Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor.

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA (1)(2)(3). The two mature receptor proteins differ by the presence or absence of 12 amino acids at the C terminus of the extracellular ␣ subunit; this insertion is encoded by the 36-nucleotide exon 11 of the receptor gene (4). The two isoforms have been reported to have different tissue distributions (3,5) and to exhibit different functional properties (6 -8).
The B isoform with the 12-amino acid insertion is the predominant form expressed in the classic insulin target tissues responsible for glucose homeostasis, i.e. fat, muscle, and liver, whereas the A isoform without the insertion predominates in non-classic target tissues, e.g. the pancreatic ␤ cell and neural tissue (3,5). The B isoform has been reported to signal more efficiently in response to insulin binding despite having a 2-fold lower affinity for insulin (6 -9). A switch in isoform expression from A to B in hepatoma cells mediated by dexamethasone treatment leads to decreased insulin sensitivity (8). In addition, the aberrant regulation of alternate splicing observed in myotonic dystrophy leads to overexpression of the A isoform of the receptor in insulin target tissues and results in insulin resistance (10). Divergent isoform-dependent signaling mechanisms have been demonstrated in the pancreatic ␤ cell, with differential activation of different phosphatidylinositol3 kinase and protein isoforms in response to insulin (11).
The ligand binding properties of the two isoforms also differ significantly. The A isoform has a 2-fold higher affinity for insulin than the B isoform. In addition, the kinetics of insulin binding to the two isoforms differ (6,7). The A isoform has a higher affinity for insulin-like growth factor (IGF) 1 -I and -II than the B isoform (7,12). Indeed, it has been proposed that it might be a physiological receptor mediating the growth-promoting effects of IGF-II (13). Taken together, these findings suggest that the structure of the ligand binding sites and the ligand receptor interface may differ.
Recently, a high resolution crystal structure of an Nterminal fragment of the homologous insulin-like growth factor I receptor (amino acids 1-460) has been reported (14). The molecule is composed of an extended bilobed structure composed of the two globular L domains flanking the cysteinerich domain with dimensions of 40 ϫ 48 ϫ 105 Å. The Nterminal globular domain contacts the cysteine-rich domain along its length. In contrast, there is minimal contact between the C-terminal domain and the cysteine-rich domain. In the crystal structure, L1 and L2 occupy very different positions relative to the cysteine-rich domain. However, this could be an artifact of crystal packing in this fragment, and the position of L2 may be very different in the native molecule. It is possible that it is rotated into a position similar to that of L1 in relation to the cysteine-rich domain. However, irrespective of this, a cavity of ϳ30 Å diameter occupies the center of the molecule and possibly represents a binding pocket.
Each L domain resembles a loaf of bread with dimensions 24 ϫ 32 ϫ 37 Å and is formed from a single right-handed ␤ helix capped at the ends by short ␣ helices and disulfide bonds. The base of the loaf is formed from a six-stranded ␤-sheet five residues in length. Both sides are formed from ␤-sheets 3 amino acids in length, and the top is composed of irregular loops connecting the short ␤-strands. As predicted from sequence comparisons, the cysteine-rich domain is composed of repetitive modules resembling parts of laminin and the tumor necrosis factor receptor (15). These form a rod-like structure connecting the two globular L domains from which a large mobile loop projects into the putative binding pocket.
Although this fragment and the corresponding one from the insulin receptor are devoid of ligand binding activity (14,16), mutagenic studies indicate they contain amino acids whose side chains are involved in ligand contacts (17,18). Affinitylabeling studies and alanine-scanning mutagenesis have also demonstrated that a C-terminal peptide of the ␣ subunit, amino acids 705-715, is involved in insulin binding (19,20). Further, fusion of this C-terminal fragment to the N-terminal 470 amino acids results in a recombinant protein that binds insulin with an affinity similar to that of the full-length secreted recombinant extracellular domain (21)(22)(23), suggesting that these elements are sufficient to form an intact ligand binding site. Recently Kristensen et al. (24) have reported that an insulin binding site can be reconstituted from a fragment of the N terminus of the receptor encompassing amino acids 1-255 and the C-terminal peptide of the ␣ subunit (amino acids 1-255), indicating that these elements are its minimal determinants.
