In 1989, Shier and Watt (1) identified a gene whose sequence predicted that it encoded a receptor (called the insulin receptor-related receptor or IRR) ()that was homologous to the insulin and insulin-like growth factor (IGF)-I receptors. Subsequent studies by polymerase chain reaction, RNase protection, and Northern analysis have demonstrated low amounts of mRNA for IRR in a variety of tissues including kidney, stomach, and thymus (2, 3, 4, 5, 6, 7) . In situ hybridization studies revealed that the IRR mRNA was most abundantly expressed in rats in sympathetic and sensory neurons of the trigeminal and dorsal root ganglia and in renal distal tubule cells(8) . The expression of the IRR mRNA in these tissues appeared to closely correlate with the presence of the mRNA for the nerve growth factor (NGF) receptor, called TRK(9) . However, the endogenous full-length IRR protein has not yet been demonstrated in any tissue or cell line. Characterization of the protein encoded by the IRR gene has been performed by utilizing recombinant DNA techniques to express either chimeric receptors containing portions of the IRR molecule in the backbone of the insulin receptor (4) or, more recently, by expression of a full-length cDNA encoding the complete IRR protein (6) . These studies have demonstrated that IRR does not bind insulin, IGF-I, IGF-II, proinsulin, relaxin, or several insulin-related molecules from lower organisms including bombyxin and mollusk insulin-related molecule. In addition, they have demonstrated that IRR, like the insulin and IGF-I receptors, has an intrinsic tyrosine kinase activity that appears to phosphorylate endogenous proteins with a specificity very similar to that of the other two receptors in this family(4, 5) . For example, a chimeric receptor with the entire cytoplasmic domain of IRR and the extracellular domain of the insulin receptor exhibited an insulin-stimulated kinase activity, which resulted in the tyrosine phosphorylation of insulin receptor substrate-I and the GAP-associated p60 and Shc, three substrates of the insulin receptor kinase(4, 5) .

To enable us to study the endogenous IRR, we have now produced several monoclonal antibodies to the extracellular domain of the human IRR by injection of whole cells expressing the chimeric receptor. In this paper, we report the characteristics of these antibodies and utilize these antibodies to demonstrate for the first time the expression and properties of the endogenous IRR protein.

EXPERIMENTAL PROCEDURES

Materials

Production of the Monoclonal Anti-IRR Antibodies

Epitope Mapping of the Monoclonal Anti-IRR Antibodies

To further define the epitope recognized by each antibody, we utilized various chimeric receptors that contained different portions of IRR in the backbone of the insulin receptor. COS-7 cells were transiently transfected by the calcium phosphate precipitation method with 10 µg of either the vector cDNA or cDNAs encoding native insulin receptor, the chimeric receptors IRR/IRK, IRRexon2,3/IR, or IRRexon3/IR(4, 10, 11) . After 48 h, the cells were lysed, and the lysates were added to microtiter wells previously coated with one of the four monoclonal anti-IRR antibodies. After an overnight incubation, the amount of immunocaptured receptor was quantified using I-labeled monoclonal antibody 29B4. Binding of receptor to the wells coated with 17A3 (14) was used to measure the level of expression of the various chimeras in COS-7 cells.

