Synthesis and Characterization of Photoreactive Insulin-like Growth

Recombinant human insulin-like growth factor 1 (hIGF-1) was reacted with ... iodination of the three monoacylated photoreactive IGF-1 derivatives, the...
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Bioconjugate Chem. 1999, 10, 200−205

Synthesis and Characterization of Photoreactive Insulin-like Growth Factor 1 Derivatives for Receptor Analysis Marlies Fabry* Deutsches Wollforschungsinstitut an der RWTH Aachen e.V., 52062 Aachen, Germany. Received May 19, 1998; Revised Manuscript Received September 23, 1998

Recombinant human insulin-like growth factor 1 (hIGF-1) was reacted with 4-azidosalicylic acid (Asa) to synthesize photoreactive IGF-1 derivatives. Depending on the time of reaction and the Asa/IGF-1 ratio, a mixture of mono-, bis-, and trisacylated derivatives and one tetraacylated derivative was produced, which was separated by reversed-phase HPLC. HPLC, PAGE, and MALDI mass spectrometry were used to determine the degree of the acylation and location of the photolabel insertion. After iodination of the three monoacylated photoreactive IGF-1 derivatives, the specific labeling of the receptor could be proved. Together with a comparative investigation in which B29-Asa-insulin was used, the results suggest corresponding contact areas for IGF-1 and insulin with the insulin receptor ectodomain.

INTRODUCTION 1

Insulin-like growth factor 1 (IGF-1) is a growth factor that exhibits structural and functional homology to insulin. The single-chain protein consists of 70 amino acids forming four peptide domains: the B (1-29), C (3041), A (42-62), and D domains (63-70). The A and B domains are structurally homologous to the insulin A and B chains; the C domain corresponds to the C peptide of proinsulin, while the D domain does not have an equivalent in proinsulin or insulin. As in insulin, six cysteine residues form three disulfide links (Rinderknecht and Humbel, 1978; Blundell et al., 1983). Whereas IGF-1 has a key function in the regulation of cellular proliferation and differentiation, insulin regulates a broad spectrum of metabolic processes. The action of both polypeptide hormones is mediated by the interaction with their specific membrane receptors. Like their ligands, the insulin and IGF-1 receptor exhibit a remarkable homology with regard to their amino acid sequence and molecular structure. Both receptors have the same heterotetrameric R2β2 structure. The ligand-binding R-domains are completely extracellular and have a cysteine-rich domain. The β-subunits cross the plasma membrane and contain the cytoplasmatic phosphotyrosine kinase domain. Ligand binding induces autophosphorylation of the receptors and subsequently phosphorylation of cytoplasmatic proteins (Ullrich et al., 1985, 1986; Ebina et al., 1985). Despite the high degree of homology of the ligands as well as the receptors, insulin and IGF-1 exhibit only weak cross reactivity. The affinity of binding of each ligand to * To whom correspondence should be addressed: Deutsches Wollforschungsinstitut, Veltmanplatz 8, D-52062 Aachen, Germany. E-mail: [email protected]. Fax: ++49 (0)241 44 69 100. Phone: ++49 (0)241 44 69 146. 1 Abbreviations: Asa, 4-azidosalicylic acid; DMF, dimethylformamide; IGF-1, insulin-like growth factor 1; IR921, insulin receptor ectodomain; MALDI mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry; OSu, N-hydroxysuccinimidyl; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TPCK, tosylphenylchloromethyl ketone.

the receptor of the other ligand is about 1% (Kjeldsen et al., 1991; Schumacher et al., 1991). Nevertheless, cross reactivity and homology between ligands and receptors suggest an analogous mechanism of ligand-receptor interaction for IGF-1 and insulin. Photoaffinity labeling is a powerful chemical approach in structural biology (Kojro et al., 1993; Zhou et al., 1997) and has already proven to be very useful in the identification of the area of binding of insulin to its receptor (Wedekind et al., 1989; Yip, 1992; Fabry et al., 1992; Whaugh et al., 1989; Kurose et al., 1994). Analogous studies with the IGF-1 receptor should now provide additional information about the ligand specificity. In this paper, the preparation and characterization of photoreactive IGF-1 derivatives are reported. These derivatives were used to label IGF-1 receptors and to further investigate the IGF-1 and insulin receptor systems. MATERIALS AND METHODS

