Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.     

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors.

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.     

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.     

Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers

In both NIH3T3 cells and HepG2 cells, insulin-like growth factor I (IGF-I) receptors possess two beta-subunits that display different electrophoretic mobilities. Increasing concentrations of IGF-I stimulated the phosphorylation of both beta-subunits to a similar extent, whereas insulin stimulated the phosphorylation of both subunits only at elevated concentrations. Both beta-subunits were immunoprecipitated with p5, an insulin receptor-specific anti-peptide antibody, or with A410, a polyclonal anti-insulin receptor antisera. However, if the tetrameric IGF-I receptor was first dissociated into alpha-beta heterodimers with 1 mM dithiothreitol, only the lower molecular weight beta-subunit was immunoprecipitated. These results suggested that p5 and A410 specifically recognized the lower molecular weight beta-subunit but immunoprecipitated the higher molecular weight beta-subunit because it was present in the same disulfide linked tetramer. Similarly, alpha-IR-3, an antibody specific for the alpha-subunit of the IGF-I receptor, immunoprecipitated both types of beta-subunit from the intact tetramer but only the higher molecular weight beta-subunit from the dissociated heterodimers, suggesting that there are two types of alpha-subunits in the same tetramer and that the alpha-subunit recognized by alpha-IR-3 is only associated with the higher molecular weight beta-subunit. Tryptic phosphopeptide maps of the lower molecular weight beta-subunit of IGF-I receptor were different from the higher molecular weight beta-subunit, but were similar to those of the insulin receptor beta-subunit. Thus, by immunochemical cross-reactivity and structural criteria, the lower molecular weight beta-subunit of the IGF-I receptor was similar to the beta-subunit of insulin receptor. These data suggest that there exists a species of IGF-I receptor that is a hybrid composed of an insulin receptor alpha-beta heterodimer and an IGF-I receptor alpha-beta heterodimer. The existence of such a hybrid receptor could have important functional consequences.     

Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.     

Identification of domains of the insulin-like growth factor I receptor that are required for protection from apoptosis.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.     

The insulin-like effect of growth hormone on insulin-like growth factor II receptors is opposed by cyclic AMP. Evidence for a common post-receptor pathway for growth hormone and insulin action.

The counter-regulatory effects of beta-adrenergic stimulation and cyclic AMP on the insulin-like action of growth hormone (GH) on the subcellular distribution of insulin-like growth factor II (IGF-II) receptors were studied in fat cells from hypophysectomized (Hx) and sham-operated rats. For comparison, the effect of insulin on this process was also studied. Basal IGF-II binding was increased by approx. 2-fold in cells from Hx as compared with sham-operated animals. The stimulatory effect of insulin was decreased in Hx cells, mainly due to a basal redistribution but also to a reduced total number of receptors. GH exerted an acute insulin-like effect in cells from Hx rats and stimulated the translocation of IGF-II receptors from an intracellular pool to the plasma membrane. beta-Adrenergic stimulation with isoprenaline or addition of the non-metabolizable cyclic AMP-analogue N6-monobutyryl cyclic AMP induced a cellular resistance to both GH and insulin and also reduced the responsiveness to these hormones. Adenosine exerted a modulatory effect on both hormones. Binding of 125I-labelled GH to its receptors was not significantly changed by any of these factors. It is concluded that: (1) beta-adrenergic stimulation and cyclic AMP induce a cellular GH resistance at a level distal to the GH-binding site, and (2) the insulin-like effect of GH shares a common pathway with insulin which occurs at the post-binding level.     