Activation of the Intrinsic Kinase Activity of IRR/IRK

Down-regulation of IRR/IRK by the Antibodies

Detection of IRR in Neuroblastomas and IMR-5 Cells

The IMR-5 human neuroblastoma cells were maintained in RPMI 1640 medium with 10% fetal calf serum. For analyses of IRR, the cells in a confluent 100-mm Petri dish were lysed as described above for the CHO-IRR/IRK cells, and the lysates were immunoprecipitated with 10 µg of the pool of monoclonal anti-IRR antibodies, normal immunoglobulin, monoclonal antibodies to TRK, the IGF-I receptor (IR3), or the insulin receptor (29B4). The precipitates were analyzed by SDS-PAGE and immunoblotting with the polyclonal antibody to the IRR subunit. To measure the activation of IRR kinase in these cells, the cells were incubated with serum-free medium for 1 h at 37 °C and then buffer, 100 nM insulin, 13 nM IGF-I, 2 nM NGF, 100 nM normal mouse IgG, or 100 nM monoclonal antibody 3B1 was added. After a 10-min incubation of cells with insulin, IGF-I, or NGF or a 30-min incubation with antibody at 37 °C, the cells were lysed, and the lysates were immunoprecipitated with either control Ig or the pool of monoclonal antibodies to IRR (times were chosen to be optimal for the ligand used). After an overnight incubation at 4 °C, the immunoprecipitates were washed and assayed for kinase activity as described above except that no preactivation step was utilized, and the exogenous substrate utilized was 1 mM of a synthetic peptide (KKHTDDGYMPMSPGVA) from the sequence of IRS-1, which had been previously shown to be a good substrate of the insulin receptor kinase(16) . To measure the radioactivity incorporated into this peptide, the reaction mixtures were spotted on P81 phosphocellulose paper strips; the strips were washed twice with 75 mM phosphoric acid (10 ml/strip) and counted.

MAP Kinase Assays

RESULTS

To generate monoclonal antibodies to IRR, mice were injected with CHO cells that were genetically engineered to overexpress a chimeric receptor containing most of the extracellular domain of human IRR (residues 7-892) in the backbone of the human insulin receptor (called IRR/IRK). The spleen cells of one mouse were fused to the mouse myeloma cell line SP2/0, and the resulting hybridomas were screened for the production of monoclonal antibodies capable of capturing the chimeric IRR/IRK in a microtiter plate assay. Of the 240 hybridomas screened, 16 were initially identified as positive in this assay; 4 (called 3B1, 10B4, 10B5, and FA4) were successfully cloned and grown up, and their antibodies were characterized. Three of these (3B1, 10B4, and 10B5) captured comparable amounts of receptor as a previously described monoclonal antibody to the cytoplasmic domain of the insulin receptor (17A3) (14) while one (FA4) bound considerably less receptor but still more than the control Ig (Fig. 1). The ability of each of these monoclonal antibodies to precipitate the IRR/IRK chimera from lysates of CHO cells overexpressing this receptor was confirmed in a standard precipitation assay ( Fig. 2and data not shown). A pool of all four monoclonal antibodies was found to be most effective in precipitating the IRR chimera (data not shown). In contrast, this pool of antibodies to IRR did not precipitate either the human insulin receptor or the receptor for insulin-like growth factor-I from CHO cells genetically engineered to overexpress these proteins (Fig. 2)(18, 19) . To determine whether these four antibodies were recognizing the same epitope on IRR, each antibody was tested for its ability to capture receptor that had first been incubated with an excess of each of the other three antibodies. Two of the antibodies (3B1 and 10B5) were found to compete to a large extent with each other's binding to the receptor (data not shown), indicating that they were directed to nearby or identical epitopes. Further characterization of the epitopes recognized by these antibodies was performed by examining their ability to recognize chimeric receptors containing different portions of the extracellular domain of IRR. All four of the antibodies recognized a chimeric receptor containing only residues encoded by exons 2 and 3 of the IRR gene about as well as the chimeric receptor IRR/IRK, indicating that all four of these antibodies recognize an epitope in residues 7-288 (Fig. 3). All four of the antibodies also recognized a chimeric receptor containing only residues encoded by exon 3 of the IRR gene, although this chimera was only recognized approximately 10-40% as well as chimera IRR/IRK (Fig. 3), possibly indicating that these antibodies recognize an epitope that is partially contained within residues 188-288.

Figure 1: The ability of the monoclonal antibodies to immunocapture the chimeric IRR/IRK in the plate assay. Microtiter plates coated with 10 µg/ml of the indicated monoclonal antibodies to IRR (3B1, 10B4, 10B5, FA4), the cytoplasmic domain of the insulin receptor (17A3), or control mouse Ig (NIg) were incubated with lysates of CHO cells overexpressing the chimera IRR/IRK and washed; the presence of bound chimera was detected by the use of an iodinated monoclonal antibody to a distinct epitope of the cytoplasmic domain of the insulin receptor. Results shown are means of three experiments each done in duplicate. Pptn. Ab, precipitating antibody.

