Comparison of Insulin-like Growth Factor I Receptor and Insulin Receptor Purified from Human Placental Membranes*

Insulin-like growth factor (1GF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, s., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chern. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (@-a-ct-@), apparent sizes of deglycosylated a (Mr = -88,000) and @ (Mr = -67,000) subunits, and amino acid composition of the subunits. each kinase a@ of appears to the


subunits, and amino acid composition of the subunits.
Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric a@ form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their ct subunits, which apparently show 60% identity. The IGF-I receptor ct subunit lacks residues corresponding to the N-terminal4 amino acids of the insulin receptor ct subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.
The insulin-like growth factor (IGF)'-I, which elicits biological responses similar to insulin, binds to a specific receptor distinct from the insulin receptor (1). The IGF-I receptor has been studied by means of affinity cross-linking methods and/ or immunoprecipitation methods using a monoclonal antibody designated aIR-3, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis (2-5). These studies have suggested that the receptor is structurally quite similar to the insulin receptor. The IGF-I receptor is a disulfide-linked heterotetramer with M , = -300,000 composed of two sets of a subunits ( M , = -120,000) and /3 subunits ( M , = -90,000). IGF-I stimulates phosphorylation of the *This work was supported by Research Grants AM29770 and AM34427 (to Y. F.-Y.) and CA16417 (to J. R.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. I The abbreviations used are: IGF-I, insulin-like growth factor I; WGA, wheat germ agglutinin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. receptor 0 subunit in both intact and broken cells (5-7). Phosphorylation oc,curred a t tyrosine residues when the receptor partially purified by wheat germ agglutinin (WGA)-Sepharose chromatography and immunoprecipitation with aIR-3 was incubated with [Y-~'P]ATP, indicating that the tyrosine-specific kinase activity is probably intrinsic to the receptor (5).
The IGF-I receptor thus shares structural and functional homology with the well-characterized insulin receptor. We have now purified the IGF-I receptor from human placental membranes by sequential affinity chromatography on WGAand aIR-3-Sepharose (8). In this article, we report characterization of the purified IGF-I receptor in terms of binding and kinase activities, immunogenicity, subunit structure, amino acid composition, and N-terminal amino acid sequence in order to determine structural and functional homology to the insulin receptor.

EXPERIMENTAL PROCEDURES
Materials-IgG class of monoclonal antibodies aIR-1 and aIR-3, which are directed against the insulin receptor and the IGF-I receptor, respectively, were kindly supplied by Dr. Steven Jacobs, The Wellcome Research Laboratories (9). The IgG fraction of rabbit antiinsulin receptor antibody (39E) was prepared as described (10). IGF-I was purchased from Amgen. Crystalline porcine insulin was kindly provided by Eli Lilly Co. A synthetic peptide resembling the tyrosyl phosphorylation site of pp60"" (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly) was purchased from Peninsula Laboratories. N-Glycanase (peptide: N-glycosidase F) was obtained from Genzyme Co. NalZ5I and [y3'P]-ATP were from New England Nuclear. Molecular weight markers for SDS-PAGE were from Bio-Rad.
Purification of IGF-Z and Insulin Receptors-IGF-I receptor was purified from Triton X-100-solubilized human placental membranes by WGA-Sepharose chromatography followed by immunoaffinity chromatography using aIR-3, a monoclonal antibody directed against the IGF-I receptor as described previously (8,11). Briefly, an a-IR-3-Sepharose column was equilibrated with 50 mM Tris-HC1 buffer, pH 7.4. After the WGA-Sepharose eluate was applied, the column was extensively washed with the equilibration buffer and then eluted under acidic conditions (50 mM acetate buffer, pH 5, followed by 1 M glycine buffer, pH 2.2). All the buffers used contained 1 M NaC1, 0.1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM Na-benzoyl-L-arginine ethyl ester. Receptor preparations eluted at pH 5 or pH 2.2 (Fraction I or I1 as described in Ref. 8) were used for the present studies; Fraction I for binding and kinase assays and Fraction I1 for structural studies.
Insulin receptor was purified 2400-fold from Triton X-100-solubilized human placental membranes by sequential affinity chromatography on WGA-and insulin-Sepharose as described (11).
Amino Acid Analyses and N-terminal Sequence Analysis-IGF-I receptor preparations obtained by affinity chromatography on aIR-3-Sepharose columns were precipitated by the methanol/chloroform procedure of Wessel and Flugge (12). The precipitate was redissolved in 50 mM Tris-HC1, pH 6.8, containing 2% SDS and 20 mM DTT and heated at 60 "C for 30 min. This material was subjected to SDS-PAGE (13) in 7% polyacrylamide gels in the presence of 0.1 mM thioglycolate. After electrophoresis, a guide strip of the gel was stained with Coomassie Blue and the bands corresponding to M, = -120,000 and -90,000 were electroeluted by the procedure of Hunkapiller et al. (14).
The purified CY and subunits obtained by electroelution were precipitated by the methanol/chloroform procedure and redissolved in 0.1 ml of 70% formic acid. Aliquots were subjected to hydrolysis in constant boiling HCI at 110 "C for 24 h and the amino acid composition was determined on a Beckman 6300 Analyzer. The rest of the sample was used for determining the N-terminal sequence using the Applied Biosystems Protein Sequencer 470B equipped with PTH Analyzer 120A.
Iodination of the Recept~rs-Na~~~I (-3 mCi in 5 pl) was added to 100 p1 of 50 mM Tris-HC1 buffer, pH 7.4, containing 0.1% Triton X-100 and -2 pg of purified IGF-I (or insulin) receptor. Freshly prepared chloramine T (0.2 pg in 5 pl) was then added. After 5 min at 25 "C, the amount of 1251 incorporated into the receptor was determined by trichloroacetic acid-precipitation (final 5%) of an aliquot diluted in the Tris-HC1 buffer containing bovine serum albumin (5 mg/ml).
When 40% of the radioactivity was incorporated, the reaction was terminated by addition of Na-metabisulfite (0.5 pg in 5 pl). The iodinated receptor was isolated by gel filtration on a 2-ml column of Sephadex G-25 equilibrated with the Tris-HC1 buffer containing 0.1% Triton X-100 and mixed with the same volume of the buffer containing 0.1% bovine serum albumin. Other Methods-Insulin and IGF-I binding were measured by incubating the '251-labeled ligand and its purified receptor at 4 "C for 16 h followed by polyethylene glycol precipitation as previously described (8,ll). Phosphorylation of the 0 subunit and the src-related peptide was performed as described (15). SDS-PAGE was performed according to the method of Laemmli (13) and the fixed gels were stained with silver (16). Myosin, 0-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin were used as standards to estimate molecular weights.