Chemicals. Recombinant human insulin-like growth factor 1 (hIGF-1) was a donation from Lilly Research Laboratories (Indianapolis, IN). HPLC-grade acetonitrile, TFA, and TPCK-trypsin were from Merck. Carrier-free 125 I was purchased from Amersham. 4-(Azidosalicyloyl)N-hydroxysuccinimide ester was synthesized as described elsewhere (Fabry and Brandenburg, 1992). Human IGF-1 receptor was isolated from transfected NIH-3T3 fibroblasts (Lammers et al., 1989). Clones of transfected CHOK1 cells that overexpress the IR921 protein were selected. This is the soluble ectodomain of the human insulin receptor in which the heterotetrameric (Rβ0)2 receptor was truncated following residue 921 (Schaefer et al., 1992). Preparation of Asa-IGF-I Derivatives. Asa-OSu was dissolved in DMF (90 µg/10 µL) and added in 4 portions to a 70 µL solution containing 500 µg of IGF-1 dissolved in 0.1 M sodium phoshate buffer (pH 7.4) which was being stirred at room temperature protected from light. After 5-10 min, the reaction was quenched with 10 µL of ethanolamine. The mixture was separated on a Sephasil C-18 SC 2.1/10 column using the SMART system (Pharmacia) and a gradient from 20 to 48%

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Receptor Analysis with Photoreactive Ligands

acetonitrile in 0.1% TFA over the course of 90 min. The proteins were detected by measuring the absorbance at 214, 256, and 280 nm. Fractions were collected automatically into 0.5 mL Eppendorf tubes and lyophilized by SpeedVac vacuum centrifugation. Degree of Acylation and Location of the Photolabel. The different Asa-IGF-1 derivatives separated by reversed-phase HPLC were analyzed by acid-urea PAGE with 8 M urea and 0.9 M acetic acid at pH 4 using a 15% polyacrylamide gel (Poole et al., 1974) and MALDI mass spectrometry to determine the degree of acylation. The mass spectra were obtained on a Bruker Biflex III TOF instrument (Bruker-Franzen Analytik, Bremen, Germany) equipped with a 337 nm nitrogen laser using sinapinic acid as the matrix, acetonitrile/TFA as the solvent, and standard experimental parameters (3 ns pulse duration, linear mode, 100-150 laser spots). To locate the position of the photolabel, IGF-1 and the monoacylated products were digested with trypsin after reduction and carboxymethylation (Honegger and Humbel, 1986). The digests were subjected to MALDI mass spectrometry to evaluate the change of the fragmentation pattern compared to that of unlabeled IGF-1. For reduction and carboxymethylation of disulfide bonds, the lyophilized proteins (10 µg) were dissolved in 20 µL of 6 M guanidinium hydrochloride, 0.5 M Tris, and 5 mM EDTA (pH 8.5) containing 40 nmol of dithiothreitol, flushed with nitrogen, and kept at room temperature in darkness for 3 h. Then, 400 nmol of iodoacetic acid was added, and the solution was further incubated under nitrogen for 2 h. The reaction was stopped by addition of 2 µL of mercaptoethanol. The proteins were desalted by reversed-phase HPLC with the same column using a 90 min gradient from 16 to 48% acetonitrile in 0.1% TFA at a flow rate of 100 µL/min and dried. The reduced products were identified by acid-urea PAGE. The enzymatic cleavage was performed by addition of 0.5 µg of TPCKtrypsin to the proteins dissolved in 30 µL of 0.1 M ammonium bicarbonate (pH 8), followed by incubation at room temperature for 18 h. To stop the reaction, 0.1% TFA was added and proteins were dried by SpeedVac vacuum centrifugation and washed with water twice. The digestion products were then studied with MALDI mass spectrometry. Radioiodination/Iodine Analysis. IGF-1 and the Asa-IGF-1 derivatives were iodinated according to the chloramine T method (Yip et al., 1993) with the following modification of reaction conditions; 4 µg of IGF-1 or the derivative was dissolved in 20 µL of 0.4 M sodium phosphate buffer (pH 7.5), and then 100 µCi of carrierfree 125I (IMS 30, Amersham) was added. Iodination was started by addition of 5 µL of chloramine T (100 ng/1 µL of 0.04 M sodium phoshate buffer) and continued for 3 min at room temperature. The reaction was stopped by adding 10 µL of tyrosine (0.2 mg/mL) and KI (1 mg/mL). The reaction mixture was applied to a SepPak C-18 cartridge (Waters Corp.) that had been equilibrated with 0.1% TFA/acetronitrile (90/10 v/v). After washing, the iodinated derivatives were eluted with 60% acetronitrile in 0.1% TFA into 50 mM ammonium bicarbonate, lyophilized, and stored at -20 °C. To define the 125I position, approximately 50 000 cpm carboxymethylated protein was digested with TPCK-trypsin as described above, and the digest was separated by reversed-phase HPLC with a gradient of 15 to 40% acetonitrile in 0.1% TFA over the course of 100 min at a flow rate of 100 µL/min. Assay of IGF-1 Receptor Binding. Binding of [125I]IGF-1 to plasma membranes of NIH-3T3 cells (50 µg of protein/sample) and competition with IGF-1 and the