In the present study, to further evaluate the molecular mechanisms responsible for the reported differences in insulin binding properties, we have compared their functional epitopes. We have used structure-directed alanine-scanning mutagenesis of the L1 domain and alanine-scanning mutagenesis of amino acids 705-715 for this purpose. The results of these studies reveal that previously unidentified amino acids in L1 are determinants of ligand binding of both isoforms. Mutation of Asp 707 and Val 713 result in compromises in affinity that are restricted to the A isoform. Mutations of amino acids Asn 15 , Phe 64 , Tyr 708 , Leu 709 , and Phe 714 have effects on receptor affinity that are isoform-specific.

EXPERIMENTAL PROCEDURES
General Procedures-All molecular biological procedures, including agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation, and DNA sequencing were performed by standard methods (25). All oligonucleotides were purchased from DNA Technology (Aarhus, Denmark). Restriction and modifying enzymes were from New England Biolabs (Beverly, MA). Recombinant human insulin and HPLC-purified monoiodinated [ 125 I-Tyr A14 ]insulin were from Novo Nordisk A/S. Protease inhibitors were from Roche Molecular Biochemicals. Medium and serum for tissue culture were from Invitrogen. PEAK Rapid cells (293 cells constitutively expressing SV40 large T antigen) were purchased from Edge Biosystems (Gaithersburg, MD). The mammalian expression vector pcDNA3-zeo(ϩ) was from Invitrogen. The hybridoma secreting monoclonal antibody 18 -44 directed toward the insulin receptor ␣ subunit (26) was a generous gift from Drs. M. Soos and K. Siddle (University of Cambridge, UK). Protein A-purified IgG (27) from the hybridoma medium was kindly provided by Dr. P. Jorgensen (Novo Nordisk A/S, Bagsvaerd, Denmark). cDNAs encoding the B isoform of a C-terminal FLAG-tagged recombinant secreted extracellular domain of the insulin receptor (28) were generously provided by Drs. C. Kristensen and J. Brandt, (Novo Nordisk A/S, Bagsvaerd, Denmark), and a cDNA encoding the A isoform of the insulin receptor was provided by Dr. Donald Steiner (University of Chicago).
Oligonucleotide-directed Mutagenesis-Oligonucleotide-directed mutagenesis was performed by a modification of the method of Kunkel (29,30) using uracil containing single-stranded DNA prepared from phage rescued from Escherichia coli CJ236 transformed with a cDNA encoding the full-length insulin receptor cloned into the phagemid pTZ18U as template. Restriction sites were deleted or introduced with the specific mutation to facilitate screening of the mutants. Successful mutagenesis was confirmed by DNA sequencing.
Expression of Mutant Receptor cDNAs-Recombinant mutant secreted insulin receptor cDNAs were reconstructed in the plasmid pcDNA3-zeo (ϩ) for expression. DNA for transfection was prepared from 10 ml of overnight cultures by a boiling decyltrimethyl-ammonium bromide (CTAB) method (31) followed by purification using QIAwell strips. The mutant receptor cDNAs were expressed transiently in Peak Rapid cells (293 cells constitutively expressing SV40 large T antigen) by transfection using FuGene 6 (Roche Molecular Biochemicals) according to the manufacturer's directions. Conditioned medium was harvested 4 days post-transfection and, if necessary, concentrated prior to assay using Centripep 30 centrifugal concentrators (Millipore, Bedford, MA).
Receptor Binding Assays-Soluble insulin receptor binding assays were performed using a modification of the microtiter plate antibody capture assay that we have described previously (17,18). Microtiter plates (Nunc Maxisorb, Roskilde, Denmark) were incubated overnight at 4°C with anti-insulin receptor antibody 18 -44 IgG (26) (100 l/well of 50 g/ml solution in phosphate-buffered saline). Washing, blocking, and receptor binding were as previously described. Competitive binding assays with labeled and unlabelled insulin were carried out as previously, except that the incubation was for 16 h at 25°C. Binding data were analyzed by computer fitting to a one-site model to obtain the K d of the expressed protein.
Insulin Receptor Enzyme-linked Immunosorbent Assay-Microtiter plates were incubated overnight at 4°C with anti-insulin receptor antibody 18 -44 IgG (26). Plates were then washed and blocked as for the receptor binding assay and incubated with varying dilutions of conditioned medium from transfected cells to immobilize the receptor. They were then incubated with biotinylated anti-FLAG M2 antibody (5 g/ul) for 60 min at room temperature. After washing, plates were incubated with a 1:16,000 dilution of streptavidin horseradish peroxidase conjugate for a further 60 min at room temperature and then assayed spectrophotometrically.