Figure 2: Specificity of the monoclonal anti-IRR antibodies. CHO cells overexpressing the chimeric IRR/IRK, the human insulin receptor (IR), or the IGF-I receptor (IGFR) were lysed, and the lysates were incubated with protein G-Sepharose beads coated with control mouse immunoglobulin (N), a monoclonal antibody to the insulin receptor (29B4), a monoclonal antibody to IRR (10B4), a monoclonal antibody to the IGF-I receptor (IR3), or a pool of the four monoclonal antibodies to IRR (P). The beads were washed, and the bound proteins were eluted and analyzed by SDS-gel electrophoresis and Western blotting with either an antibody to the subunit of the insulin receptor (IR) or the IGF-I receptor (IGFR). The positions of the chimeric subunit (chim ), insulin receptor subunit, and IGF-I receptor subunit (IGFR) are indicated. Molecular masses (in kDa) of marker proteins are also indicated. Pptn. Ab, precipitating antibody.

Figure 3: Mapping of the epitopes recognized by the monoclonal anti-IRR antibodies. CHO cells expressing the chimeric IRR/IRK, IRRexon2,3/IR, or IRRexon3/IR were lysed, and the lysates were precipitated with either the monoclonal antibodies indicated or a monoclonal antibody to the cytoplasmic domain of the insulin receptor (17A3). Results shown for IRRexon2,3 (filledbar) and IRRexon3 (openbar) have been normalized to the amount of IRR/IRK chimera receptor precipitated with each antibody. The expression of the three different chimeras was found to be comparable by the use of the 17A3 antibody (for IRR/IRK, IRRexon2, 3, and IRRexon3/IR, the counts bound were 6,170, 5,670, and 6,070, respectively). Pptn. Ab, precipitating antibody.

To test whether these monoclonal antibodies could potentially serve as agonists for IRR, they were each tested for their ability to activate the tyrosine kinase activity of the chimera IRR/IRK. Intact CHO cells overexpressing this receptor were incubated with various concentrations of these antibodies, the cells were lysed, and the receptor was immunocaptured on microtiter wells and tested for tyrosine kinase activity. Two of the antibodies (3B1 and 10B5) were found to activate the enzymatic activity IRR/IRK of the chimeric receptors approximately 2-2.5-fold (Fig. 4). A further 2-fold increase in this activity was observed with these antibodies if the cells were incubated with the monoclonal antibody in the presence of an anti-mouse Ig (data not shown), presumably due to a further aggregation of the receptor. No activation of tyrosine kinase activity was observed in cells overexpressing the native human insulin receptor (data not shown), further confirming the specificity of these antibodies.

Figure 4: Activation of the IRR/IRK intrinsic tyrosine kinase activity by the monoclonal anti-IRR antibodies. CHO cells overexpressing IRR/IRK were incubated with the indicated concentrations of monoclonal anti-IRR antibodies or control mouse immunoglobulin (NIg) for 30 min and lysed; the IRR/IRK was precipitated from the lysates and tested for its ability to phosphorylate poly(Glu:Tyr). Results shown are the means of a representative experiment performed in duplicate.

Another potential property of monoclonal anti-receptor antibodies is their ability to down-regulate the receptor(20) . To test these antibodies for this property, CHO cells overexpressing IRR/IRK were incubated with different concentrations of each of the antibodies for 18 h at 37 °C, the cells were lysed, and the receptor was immunoprecipitated and quantitated by Western blotting. Monoclonal antibodies 10B5 and 3B1 were both found to cause a 45-55% decrease in the levels of the receptor in these cells (Fig. 5).

Figure 5: Down-regulation of IRR/IRK by the monoclonal antibodies to IRR. CHO-IRR/IRK cells were incubated with the indicated concentrations of monoclonal anti-IRR antibodies for 18 h and lysed; the IRR/IRK was precipitated from the lysates and quantitated by Western blotting with an anti-receptor antibody and iodinated protein A. Results are expressed as the percent of IRR/IRK remaining with no antibody addition.