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Figure 1. Reversed-phase HPLC separation of the reaction mixture of 4-(azidosalicyloyl)-N-hydroxysuccinimide (Asa-OSu) with IGF-1. Fifty micrograms of the reaction mixture was loaded onto a C-18 column and separated with a flow rate of 100 µL/ min using a 20 to 48% acetonitrile gradient over the course of 90 min. Proteins were monitored at 215, 256, and 280 nm. IGF-1 and the Asa derivatives were identified by acid-urea PAGE (Figure 2) and MALDI mass spectrometry (Table 1).

Asa-IGF-1 derivatives were assessed as described previously (Schumacher et al., 1991). Plasma membranes were prepared according to a previously published procedure (Breiner et al., 1993). Photoaffinity Labeling. Plasma membranes (protein content of at least 30 µg) prepared from NIH-3T3 fibroblasts or IR921 were incubated with the photoreactive IGF-1 derivatives (approximately 500 000 cpm, 2.95 nM) in 50 µL of KRH buffer [50 mM Hepes, 13 mM NaCl, 5.1 mM KCl, and 1.3 mM Mg2SO4 (pH 7.8)] at 15 °C for 90 min. Membrane pellets were washed once with KRH buffer and photolyzed by irradiation with three flashes (each 1000 Ws) of an UV flash apparatus (“Lizzy”, Raytest) in 20 µL of KRH buffer. When the soluble IR921 was labeled, noncovalently bound ligand was removed by precipitation with polyethylene glycol and γ-globulin after photolysis (Fabry et al., 1992). The membrane pellets were solubilized in SDS sample buffer in the presence of dithiothreitol and analyzed by SDS-PAGE (7.5%). Gels were dried prior to autoradiography. Time Course of the Tryptic Digestion of the Photoaffinity-Labeled Ectodomain. IR921 was labeled with B29-Asa-insulin and B27-Asa-IGF-1. Digestion was carried out with 30 µg/mL TPCK-trypsin at room temperature (reaction times indicated). Aliquots were taken, and the reaction was stopped by addition of Laemmli sample buffer and heating for 5 min at 95 °C and analyzed by SDS-PAGE (12.5%). RESULTS AND DISCUSSION

Preparation of Asa-IGF-1 Derivatives. By reaction of the photoreagent, the N-hydroxysuccinimide ester of 4-azidosalicylic acid, with unprotected IGF-1, four free amino groups, the R-amino group in B1 and the three -amino groups of B27-, D65-, and D68-lysine, can be substituted. The resulting mixture of reaction products, varying in degree and site of acylation, can be separated by reversed-phase HPLC due to differences in charge and hydrophobicity (Figure 1). Proteins were detected at the leading wavelength of 214 nm. Two secondary wavelengths at 256 and 280 nm were of interest for the detection of aromatic or modified amino acids. Since the photolabel also absorbs at 256 and 280 nm, the absorbance ratios A214nm/A256nm and A214nm/

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Table 1. Absorbance Ratios of the HPLC Fractions for Reaction of Asa-OSu with IGF-1 (Figure 1)a HPLC fraction

A214nm/ A256nm

A214nm/ A280nm

A256nm/ A280nm

assumed number of photogroups

9 11 13 15 17 19 21 23 25

45.0 12.9 16.2 13.3 8.5 8.2 7.8 5.2 5.6

28.0 8.1 10.4 8.0 6.5 5.0 5.0 3.1 3.5

0.63 0.7 0.64 0.6 0.62 0.63 0.63 0.63 0.62

0 1 1 1 2 2 2 3 3

a Absorptions were simultaneously monitored at 214, 256, and 280 nm.