RESULTS
Alanine Mutagenesis-We have constructed cDNAs encoding alanine mutants of the ligand-accessible amino acid side chains in the regions of the A and B isoforms of the human insulin receptor, the L1 domain (14), amino acids 1-150, and amino acids 705-715 at the C terminus of the ␣ subunit (1, 2) that have been shown to be the minimal essential structural components of one of its major ligand binding sites (24). These were expressed transiently as recombinant secreted receptors with FLAG epitope tags in 293 Peak Rapid cells.
To evaluate receptor expression, receptor in conditioned media was quantitated by enzyme-linked immunosorbent assay in a sandwich assay in which receptor was immobilized from conditioned medium of transfected cells by the anti-receptor monoclonal antibody 18 -44 that is directed toward a linear epitope in the N terminus of the ␤ subunit of the receptor (26) and then detected with biotinylated anti-FLAG antibody and streptavidin-horseradish peroxidase. We found that, with the exception of the R86A and F96A mutants of both isoforms of the insulin receptor that were not secreted, all mutants were present in conditioned medium in approximately the same concentrations as those of control wild type receptor A and B isoforms (data not shown); we failed to detect R86A and F96A mutant receptors in conditioned medium even after 100-fold concentration of the medium. We have previously shown that the secretion of recombinant insulin receptor mutants, including the F96A mutant, is exquisitely sensitive to the integrity of the structure of the receptor protein (17). Thus it is probable that alanine mutations in these positions disrupt the structure of the receptor. This is supported by the report that a proline mutation of Arg 86 of the insulin receptor was the causative mutation in a patient with extreme insulin resistance (32,33).
In that patient, processing of the full-length receptor was impaired with inefficient insertion in the plasma membrane.
Equilibrium binding studies were performed on conditioned media from transfected cells to characterize insulin binding to both isoforms of wild type and mutant receptors. As previously described, insulin binding to both isoforms of the recombinant secreted receptor displayed simple kinetics and could be best fitted to a single-site model (data not shown). Computer analysis indicated a single population of binding sites with a K d of 2.1 Ϯ 0.2 ϫ 10 Ϫ9 M for the A isoform and 1.8 Ϯ 0.2 ϫ 10 Ϫ9 M for the B isoform (mean Ϯ S.E., n ϭ 8). It should be noted that these values are higher than we have previously reported and probably reflect changes in assay conditions and computerized analysis of binding data (17,20). Because studies utilizing alanine-scanning mutagenesis have demonstrated that meaningful changes in affinity, produced by a single alanine substitution, range from 2-to 100-(34), in the experiments described below we regarded any mutant with a greater than 2-fold increase in K d , i.e. K d greater than 4.2 ϫ 10 Ϫ9 M for the A isoform and 3.6 ϫ 10 Ϫ9 M for the B isoform, as exhibiting a significant disruption of insulin-receptor interactions.
The effects of the individual alanine mutations on the K d for insulin of the A and B isoforms of the receptor are summarized in Fig. 1. In the L1 domain of the B isoform of the receptor, mutations of Asp 12 , Leu 36 , Leu 87 , Phe 88 , Phe 89 , Asn 90 , Tyr 91 , Glu 97 , and Glu 120 produced a 2-to 10-fold increase in K d and mutations of Asn 15 , Gln 34 , Leu 37 , and Lys 121 a 10-to 65-fold increase (Fig. 1A). In addition, alanine mutations of Arg 14 and Phe 64 resulted in proteins with an affinity for insulin that was too low to be accurately determined (Fig. 1A). In the L1 domain of the A isoform, with the exception of mutants of Asn 15 and Leu 37 , similar effects were observed for all alanine mutations. Alanine mutation of Asn 15 resulted in a receptor with unmeasurable affinity for insulin, and mutation of Leu 37 had an effect that was half that in the B isoform (Fig. 1A). In the cysteinerich domain, alanine mutations of His 247 , Phe 248 , Gln 249 , and Asp 250 failed to produce any significant compromise in the affinity of either the A or B isoform for insulin (data not shown). At the C terminus of the ␣ subunit of the B isoform of the receptor, alanine mutations of Phe 705 , Glu 706 , Tyr 708 , His 710 , and Asn 711 resulted in receptors with insulin binding that was too low to be accurately determined, and alanine mutations of Leu 709 and Phe 714 produced increases in K d for insulin of 40and 80-fold, respectively (Fig. 1B). In the A isoform, alanine mutations of Phe 705 , Glu 706 , and His 710 had the same profound effects on insulin binding as they did in the B isoform. Mutations of Asp 707 , Val 713 , and Val 715 that were without effect on the B isoform produced increases of 7-to 16-fold in the K d for insulin of the A isoform. Also, mutation of Leu 709 that resulted in an 80-fold increase in the K d of the B isoform produced an A isoform receptor devoid of insulin binding activity (Fig. 1B). In contrast, mutation of Asn 711 , which had a similar effect on the B isoform receptor, only produced a 45-fold increase in the K d of the A isoform (Fig. 1B). DISCUSSION In the present study we have used alanine-scanning mutagenesis to compare the functional epitopes of the A and B isoforms of the insulin receptor. In contrast to previously reported studies of insulin (6, 7), we have found that there is no significant difference between the affinities of the two isoforms for insulin. This is probably because of the different forms of the receptor that were studied. In previous studies, binding experiments were performed on native receptors in intact cells or solubilized native receptors, whereas in the present study they were performed on soluble recombinant receptors. These two types of preparation differ significantly in their binding properties (35,36); the secreted receptors exhibit lower affinity for insulin and no negative cooperativity. These differences have been attributed to differences in the molecular mechanisms underlying binding; thus it is not surprising that the presence or absence of the alternately spliced exon should impact the binding properties of the two forms of the receptor differently. However, point mutations appear to have the same relative effects on both forms of the receptor (17).