RESULTS
Subunit Structure-The purified IGF-I receptor preparation was composed of three major disulfide-linked complexes whose molecular weights were estimated to be 320,000, 285,000, and 270,000 ( Fig. 1, lune 1). Corresponding insulin receptor species with M , = 320,000, 300,000, and 280,000 are also shown in Fig with increasing concentrations of DTT revealed patterns similar to those of the insulin receptor (Fig. 1); M , = -195,000 and M, = -150,000 forms of the IGF-I receptor appeared at concentrations of 0.5 mM DTT. Some variation in the sensitivity to DTT in terms of formation of these forms was noticed with different receptor preparations. With fresh preparations of the receptor the concentration of DTT required for conversion of higher molecular weight forms to these forms was significantly lower (0.05 mM, see Fig. 6C). These complexes were completely reduced to two major subunits, CY and 0, by treatment with 10 mM DTT (Fig. 1, lune 5). Two-dimensional SDS-PAGE gel electrophoresis of the IGF-I receptor in the absence and presence of DTT indicated a result similar to that obtained for the insulin receptor (17) although the detection of the P subunit was very difficult due to degradation and lower intensity of the silver stain (data not shown). These results suggest that an intact IGF-I receptor is composed of the CY and / 3 subunits which are linked in a P-a-a-P form by disulfide bridges. Based on the analogy with insulin receptor, the IGF-I receptor P subunit also appears to be susceptible to proteases, producing the P1-like subunit with M , = -50,000 (17) which has not been clearly detected by silver staining but is visible when labeled with 1251 (Fig. 3). The three disulfidelinked complexes shown in Fig. 1, lune 1, are, thus, most likely to be 432, a2PP1, and a2(Pl)2.
Molecular Size and Glycoprotein Nature of the Subunits-Although the CY and P subunits of the IGF-I receptor appear to be similar to the insulin receptor subunits, the IGF-I receptor a subunit was seen to be much broader on SDSpolyacrylamide gels (see Fig. 1, lunes 5 and 10). When the receptors were treated with N-glycanase, the CY and P subunits of both receptors moved faster than the corresponding native subunits on SDS-polyacrylamide gel under reducing conditions (Fig. 2). Both deglycosylated CY subunits moved the same distance although they were broad which may indicate incomplete digestion. Molecular weights of deglycosylated CY and P subunits of both receptors were estimated to be 88,000 and 67,000, respectively. Due to the poor staining of the P-subunit, it is difficult to detect the deglycosylated form of this subunit. These values agree with molecular weights of the protein portion of the insulin receptor subunits which have been calculated from the amino acid sequence deduced from its cDNA (18), indicating that the protein part of both receptors is similar in size. Immunoreactiuity-Purified IGF-I and insulin receptors were iodinated (Fig. 3, A15 and B15)' and examined for their reactivity with mouse monoclonal antibodies, CYIR-1 and aIR-3, as well as with rabbit anti-insulin receptor antibody 39E. As shown in Fig. 3, CYIR-1 (monoclonal antibody against the insulin receptor) precipitated the insulin receptor even at a 1/10,000 dilution, whereas it was much less effective in precipitating the IGF-I receptor (Fig. 3, lunes 7-9). aIR-3 (monoclonal antibody against the IGF-I receptor) reacted well with the IGF-I receptor even at 1/10,000 dilution but significantly with the insulin receptor only at a 1/100 dilution (Fig. 3, lunes  10-12). The IGF-I receptor cross-reacted with rabbit antiinsulin receptor antibody at a dilution of 1/100 (Fig. 3, lune  4), although its reactivity was less than that of the insulin receptor (Fig. 3, lunes 4 and 5).
Amino Acid Analysis of the IGF-I Receptor Subunits-The In this particular IGF-I receptor preparation, the 0 subunit was hardly seen, instead the p1 subunit was detected, which is most likely due to the degradation of the subunit. In both iodinated receptor preparations even after immunoprecipitation, a band with M, = -70,000 was seen. The nature of this band is not known although it seems that an excess amount of bovine serum albumin added to the receptor preparations may have caused it.  Table I together with the composition of electroeluted insulin receptor subunits (17). Both receptors seem quite similar in terms of amino acid composition, except for a somewhat higher leucine content observed in the IGF-I receptor p subunit.

