A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

The metabolic and mitogenic effects of both insulin and insulin-like growth factor are enhanced by transfection of insulin receptors into NIH3T3 fibroblasts

Insulin and insulin-like growth factor (IGF-I) have 50% sequence homology and regulate similar cellular functions. Their membrane receptors also share 84% homology in a tyrosine kinase domain essential to transmembrane signaling and may thus share common postreceptor paths. To probe action mechanisms for these related hormones, we examined the receptor and postreceptor overlap of responses stimulated by insulin and IGF-I. NIH3T3 mouse fibroblasts have few endogenous insulin receptors and are insensitive to insulin; they have IGF-I receptors and are responsive to IGF-I. Stable transfection of these cells with cDNA for the human insulin receptor yielded a cell line (3T3/HIR) expressing greater than 6 x 10(6) receptors/cell that was highly sensitive and responsive to insulin for stimulation of deoxy[14C]glucose uptake and [3H]thymidine incorporation. The cells also showed increased responses to IGF-I, although the sensitivity was less than that for insulin. The receptor specificity of such responses was examined with a monoclonal antibody MA10 that bound to insulin receptors, but elicited no responses. When 3T3/HIR cells were preincubated with MA10, subsequent insulin- or IGF-I-stimulated deoxy[14C]glucose uptake was markedly inhibited. Likewise, the presence of MA10 caused a 10-fold increase in the concentration of insulin needed to stimulate half-maximal incorporation of [3H]thymidine and also led to diminished IGF-I-stimulated responses. These results showed that the transfected human insulin receptors coupled readily with existing effector pathways in the mouse fibroblasts and mediated metabolic and mitogenic responses to both insulin and IGF-I. Such findings indicate that insulin and IGF-I regulate common cellular functions using both overlapping receptor and postreceptor signaling pathways.     

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.     

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.     

Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.     

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

The structure and biological activities of two disulphide isomers of a C-region deletion mutant of insulin-like growth factor-I (IGF-I) which has an Asn--Gly link engineered at the junction of the A- and B-regions were studied before and after chemical cleavage. Circular dichroism (CD) spectra and binding affinity to IGF binding protein 3 (IGFBP3) indicated that the treatment with hydroxylamine did not disrupt the overall tertiary fold of the hormones. Cleavage restored some binding affinity for the IGF-I receptor in both isomers and weakly restored the ability to stimulate incorporation of tritiated thymidine into DNA in NIH 3T3 fibroblasts transfected with the human IGF-I receptor. Cleavage also restored metabolic capacity, as measured by the ability of the isomers to promote lipogenesis in isolated rat adipocytes through the insulin receptor. These results are consistent with the theory that binding of IGF-I to the IGF-I receptor requires a conformational change similar to that involved in insulin binding the insulin receptor. The weak affinity for the IGF-I receptor after cleavage is consistent with the belief that residues in the C-region interact with the IGF-I receptor. This structural difference between insulin and IGF-I gives each a higher binding affinity for its own receptor.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells.

Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

Identification of a monoclonal antibody which can distinguish between two distinct species of the type I receptor for insulin-like growth factor

A monoclonal antibody was identified which equally inhibits 125I-labeled insulin and insulin-like growth factor I (IGF-I) binding to their respective receptors in human IM-9 lymphoid cells and solubilized placenta receptor preparations. In contrast, this monoclonal antibody inhibits insulin but not IGF-I binding to human hepatoma (HepG2) cells, fibroblasts and muscle cells. These results indicate that there are two distinct species of the type I insulin-like growth factor receptor (which we have named type IA and type IB) and suggest that this monoclonal antibody may be useful in determining whether different biological effects are mediated through these two receptors.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.     

Insulin receptor is phosphorylated in response to treatment of HepG2 cells with insulin-like growth factor I.

1. Binding of insulin and insulin-like growth factor I (IGF-I) to HepG2 cells was analysed with regard to competition by both insulin and IGF-I. At concentrations of insulin that caused maximal phosphorylation of the insulin receptor, virtually no displacement of IGF-I binding was observed. Similarly, at concentrations of IGF-I that caused maximal phosphorylation of the IGF-I receptor, no displacement of insulin binding was observed. 2. When the phosphorylation of both receptors was examined individually by using specific monoclonal antibodies to immunoprecipitate the receptors, phosphorylation of the insulin receptor was found to increase on both serine and tyrosine residues in cells treated with 100 ng of IGF-I/ml. In contrast, no increased phosphorylation of IGF-I receptor was observed in cells treated with 100 ng of insulin/ml. 3. The increase in phosphorylation of insulin receptor in response to IGF-I correlated with the dose-response of IGF-I-stimulated phosphorylation of the IGF-I receptor. 4. The IGF-I-stimulated phosphorylation of the insulin receptor could be blocked by preincubation with a monoclonal antibody that blocks IGF-I binding to the IGF-I receptor.     

Identification of Serotonin Receptors Recognized by Anti-Idiotypic Antibodies

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.     

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.     

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hen's eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.     

Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1.

The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, except that bovine IGF-1 showed a severalfold greater affinity for the monoclonal antibody than for either polyclonal antiserum. Membranes isolated from human placenta, sheep placenta and foetal-human liver were used as sources of cellular receptors. In human placental membranes, most of the binding of IGF-1 tracers could be attributed to a type-1 receptor, because insulin inhibited up to 65% of tracer binding. The other two tissues apparently contain only type-2 receptors, as evidenced by the very low potency of bovine or human IGF-1 in competing for binding with IGF-2 tracers and the absence of any competition by insulin. In competition for binding with labelled bovine or human IGF-1 to human placental membranes, bovine IGF-1 had a similar potency to human IGF-1, whereas bovine IGF-1 was more potent in binding studies with tissues rich in type-2 receptors. Rat IGF-2 was considerably less effective than human IGF-2 in competition for receptors on any of the membrane preparations.