The present study has identified a number of new mutations in the L1 domain (14) that compromise the affinity of both receptor isoforms for insulin. Two of these, alanine mutations of Leu 37 and Glu 97 , were examined in our previous alaninescanning study of the N-terminal binding domain of the B isoform of the receptor (17). In that study we reported that secretion of these mutant receptors was impaired and their affinity for insulin was too low for accurate determination. However, receptor secretion was not completely abolished as FIG. 1. Alanine-scanning mutagenesis of the A and B isoforms of the insulin receptor. 293 PEAK cells were transfected with cDNAs encoding alanine mutants of ligand-accessible amino acids of the L1 domain (A) and of amino acids 705-715 (B) of the A and B isoforms of the recombinant secreted insulin receptor prepared by oligonucleotidedirected mutagenesis. Four days postinfection, conditioned medium from the cells was harvested, and insulin binding of the mutant receptors was evaluated as described under "Experimental Procedures." The dissociation constant was determined by computer fitting to a singlesite model. The dissociation constants of the wild type receptors determined under these conditions were 2.1 Ϯ 0.2 ϫ 10 Ϫ9 M for the A isoform and 1.8 Ϯ 0.2 ϫ 10 Ϫ9 M for the B isoform (mean Ϯ S.E., n ϭ 8). The results are expressed as the ratio of the dissociation of the mutant to that of the wild type and represent the mean Ϯ S.E. of four independent determinations. The amino acids mutated to alanine are designated by the single letter code. Alanine mutants of Arg 86 and Phe 96 were not secreted in detectable quantities. Those mutants with an affinity for insulin that was too low to be determined accurately were assigned a value for K d MUT/KdWT of 100. observed for the alanine mutants of Arg 86 and Phe 96 discussed above. It is likely that low receptor affinity combined with low transfection efficiency was the reason for our inability to determine the affinity of these mutants; the expression system used in the present study gives significantly higher yields of receptor (data not shown). Alanine mutations of Leu 87 , Glu 120 , and Lys 121 were not examined in our previous study of the B isoform of the receptor (17) as a consequence of the scanning strategy that was employed. These mutations compromise affinity of both isoforms of the receptor.
The results of the present study indicate that the insulin contact site on the L1 domain of both isoforms of the receptor is composed of two functional epitopes. The first of these is more extensive than we previously proposed (17) and comprises the side chains of Asp 12 , Arg 14 , Asn 15 , Gln 34 , Leu 36 , Leu 37 , Phe 64 , Glu 97 , Glu 120 , and Lys 121 . These form a continuous footprint on the ␤-sheet at the base of the domain extending from the first turn of the ␤ helix to the fifth (see Fig. 2). The residues Arg 14 , Asn 15 , and Phe 64 form a hot spot. The second is quantitatively less important and is formed from Leu 87 , Phe 89 , Asn 90 , and Tyr 91 and is located in the bulge just N-terminal to the fourth strand of the ␤-sheet at the domain base (Fig. 2). Although this functional epitope is the same for both receptor isoforms, the quantitative contributions of the side chains of Asn 15 and Leu 37 to insulin affinity differ significantly for each. The contribution of Asn 15 is greater in the A than in the B isoform. Alanine mutation in the A isoform produced a receptor completely devoid of insulin binding activity, whereas in the B isoform it only resulted in a 63-fold reduction in affinity. In contrast, Leu 37 appeared to make a greater contribution to the B isoform affinity for insulin; alanine mutations of B and A isoforms produced 40-and 20-fold reductions in affinity, respectively.