These monoclonal antibodies were then used to examine various cell lines and tissues for the presence of the endogenous IRR. Lysates were immunoprecipitated with the pool of the monoclonal antibodies to IRR, and the precipitates were tested for the presence of IRR by either measuring tyrosine kinase activity (after activation by a preincubation with ATP) or by immunoblotting the precipitates with a polyclonal antibody to the cytoplasmic domain of IRR. Materials tested included the human neuroblastoma cell lines SY5Y, SK-N-SH, and IMR-5, kidney cell lines including pig renal carcinoma line AE 6010, African green monkey epithelial line BSC-1, human Wilms' tumor G401 line, Simian monkey kidney cells COS, primary human and rat kidney samples, and primary human neuroblastoma tissues. A significant level of IRR was detected only in the IMR-5 neuroblastoma cells and several of the primary human neuroblastomas. The IRR in these samples could be detected either by kinase activity (Fig. 6) or by immunoblotting (Fig. 7). The levels of IRR found by these two methods were in good agreement for the different neuroblastoma samples and were, in general, highest in samples from stage A neuroblastomas ( Fig. 6and Fig. 7A). By immunoblotting, the subunit of the endogenous IRR from both primary neuroblastomas and the IMR-5 cells migrated on SDS gels slightly below the albumin marker protein (a position consistent with a M of 66,000) (Fig. 7). The immunoblots from both the tumor tissues and the IMR-5 cells also exhibited a specific band of approximate M of 160,000 (Fig. 7), possibly the precursor of IRR.

Figure 6: Detection of the presence of endogenous IRR in neuroblastomas by kinase activity. Samples of tumors from individual patients were lysed, and the lysates were precipitated with either control Ig or a pool of the monoclonal antibodies to IRR. The precipitates were tested for tyrosine kinase activity after preactivation with 1 mM ATP. Results shown are from two different experiments utilizing samples from 9 (shown in panelA) or 12 (panelB) distinct tumors. Samples were either from stage A (filledboxes) or stage D (openboxes) tumors.

Figure 7: Detection of the endogenous IRR by immunoblotting. A, detection of IRR in the neuroblastomas. The immunoprecipitates (utilizing either control Ig (N) or the pool of anti-IRR antibodies (P)) of the tumor samples from patients 6-9 shown in panelA of Fig. 6were analyzed by electrophoresis and immunoblotting with a polyclonal antibody to the subunit of IRR. No detectable IRR band was observed in the precipitates of the samples from patients 1-5. B, detection of IRR in the IMR-5 cells. Lysates of IMR-5 cells were precipitated with either a monoclonal antibody to the IGF-I receptor (IGFR), the NGF receptor (TRK), the insulin receptor (IR), or control immunoglobulin (NIg) or the pool of monoclonal antibodies to IRR (IRR). The precipitates were analyzed by Western blotting with a polyclonal antibody to the subunit of IRR. The positions of the subunit of IRR (IRR) and the putative precursor protein (IRRp) are indicated. Pptn. Ab, precipitating antibody.

To test whether the endogenous IRR expressed in the IMR-5 cells was also present as a hybrid receptor with the endogenous insulin and IGF-I receptors present in these cells, the lysates from these cells were also immunoprecipitated with antibodies specific to these receptors. The IRR subunit was detected to a small degree in these precipitates but not in control Ig precipitates (Fig. 7B). The reverse experiment was also performed; precipitates of IMR-5 cell lysates with antibodies to IRR were also found to contain low levels of insulin and IGF-I receptors (data not shown). However, the IRR subunit band could not be detected in precipitates with antibodies to the NGF receptor, TRK (Fig. 7), although these cells do contain a high level of this receptor. TRK and IRR were not found to associate even when the cells were first treated with NGF. Attempts to detect TRK by Western blotting in anti-IRR immunoprecipitates were also negative.