Figure 3. MALDI mass spectrum of B27-Asa-IGF-1 (calculated mass of 7810.3 Da; fraction 11 of the reversed-phase HPLC separation, Figure 1). In addition to the observed mass of 7813.3 Da, a second peak with a mass 28.6 Da lower and the corresponding doubly protonated proteins were detected. The spectrum was recorded using sinapinic acid as the matrix.

Figure 2. Acid-urea PAGE of the HPLC fractions, and reaction of Asa-OSu with IGF-1 (Figure 1). Msc-insulins are a standard mixture of differently charged insulins. Fraction 9 contains unmodified IGF-1 with the same mobility as the standard IGF-1. Fractions 11, 13, and 15 correspond to monoacylated, fractions 17, 19, and 21 to bisacylated, and fractions 23 and 25 to trisacylated Asa-IGF-1, and fraction 29 corresponds to tetraacylated Asa-IGF-1.

A280nm change characteristically with the degree of acylation (Table 1). Thus, a first classification of the products according to their degree of acylation was possible. Fraction 9 exhibits the typical absorption characteristics of native IGF-1 (45A214nm/A256nm and 28A214nm/A280nm). The acid-urea PAGE (Figure 2) confirmed that fraction 9 contained IGF-1 and fractions 11, 13, and 15 the monoacylated products, followed by three bisacylated IGF-1 derivatives (fractions 17, 19, and 21), and two trisacylated products (fractions 23 and 25). When the reaction was carried out with a 5-fold excess of active ester and a reaction time of 5 min, 29% of the IGF-1 was not modified, 41% monoacylated, 23% bisacylated, and 4% trisacylated. With the same Asa-OSu/IGF-1 ratio and a longer reaction time, there was actually less educt, but the amount of more highly acylated products increased considerably: 4% nonacylated IGF-1 and 11% monoacylated, 33% bisacylated, 46% trisacylated, and 7.5% tetraacylated IGF-1 derivatives (see Figure 2, fraction 29). For native IGF-1, the mass determined by MALDI mass spectrometry corresponds to the calculated mass. For the Asa-IGF-1 derivatives, mass shifts of 1-3-fold minus 28 Da were found in addition to the expected data.2 The mass spectrum for B27-Asa-IGF-1 is given in Figure 3. This mass shift can be explained by a partial photoactivation of the aryl azides effected by the laser energy of the spectrometer during measurement leading to the formation of nitrene with elimination of nitrogen and possibly an addition of two atoms of hydrogen. Normally, this reaction leads to a covalent binding to the receptor. Location of the Photolabel. Tryptic digestion of reduced IGF-1 leads to the formation of six fragments, T1-T6 (Rinderknecht and Humbel, 1978; Honegger and Humbel, 1986). The substitution sites of the photolabel at B1-glycine, B27-lysine, or D65/D68-lysine are within

tryptic fragments T1, T2, or T5, respectively (Figure 4). The mass values of the fragments are changed characteristically due to substitution. IGF-1 and the three monoacylated IGF-1 derivatives were reduced, carboxymethylated, and treated with trypsin after reversed-phase HPLC purification. The peptides were analyzed by MALDI mass spectrometry. Under the conditions used, trypsin cleaved IGF-1 and its derivatives completely and as expected.3 Additionally, at the sequence position SC35-RC36-RC37-AC38, a shifting of the cleavage occurred. As a result, tryptic fragment T2 with two C-terminal arginines and tryptic fragment T3 without the N-terminal arginine were found. At the second position with two arginine residues in sequence, LA54-RA55-RA56-LA57, cleavage occurred only between the two arginines. The mass analyses of the tryptic fragments of the three monoacylated IGF derivatives confirmed the complete elimination of nitrogen from the photofragments. In fraction 11, a mass shift of the tryptic fragment T2 from 1668.78 to 1803.76 Da was observed, corresponding to the activated photogroup (Figure 5). In fraction 15, this caused a shift of the mass of fragment T1 from 2310.28 to 2445.07 Da. Due to the acylation at D68-lysine, no tryptic cleavage of the peptide of fraction 13 was possible.