N-terminal Amino Acid Sequence of the IGF-I Receptor a
Subunit-N-terminal amino acid sequence of the isolated a subunit was determined by advanced Edman degradation. From 25 cycles of degradation, 22 amino acids were identified. Traces of amino acid residues derived from the insulin receptor were also detected along with the IGF-I receptor sequence, which in fact provided internal controls.
The N-terminal amino acid sequence of the IGF-I receptor a subunit is compared with that of the insulin receptor a subunit in Fig. 4. The sequence is quite homologous to, and yet significantly different from that of the insulin receptor. The first 4 amino acids from the N terminus of the insulin receptor are missing from the IGF-I receptor a subunit; however, alignment of the IGF-I receptor starting from the fifth amino acid of the insulin receptor revealed 52% homology. If, as seems probable, the 3 residues (designated as X in Fig. 4) unidentified in the IGF-I receptor sequence are also identical to their counterparts in the insulin receptor a subunit, the homology increases to 64%.
Binding Actiuity of the Purified IGF-I Receptor-Competition of unlabeled IGF-I and insulin for binding of '2sI-IGF-I to purified IGF-I receptor is shown in Fig. 5A. Similar studies using purified insulin receptor are shown in Fig. 5B. The results clearly demonstrate preferential binding of IGF-I to the IGF-I receptor and of insulin to the insulin receptor. The difference in affinity between insulin and IGF-I appears in both cases to be at least lOO-fold, based on estimates of the concentration required to displace 50% of the bound radioactive ligand. These results provide evidence that ligand specificity of the purified IGF-I receptor is different from that of the insulin receptor, as previously reported for crude receptor preparations (19). The pH optimum for the binding activity was found to range from pH 7 to 8 as observed for the insulin receptor (data not shown).
Effect of DTT Treatment on Kinase Actiuity of the Purified IGF-I Receptor-The relationship between the structure of the IGF-I receptor and its kinase activity was studied using the purified receptor treated with different concentrations of DTT, since our previous studies on the purified insulin receptor treated with different concentrations of DTT suggested that the a@ form of the receptor exhibits much higher kinase activity than the intact receptor in the a2@2 form (15). Kinase activity as measured by phosphorylation of both the srcrelated peptide and the @ subunit was significantly enhanced after treatment with 0.01-0.5 mM DTT (Fig. 6, A , B, and D).  (17) are derived from actual analysis of electroeluted receptor subunits. The values are consistent with the amino acid compositions calculated from the cDNA data (18) except Gly which is overestimated in electroeluted proteins due to a trace of Tris-Gly buffer.