To test whether the endogenous IRR protein could be activated in the IMR-5 cells, the cells were treated with the agonist monoclonal antibody 3B1 (in the presence or absence of anti-mouse Ig), insulin, IGF-I, or NGF. The cells were then lysed, and the lysates were immunoprecipitated with the pool of anti-IRR antibodies; these precipitates were tested for kinase activity. The monoclonal antibody 3B1 in the presence of anti-mouse Ig stimulated the kinase activity of IRR to the greatest degree, approximately 3-fold (Fig. 8). No significant stimulation of the kinase activity of IRR was observed with insulin, IGF-I, or NGF (Fig. 8), although precipitates of the respective receptors for each of these ligands demonstrated that the kinase activity of their own receptors were activated by these treatments (data not shown). To test whether the increase in IRR kinase activity had any functional consequences, the monoclonal antibody 3B1 was tested for its ability to stimulate MAP kinase activity in the IMR-5 cells. Treatment of these cells with either 3B1 alone or 3B1 in the presence of rabbit anti-mouse Ig was found to cause a 2- or 3-fold increase, respectively, in MAP kinase activity in these cells (Fig. 9). The stimulation observed with 3B1 in the presence of the anti-mouse Ig antibodies was comparable with that observed with 2 nM NGF (data not shown) and greater than that observed with 1 µM insulin (Fig. 9).

Figure 8: Activation of the tyrosine kinase activity of the endogenous IRR in IMR-5 cells. Intact IMR-5 cells were incubated 10 min with either 2 nM NGF, 100 nM insulin, 13 nM IGF-I, or 100 nM normal mouse Ig (NIg) or 100 nM monoclonal anti-IRR 3B1 in the presence or absence of anti-mouse Ig (anti-mIg) (2 µg/ml). The IRR was immunoprecipitated with the pool of monoclonal anti-IRR antibodies and tested for its ability to phosphorylate the IRS-1 peptide. Results shown are the means of three experiments ± S.E., each normalized to control cells incubated with either buffer or normal mouse Ig.

Figure 9: Activation of MAP kinase in IMR-5 cells is mediated via the IRR. Intact IMR-5 cells were treated with either buffer (BUFF), 1 µM insulin (INS), 100 nM normal mouse Ig (NIg), 100 nM 3B1 in the presence or absence of rabbit anti-mouse Ig (RM). The cells were lysed, and MAP kinase was specifically immunoprecipitated and tested for its ability to phosphorylate myelin basic protein.

DISCUSSION

In the present studies, we describe the generation of four monoclonal antibodies to the extracellular domain of the human IRR. These antibodies were generated by immunizing mice with intact CHO cells that were genetically engineered to overexpress the human IRR extracellular domain (residues 7-892) in the backbone of the human insulin receptor(4) . The advantages of this technique are that the receptor protein does not have to be first purified before immunization, and the chances of generating an antibody to the non-denatured form of the extracellular domain are greatly increased, thereby increasing the likelihood of obtaining monoclonal antibodies that are either agonists or antagonists. The ease in generating monoclonal antibodies to the human insulin and IGF-I receptors by this whole cell approach has been previously demonstrated(21, 22) . One disadvantage of this technique is that the antibodies generated are unlikely to recognize the denatured protein; indeed, none of these four monoclonal antibodies recognize the receptor on Western blots. However, all four of these antibodies appear to specifically precipitate the nondenatured IRR and not recognize the related insulin and IGF-I receptors (Fig. 2). One explanation for these findings may be that these four antibodies appear to recognize an epitope in residues 7-288 of IRR (Fig. 3), a region that is poorly conserved between these receptors(1) . The finding that these antibodies only partly recognize the IRR chimera containing only residues 188-288 (Fig. 3) could indicate that the epitope they bind is partly formed from these amino acids and partly from the amino-terminal residues 7-188. Alternatively, residues 188-288 of IRR may not be able to form their native conformation in the absence of IRR residues 7-188.

The finding that two of these monoclonal antibodies are capable of activating the intrinsic tyrosine kinase activity of IRR/IRK (and the native IRR) ( Fig. 4and Fig. 8) as well as stimulating the down-regulation of this receptor (Fig. 5) may be useful for future studies. At the present time, no ligand has been identified that stimulates the kinase activity of IRR. The availability of a monoclonal antibody with this property allows one to test the function of this receptor in stimulating various biological responses (for example, see below). Similarly, the availability of a monoclonal antibody that can down-regulate this receptor may also be useful in testing the role of this receptor and its ligand in various physiological processes.