2 Interested investigators can request molecular mass data from [email protected].

3 Molecular mass data also available via e-mail from Fabry@ dwi.rwth-aachen.de.

Figure 4. Amino acid sequence of IGF-1 showing tryptic peptides T1-T6, the acylation sites of the photolabel Asa (black), and the positions of iodination (gray).

Receptor Analysis with Photoreactive Ligands

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Figure 5. MALDI mass spectrum of the tryptic peptide map of B27-Asa-IGF-1. In addition to the native tryptic peptides, a peak at m/z 1803.76 corresponding to the photofragment T2 with Asa in position B27 was observed. The spectrum was recorded using R-cyano-4-hydroxycinnamic acid as the matrix.

Therefore, this photofragment was enlarged not only by the mass of the activated photogroup but also by the two C-terminal amino acids serine and alanine. As a result, it could be confirmed that fraction 11 contained B27Asa-IGF-1 (tR ) 42.53 min, Figure 1), fraction 13 D68Asa-IGF-1 (tR ) 45.84 min), and fraction 15 B1-AsaIGF-1 (tR ) 48.21 min). The derivative acylated in position B27 was of special interest, because here the photogroup is directly located within the postulated binding area (Blundell et al., 1983). Radioiodination and Analysis of the Position of 125 I. To verify that after tryptic digestion of radioactive IGF-receptor conjugates the resulting binding fragments are still detectable, the insertion of iodine was investigated. Iodine can be inserted onto three tyrosine residues in positions 24, 31, and 60 (Figure 4), as well as into the photolabel itself. Under the conditions used, radioiodination of IGF-1 results in the formation of three monoiodo isomers and radioiodination of the mono-Asa-IGF-1 derivatives forms four isomers. To clarify the site of iodination, radiolabeled IGF-1 and the three mono-Asa-IGF-1 derivatives were reduced, carboxymethylated, and digested with trypsin. Since fragments T1 and T3 only contain phenylalanine, fragment T5 only contains tyrosine, and fragment T2 contains phenylalanine as well as tyrosine, typical absorbance ratios were obtained. The elution profile given in Figure 6 and the assignment were identical to the tryptic peptide map of Honegger and Humbel (1986), who determined the primary structure of the hormone isolated from bovine serum. The tryptic mapping of [125I]IGF-1 revealed the presence of three radioactive peaks. They correspond to tryptic fragment T5 with iodine in position tyrosine 60 and to fragment T2, one with iodine in position tyrosine 24 and one in position tyrosine 31 (Figure 4). When the photolabel is inserted into position B1, tryptic fragment T1 that is not radioactive without the photolabel now is radioactive. Therefore, the reversedphase HPLC separation of the tryptic map of B1-AsaIGF-1 shows a fourth radioactive peak neighboring fragment T1 (Figure 6, tR ) 74.4 min); here, 30% of the iodine was inserted into the N-terminal photolabel (Figure 6, histogram). Fragments T2 and T5 are radioactive without photolabel, but the elution time of the photofragments is much longer compared to those of the radioactive fragments without photolabel. Iodine is inserted into position tyrosine 60 of B27-Asa-IGF-1 to a small extent; iodination of tryptic fragment T2 (with two tyrosine residues and one photogroup) is strongly preferred. For fragment T5, insertion of the photolabel in D65/D68 increases the iodine uptake from 11 to 21%.

Figure 6. Reversed-phase HPLC separation of the tryptic peptide map of carboxymethylated IGF-1 (lower UV curve) with the corresponding radioactivity profile of B1-Asa-IGF-1 (upper histogram). The digest was fractionated on a C-18 column at a flow rate of 100 µL/min using a 10 to 40% acetonitrile gradient over the course of 100 min. Fractions (400 µL) were collected, and the amount of radioactivity was counted. Table 2. Receptor Binding of Native IGF-1 and Asa-IGF-1 Derivatives HPLC fraction

derivative

KD ( SD (nM)

relative biological binding affinity (%)

9 11 13 15 17 19 21 23 25 27 29

IGF-1 B27-Asa-IGF-1 D65/D68-Asa-IGF-1 B1-Asa-IGF-1 bis-Asa-IGF-1 bis-Asa-IGF-1 bis-Asa-IGF-1 tris-Asa-IGF-1 tris-Asa-IGF-1 tris-Asa-IGF-1 tetra-Asa-IGF-1