The structural basis for insulin-like growth factor I receptor high affinity binding

We have recently identified high and low affinity insulin-like growth factor I (IGF I) binding sites in solubilized human placental membranes and purified the high affinity IGF I receptor by IGF I affinity chromatography (Tollefsen, S. E., Thompson, K., and Petersen, D. J. (1987) J. Biol. Chem. 262, 16461-16469). To define the structural basis for high affinity IGF I binding, we have examined the effect of disulfide bond reduction on the binding parameters of the high affinity IGF I receptor. We find that the disulfide bonds linking the two alpha beta dimers of the IGF I receptor heterotetramer are reduced by incubation at pH 8.75 with 2 mM dithiothreitol (DTT) for 5 min at room temperature. Gel filtration chromatography on a Superose 12 fast protein liquid chromatography column indicates that the alpha beta dimers do not remain associated by noncovalent interactions after reduction. Scatchard plots of IGF I binding to the IGF I receptor incubated at pH 8.75 with or without DTT indicate that the IGF I receptor alpha beta dimers have a 6.1 +/- 1.6 (mean +/- S.D.) times lower affinity than the heterotetramer for IGF I. The total binding capacity of the IGF I receptor treated with DTT is 1.6 +/- 0.3 (mean +/- S.D.) times higher than that of an equal amount of receptor treated without DTT. These results are consistent with a model in which the heterotetramer binds a single IGF I molecule with high affinity, whereas each of the two alpha beta dimers binds an IGF I molecule with lower affinity after dissociation. We conclude that association of two alpha beta dimers is required for formation of an IGF I receptor with high affinity for its ligand.     

Regulation of insulin-like growth factor I binding in the rat uterus by growth hormone and thyroxine.

The insulin-like growth factor-I (IGF-I)-binding sites in the rat uterus were characterized further and the effects of growth hormone and thyroxine examined. The 125I-labelled IGF-I binding sites on uterine membranes demonstrated relative binding affinity of less than 20% for IGF-II, less than 1% for insulin and no affinity for an unrelated peptide, epidermal growth factor, compared with 100% for IGF-I confirming the specificity of these binding sites. Scatchard analysis of the specific binding data revealed the presence of a single class of high-affinity binding sites (Kd = 2.50 +/- 0.68 nmol l-1, with a binding capacity of 1.02 +/- 0.13 pmol mg-1 membrane protein in the uterus of the pituitary-intact ovariectomized rat. After hypophysectomy, the uteri from these rats had significantly (P less than 0.05) increased IGF-I binding sites, without significant changes in their affinity. Administration of growth hormone with or without L-thyroxine reversed this increase in IGF-I binding. Injection of thyroxine alone to the hypophysectomized ovariectomized rats had no significant effects on the uterine IGF-I binding sites. These data show that growth hormone, but not thyroxine, can regulate IGF-I binding sites in the rat uterus, possibly through regulating IGF-I production.     

Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

Ontogeny of insulin-like growth factor I and insulin receptor kinase activity in rat liver.

IGF-I and insulin receptors possess tyrosine-kinase enzymatic activity considered to be essential for signal transduction and thereby mediating the putative effects of these hormones on fetal growth and development. We investigated the ontogeny of IGF-I and insulin receptor tyrosine-kinase activity in at least 3 separate membrane preparations from liver of rats at 21 day of embryonic life (21ED), 1 and 5 day of postnatal life (1PD and 5PD respectively) and adult. Receptors purified by wheat germ agglutinin chromatography (WGA) were exposed to graded concentrations of IGF-I or insulin, and tyrosine-kinase activity was measured by quantifying incorporation of 32P into the exogenous substrate poly[Glu,Tyr; 4:1]. IGF-I stimulated tyrosine-kinase solely at 1 PD as documented by a maximal increase of 346 +/- 167% over basal kinase activity with 6.6 nmol/L IGF-I. While the lack of response in adult animals could be explained by a striking decrease in receptors at that age, 125I-IGF-I binding and affinity labelling of the WGA preparations indicated substantial IGF-I receptors were present in the liver at each of the perinatal ages. Furthermore, this dissociation between IGF-I binding and the tyrosine-kinase activity of these IGF-I receptors could not be attributed to the presence/absence of IGF-I binding proteins as judged by affinity labelling. In contrast, insulin-stimulated tyrosine-kinase activity was observed at all ages tested although it appeared greatest at 1PD. We conclude that (i) expression of IGF-I tyrosine-kinase activity is linked to developmental events and differs from that found for the insulin receptor tyrosine-kinase activity, (ii) during the perinatal period there is an apparent dissociation between ligand binding by the IGF-I receptor and receptor tyrosine-kinase activity. These observations suggest modulation of IGF-I receptor tyrosine-kinase activity may be an important regulator of IGF-I action during the perinatal period.     

Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal of receptor alpha subunit

We have investigated the role of the C-terminal of the alpha-subunit in the insulin receptor family by characterizing chimeric mini-receptor constructs comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-terminal domain from either insulin receptor (IR) (residues 704-719), IGFIR, or insulin receptor-related receptor (IRRR). The constructs were stably expressed in baby hamster kidney cells and purified, and binding affinities were determined for insulin, IGFI, and a single chain insulin/IGFI hybrid. The C-terminal domain of IRRR was found to abolish binding in IR and IGFIR context, whereas other constructs bound ligands. The two constructs with first three domains of the IR demonstrated low specificity for ligands, all affinities ranging from 3.0 to 15 nM. In contrast, the constructs with the first three domains of the IGFIR had high specificity, the affinity of the novel minimized IGFIR for IGFI was 1.5 nM, whereas the affinity for insulin was more than 3000 nM. When swapping the C-terminal domains in either receptor context only minor changes were observed in affinities (<3-fold), demonstrating that the carboxyl-terminal of IR and IGFIR alpha-subunits are interchangeable and suggesting that this domain is part of the common binding site.     

Cholecystokinin and insulin regulate insulin-like growth factor II binding to pancreatic receptors; Evidence of a role for intracellular calcium

The binding of ¹²⁵I-labeled insulin-like growth factor II (¹²⁵I-IGF II) to mouse pancreatic acini was stimulated (45%) by insulin and inhibited (30%) by cholecystokinin octapeptide (CCK₈). When CCK₈ and insulin were added together, the effect on IGF II binding was similar to that seen when CCK₈ was added alone. Two lines of evidence suggest that this effect of cholecystokinin on basal and insulin-stimulated ¹²⁵I-IGF II binding was mediated via a change in intracellular calcium: (1) the cholinergic agent carbachol inhibited IGF II binding to its receptors; (2) addition of the Ca²⁺ ionophore A23187 mimicked the effects of CCK₈ and carbachol. In contrast to its effects on IGF II binding to acini, CCK₈ had only small effects on IGF I binding and no effects on insulin binding.     

Phosphorylation of receptors for insulin and insulin-like growth factor I. Effects of hormones and phorbol esters

The phosphorylation of receptors for insulin and insulin-like growth factor I was studied by phosphoamino acid analysis and tryptic phosphopeptide maps in an attempt to determine if protein kinase C is involved in their phosphorylation in response to insulin and insulin-like growth factor I, respectively. Two cell lines were utilized, Hep G2 and IM-9 cells. sn-1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol 13-acetate (TPA), agents known to activate protein kinase C, stimulated the phosphorylation of the beta subunits of both receptors, as did their hormones. In unstimulated cells, phosphorylation of the insulin receptor occurred on seryl and to a lesser extent on threonyl residues. TPA stimulated seryl and threonyl phosphorylation that resulted in the appearance of four major phosphoserine-containing phosphopeptides which were not detected in the basal state and an increase in phosphorylation of a phosphothreonine-containing peptide which was present in the basal state. Insulin treatment resulted in the appearance of three major phosphotyrosine-containing tryptic peptides. In IM-9 cells, insulin also increased the phosphoserine and possibly the phosphothreonine content of the beta subunit. In both cells, the major phosphoserine-containing peptides that were stimulated by TPA were not detected following treatment with insulin. Very similar results, including similar peptide maps, were obtained for the insulin-like growth factor I receptor from cells treated with TPA and insulin-like growth factor I. Although not entirely conclusive, these results suggest that the insulin- and insulin-like growth factor I-stimulated phosphorylation of their receptors does not result from activation of protein kinase C.     

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.     

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.