FIG. 4. N-terminal amino acid sequence of IGF-I receptor CY
subunit. The N-terminal amino acid sequence is aligned with that of the insulin receptor 01 subunit (18). X indicated unidentified residue.
Addition of IGF-I further enhanced the phosphorylation of the /I subunit (Fig. 6, A and B ) . Furthermore, when phosphorylation of the p subunit after treatment with different concentrations of DTT were simultaneously analyzed by 5% (-DTT) and 7.5% (+DTT) SDS-polyacrylamide gels, and compared to kinase activity of corresponding DTT-treated receptor preparations, a good correlation between the appearance of an aP form and an increase in phosphorylation of the /I subunit and the src-related peptide was observed (Fig. 6). These results suggest that the cup form of the IGF-I receptor is also more active than the intact receptor.

DISCUSSION
We have purified and characterized the human placental IGF-I receptor and compared it with the well-characterized insulin receptor. Subunit structure, sizes of the protein portion of a and p subunits, and amino acid composition of the subunits of the IGF-I receptor are found to be very similar t o those of the insulin receptor.
Although each receptor was recognized by a specific monoclonal antibody, a small degree of cross-reactivity of the insulin receptor with aIR-3 monoclonal antibody was observed, indicating that the insulin receptor might be copurified with the IGF-I receptor by aIR-3-Sepharose chromatography. Indeed, N-terminal sequence analyses revealed that the isolated IGF-I receptor a subunit contained approximately 10% of the insulin receptor a subunit. The extent of contamination of the insulin receptor in the affinity-purified IGF-I receptor preparations was previously estimated to be less than 5% by competition binding studies (8). The discrepancy between the two estimates suggests that aIR-3 antibody -A The most significant difference between the two receptors is seen in their primary structures. N-terminal amino acid sequence of the IGF-I receptor a subunit revealed 52-64% homology with, as well as obvious differences from, that of the insulin receptor a subunit. Although residues 3, 20, and 21 of the N-terminal sequence of the IGF-I receptor a subunit are not identified, residues 3 and 21 are almost certainly cyst(e)ines by homology with the insulin receptor, and residue 20 is probably an asparagine with carbohydrate attached, again by homology, since the serinelthreonine substitution at residue 22 would preserve the consensus sequence for carbohydrate attachment. The N-terminal amino acid sequence of the IGF-I receptor a subunit could be aligned with the sequence starting from the fifth amino acid of the insulin receptor a subunit. It is possible that the IGF-I receptor a subunit could have been specifically cleaved so that 4 amino acids from its N terminus are missing. Herrera et al. (21) recently reported structural analyses on the IGF-I receptor using antipeptide antibodies raised against two synthetic peptides corresponding to two different regions of the insulin receptor p subunit sequence. They predicted that the C terminus of the insulin receptor p subunit is not conserved in the IGF-I receptor but that the site close to the tyrosine kinase domain is homologous to that of the IGF-I receptor. Since ligand specificity is clearly different between the two receptors, more differences would be expected in the a subunit than in the p subunit. The difference in N-terminal amino acid sequence of the two a subunits shown in this report provides direct evidence that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.

IGF-I Receptor
Tyrosine-specific protein kinase activity has been found in  10 mM. D: the purified IGF-I receptor (0.6 pg) was treated with different concentrations of DTT and assayed for kinase activity using the src-related peptide in the absence (0) or presence (0) of IGF-I as described (15). Kinase activity is expressed as a ratio of control (i.e. the activity of the IGF-I receptor without DTT/IGF-I treatment). oncogene proteins as well as growth factor receptors (22). Insulin receptor kinase purified from human placental membranes has been characterized (20,23,24). We have shown in a previous report that tyrosine-specific kinase activity was co-purified with IGF-I binding activity from human placental membranes and that the purified IGF-I receptor exhibits kinase activity comparable to that of the purified insulin receptor (8). In this report, we have further characterized IGF-I receptor tyrosine-specific protein kinase and compared it with some of the properties of the insulin receptor kinase. Present studies suggest that an a/3 form of the IGF-I receptor exhibits higher tyrosine kinase activity than the intact a& form as was observed for the insulin receptor (15). IGF-I seems to stimulate the kinase activity of the ap form of the receptor. Whether a free ap form is the more activated kinase or not remains to be investigated by isolating the a@ form.
Previous studies have shown that the IGF-I receptor is very similar to the insulin receptor in terms of subunit structure, molecular size, and its functional properties such as tyrosinespecific protein kinase activity and its stimulation by the corresponding hormone (1)(2)(3)(4)(5)(6)(7)(8). Major evidence that the two receptors are distinct entities comes from specific binding to the ligand and immunochemical studies using monoclonal antibodies against the receptors (2-5). The results presented in this article provide the first direct proof that the IGF-I receptor is different from the insulin receptor. The N-terminal sequences of the two receptors, however, show sufficient homology to suggest that they are derived from a common ancestor.