0.72 ( 0.36 6.3 ( 0.44 1.2 ( 0.14 0.61 ( 0.04 4 ( 0.36 15 ( 1.80 7 ( 0.98 12 ( 1.68 9.2 ( 1.47 18 ( 1.62 15 ( 1.05

100 11.5 59 120 18.5 4.8 10.4 6.3 7.9 4.1 4.8

As a conclusion, it can be expected that after tryptic digestion of the covalent hormone-receptor conjugate binding fragments are still detectable because the point of receptor labeling and 125I would not be separated. Receptor Binding Studies. To determine the binding characteristics of the photo-IGF-1 derivatives, saturation binding experiments were carried out with [125I]IGF-1 on plasma membranes of NIH-3T3 cells that overexpress wild-type IGF-1 receptors (Table 2). The binding affinities of the three monoacylated photo-IGF-1 derivatives differed considerably, indicating the importance of the substituted positions for receptor binding. The severest impairment of receptor binding was caused by a modification of position B27, which is within the postulated binding area. The derivative acylated in position B1 showed an even slightly higher binding affinity than native IGF-1, indicating that this position is not significantly involved in receptor binding. The binding affinity of the derivative with modification of the C-terminal D domain was intermediate. As expected, the binding affinity decreased with a further increase in the degree of acylation, with the exception of a bisacylated derivative that showed a rather low binding affinity of 4.8% compared with that of IGF-1. Photoaffinity Labeling. Figure 7A shows the results of the photoaffinity labeling of plasma membranes of NIH-3T3 cells overexpressing the human IGF-1 receptor with the three mono-Asa-IGF-1 derivatives analyzed by SDS-PAGE. Specifically labeled bands with Mrs of

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Figure 8. Time course of the tryptic digestion of the insulin receptor ectodomain labeled with B29-Asa-insulin and B27Asa-IGF-1. Receptors were photoaffinity labeled as described in Materials and Methods. At the indicated time points, aliquots were withdrawn and analyzed by SDS-PAGE (12.5%) and autoradiography.

detecting and comparing specific binding areas of ligands and their receptors. ACKNOWLEDGMENT

Figure 7. (A) Photoaffinity labeling of the IGF-1 receptor with B27-Asa-IGF-1 (1), D65/D68-Asa-IGF-1 (2), and B1-AsaIGF-1 (3). Lanes marked with + are protein labeling (control) with an excess of IGF-1 (10-5 M). (B) Photoaffinity labeling of the IGF-1 receptor (IGF-1R), insulin receptor (IR), and insulin receptor ectodomain (IR921) with B27-Asa-IGF-1 and B29Asa-insulin, as analyzed by SDS-PAGE under reducing conditions and autoradiography.

120 000 Da correspond to the hormon binding R-subunit. The B1-, B27-, and D65/D68-Asa-IGF-1 derivatives revealed no detectable difference in the degree of labeling of the receptor. The efficiency of photolabeling was approximately 10% (calculated as the extent of incorporation of radioactivity into the IGF-1 receptor R-subunit vs the total amount of radioactivity applied onto SDSPAGE). In Figure 7B, the photoaffinity labeling of insulin receptor ectodomain (IR921), native insulin (IR), and IGF-1 receptors (IGF-1R) of NIH-3T3 cells with B27Asa-IGF-1 and B29-Asa-insulin is compared. PhotoIGF-1 labels the insulin receptor and vice versa; photoinsulin also labels the IGF-1 receptor. The soluble ectodomain is strongly labeled by insulin (maximum of 40%) but only weakly by IGF-1 (approximately 5%). The tryptic peptide maps of the photoinsulin and IGF-1 IR921 conjugates are very similar (Figure 8). SDS-PAGE of the cleavage products under reducing conditions indicated for labeling with photoinsulin major fragments of 97, 72, 59, 31, 28, and 22 kDa and for labeling with photo-IGF-1 of 94, 68, 58, 32, 29, 24, and 23 kDa. For both labels, weak bands at 43 and 13 kDa were also observed. The high correspondence of the digestion pattern indicates identical fragments and thus identical binding areas. The synthesis of a series of photoreactive hormone derivatives for cross-linking described here will facilitate the construction of a detailed interaction map of the insulin and IGF-1 receptors and their ligands. Synthesis and radioiodination of the ligands as well as photoaffinity labeling of the receptors are possible within a short period of time. The method presented is a powerful tool for

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