In the cysteine-rich domain, we analyzed the insulin binding properties of alanine mutants of four candidate residues, His 247 , Phe 248 , Gln 249 , and Asp 250 . These were selected because reconstitution studies had shown that amino acids 1-250 and 704 -719 are the minimal determinants of this ligand binding site (24), and homology modeling based on the IGF-I receptor coordinates (14) showed these to be the only residues in the cysteine-rich domain between amino acids 1 to 250 that are accessible to ligand. Despite the fact that these residues correspond to residues in the IGF-I receptor cysteine-rich domain that have been implicated in IGF-I binding (18), their mutation had no significant effect on the affinity of either isoform of the insulin receptor for insulin. Thus it does not appear that insulin interactions with the cysteine-rich domain of either isoform of the insulin receptor make an energetically significant contribution to the insulin receptor interaction. This is in contrast to predictions made from a model of insulin receptor structure constructed from cryoscanning tunnelling electron microscopic studies of the quarternary structure of the insulin receptor complex (38). In this study, the side chains of His 247 , Gln 249 , and Asp 250 were reported to be contact sites for the side chains of the insulin residues, Glu B21 and Arg B22 . Alanine mutations of these residues have been reported to increase the affinity of insulin for the receptor (39). Thus it would be predicted that alanine mutations of their putative contact sites in the receptor would increase the affinity of the receptor for insulin.
In the C-terminal domain of the ␣ subunit, alanine mutations cause similar deleterious effects on ligand binding of both isoforms of the receptor and suggest that this region is the major contributor to the free energy of ligand binding for both, although certain mutants have differential effects on the A and the B isoforms. Alanine Mutations of Phe 705 , Glu 706 , and His 710 inactivate both receptor isoforms. Alanine mutations of Tyr 708 and Asn 711 selectively have a greater effect on the affinity of the B isoform than the A, whereas the reverse is seen with alanine mutations of Leu 709 and Phe 714 . Also, alanine mutations of Asp 707 , Val 713 , and Val 715 moderately compromise the affinity of the A isoform for insulin but are without effect on the B.
The finding that alanine mutations in the insulin receptor ligand binding site have differential effects on the ligand affinity on the A and B isoforms of the receptor indicates that the structures of their binding sites must be different. This would be consistent with the observation that the kinetics of association and dissociation differ for the two receptor isoforms (6). Further support for this concept is provided by the finding that IGF-I and -II that bind to receptor sites that overlap those of the insulin binding site have very different affinities for the two receptor isoforms; their affinity is at least 10-fold greater for the A isoform than for the B isoform (7).
When these findings are taken together with the observation that both isoforms have almost identical affinities for insulin, it would appear that insulin-receptor interface exhibits significant plasticity, implying that either or both of the partners in the interaction can undergo significant conformational changes to accommodate the structural changes entrained by insertion of the additional 12 amino acids just adjacent to one of the subdomains of the binding site. Such a notion is supported by the relative independence of insulin affinity of the exact posi-  (37) is shown as a ribbon representation. The amino acids mutated are shown in spacefilling representation. Color coding is according to the results for the B isoform of the insulin receptor. Alanine mutations of amino acids colored green produced a 2-to 10-fold reduction in affinity, those colored yellow a 10-to 100-fold reduction, and those colored red Ͼ100-fold. Alanine mutations of residues colored white resulted in receptors that were not secreted in detectable amounts. This figure was prepared with the Swiss PDB Viewer (37).
tioning of the C-terminal peptide observed in recombinant receptors or in reconstitution experiments (21)(22)(23). Plasticity of protein-protein interfaces is not an uncommon phenomenon in nature (for review see Ref. 40). However, it is rare to observe the preservation of affinity that is observed in this situation. This presumably has arisen as a consequence of the co-evolution of insulin and its receptor.
In summary, we have demonstrated by comparative alaninescanning mutagenesis that the A and B isoforms of the receptor have functional epitopes that differ qualitatively and quantitatively. Elucidation of the underlying bases for these differences at the atomic level will require the elucidation of the structure of both isoforms of the receptor in complex with insulin.