In the present studies, these monoclonal antibodies have been used to identify the full-length endogenous IRR for the first time (Fig. 7). The only samples examined that had a detectable level of this receptor were human neuroblastoma tissues from various patients and the human neuroblastoma cell line IMR-5. These findings are consistent with the detection of the highest levels of IRR mRNA by in situ hybridization in various neuronal cells(8, 9) . Our inability to detect the IRR protein in various rat and human kidney samples (a source for the IRR mRNA)(2, 3, 4, 5, 6, 7) and cell lines could be due to a lower level of expression of the IRR protein in these materials or possibly due to some other technical problems (the degradation of the receptor in these samples, lack of cross-reactivity with the rodent receptor, etc.). The finding that the levels of IRR were in general higher in the patients samples from stage A tumors (Fig. 6) is consistent with prior studies indicating that such tumors in general have higher levels of the NGF receptor (23) since these two receptors appear to be coexpressed in various tissues and in development(9) .

The migration of the subunit of the endogenous receptor IRR (M of approximately 66,000) in both this cell line and the tumor samples (Fig. 7) was considerably smaller than that of both the human insulin and IGF-I receptor subunits (Fig. 2) but consistent with results recently reported for a recombinantly expressed intact IRR subunit(6) . The finding of a fairly high proportion of an uncleaved precursor form of this receptor in both samples was unexpected and could possibly be due to the nature of these samples or the presence of a sequence in IRR slightly different from the insulin and IGF-I receptors at the cleavage position (IRR contains an Arg-His-Arg-Arg at the cleavage site whereas the insulin and IGF-I receptors contain Arg-Lys-Arg-Arg)(1) .

The finding of the endogenous IRR in the IMR-5 cells also allowed us to further examine the properties of this receptor. Since these cells also contain receptors for insulin, IGF-I, and NGF, we tested whether some of the endogenous IRR was present as a hybrid molecule with these other receptors. Prior studies have extensively documented that the insulin and IGF-I receptors form hybrids in multiple tissues and cells(24, 25) . In the present studies, we were able to demonstrate that a portion of the endogenous IRR is present as a hybrid with the endogenous insulin and IGF-I receptors since the IRR could be partially immunoprecipitated with monoclonal antibodies specific to these two other receptors (Fig. 7) and since the monoclonal antibodies to IRR could precipitate a portion of the other two receptors (data not shown). However, no IRR was detected in anti-TRK precipitates whether or not the cells were treated with NGF. These results indicate that IRR, although coexpressed with TRK in many tissues(9) , is unlikely to form a stable complex with this receptor.

A further examination of the functional properties of the IRR in the IMR-5 cells was performed by testing the activation of the intrinsic tyrosine kinase activity of this receptor. The monoclonal antibody 3B1 was found to stimulate the tyrosine kinase activity of the endogenous IRR, and this affect was potentiated by the addition of anti-mouse Ig (Fig. 8). In contrast, insulin, IGF-I, and nerve growth factor did not stimulate the tyrosine kinase activity of IRR. These studies further support the hypothesis that the IRR does not bind these ligands. In addition, they indicate that the endogenous IRR cannot be greatly activated by cross-phosphorylation by these other receptors, supporting the hypothesis that a distinct ligand must exist to normally activate this receptor. Finally, the monoclonal antibody 3B1 (primarily in the presence of the anti-mouse Ig) was capable of stimulating the activation of MAP kinase in the IMR-5 cells (Fig. 9). This is the first biological effect demonstrated to be mediated by the native IRR. Prior studies in other neuronal cell lines have demonstrated that activation of MAP kinase can be linked to either a proliferative response or the stimulation of differentiation(26, 27) . Additional studies of other neuronal cell lines will be required to determine which of these effects are mediated via IRR since the IMR-5 cells do not appear capable of a differentiative response, even to NGF (data not shown).

FOOTNOTES

*

This work was supported by National Institutes of Health Grant DK 45652. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom all correspondence should be addressed. Tel.: 415-723-5933; Fax: 415-725-2952.

()

The abbreviations used are: IRR, insulin receptor-related receptor; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; NGF, nerve growth factor; IGF, insulin-like growth factor; MAP kinase, mitogen-activated protein kinase.

ACKNOWLEDGEMENTS

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