptor-bound proteins) Grb7, Grb10 and Grb14 constitute a family of structurally related multidomain adapters with diverse cellular functions. Grb10 and Grb14, in particular, have been implicated in the regulation of insulin receptor signalling, whereas Grb7 appears predominantly to be involved in focal adhesion kinase-mediated cell migration. However, at least in vitro, these adapters can bind to a variety of growth factor receptors. The highest identity within the Grb7/10/14 family occurs in the C-terminal SH2 (Src homology 2) domain, which mediates binding to activated receptors. A second well-conserved binding domain, BPS [between the PH (pleckstrin homology) and SH2 domains], can act to enhance binding to the IR (insulin receptor). Consistent with a putative adapter function, some non-receptor-binding partners, including protein kinases, have also been identified. Grb10 and Grb14 are widely, but not uniformly, expressed in mammalian tissues, and there are various isoforms of Grb10. Binding of Grb10 or Grb14 to autophosphorylated IR in vitro inhibits tyrosine kinase activity towards other substrates, but studies on cultured cell lines have been conflicting as to whether Grb10 plays a positive or negative role in insulin signalling. Recent gene knockouts in mice have established that Grb10 and Grb14 act as inhibitors of intracellular signalling pathways regulating growth and metabolism, although the phenotypes of the two knockouts are distinct. Ablation of Grb14 enhances insulin action in liver and skeletal muscle and improves whole-body tolerance, with little effect on embryonic growth. Ablation of Grb10 results in disproportionate overgrowth of the embryo and placenta involving unidentified pathways, and also impacts on hepatic glycogen synthesis, and probably on glucose homoeostasis. This review discusses the extent to which previous studies in vitro can account for the observed phenotype of knockout animals, and considers evidence that aberrant function of Grb10 or Grb14 may contribute to disorders of growth and metabolism in humans.

Attractin' more attention - new pieces in the obesity puzzle?

Genetic, biochemical and pharmacological studies in humans and rodents have established that signalling through the G-protein-coupled melanocortin-4 receptor (MC4R) by pro-opiomelanocortin (POMC)-derived ligands plays a critical role in the central suppression of appetite. As a consequence, malfunction of this signalling system leads to the development of obesity. It has been shown previously that melanocortin signalling can be modulated by the type 1 transmembrane protein attractin, apparently acting as a co-receptor for the inhibitory ligand agouti. Work reported in this issue of Biochemical Journal (Haqq et al.) demonstrates that the cytosolic tail of an attractin-like protein (ALP) binds directly and specifically to the C-terminal region of MC4R, raising the possibility that proteins of the attractin family influence melanocortin receptor function through multiple mechanisms.

Microarray analysis of insulin and insulin-like growth factor-1 (IGF-1) receptor signaling reveals the selective up-regulation of the mitogen heparin-binding EGF-like growth factor by IGF-1.

Insulin and insulin-like growth factor-1 (IGF-1) act through highly homologous receptors that engage similar intracellular signaling pathways, yet these hormones serve largely distinct physiological roles in the control of metabolism and growth, respectively. In an attempt to uncover the molecular mechanisms underlying their divergent functions, we compared insulin receptor (IR) and IGF-1 receptor (IGF-1R) regulation of gene expression by microarray analysis, using 3T3-L1 cells expressing either TrkC/IR or TrkC/IGF-1R chimeric receptors to ensure the highly selective activation of each receptor tyrosine kinase. Following stimulation of the chimeric receptors for 4 h, we detected 11 genes to be differentially regulated, of which 10 were up-regulated to a greater extent by the IGF-1R. These included genes involved in adhesion, transcription, transport, and proliferation. The expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen, was markedly increased by IGF-1R but not IR activation. This effect was dependent on MAPK, but not phosphatidylinositol 3-kinase, and did not require an autocrine loop through the epidermal growth factor receptor. HB-EGF mitogenic activity was detectable in the medium of 3T3-L1 preadipocytes expressing activated IGF-1R but not IR, indicating that the transcriptional response is accompanied by a parallel increase in mature HB-EGF protein. The differential abilities of the IR and IGF-1R tyrosine kinases to stimulate the synthesis and release of a growth factor may provide, at least in part, an explanation for the greater role of the IGF-1R in the control of cellular proliferation.

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies.

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 amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.