Biol. Chem. Hoppe-Seyler Vol. 373, pp. 915-923, September 1992
Analysis of the Human Insulin Receptor Marlies FABRY and Dietrich BRANDENBURG Deutsches Wollforschungsinstitut an derTechnischen Hochschule Aachen
(Received 5 June 1992)
Summary: The insulin derivative 4-azidosalicyloyl[Bl-biocytin-B2-lysine]insulin was used to photoaffinity-label the highly purified insulin receptor from human placenta. As shown by SDS-polyacrylamide gel electrophoresis, the 5 monoiodo isomers, with iodine in positions B l, B16, B26, A14 orA19, gave different labelling patterns. After complete tryptic digestion of the covalent receptor complex with 12:>I-Asa[BctB1,LysB2]insulin, a stable fragment of 18 kDa was
isolated, which was further purified by HPLC. This tryptic fragment of the intact receptor corresponds, according to HPLC,Tricin-SDS-PAGE and 2D-electrophoresis, to the similarly labelled sequenced domain of the receptor ectodomain (Fabry, M. et al. (1992) J. Biol. Chem. 267, 8950-8956). We thus conclude that insulin is bound to identical contact sites of native receptor and truncated ectodomain.
Analyse des humanen Insulinrezeptors Zusammenfassung: Das Insulinderivat 4-Azidosalicyloyl[Bl-biocytinyl-B2-lysin]insulin wurde zur Photoaffinitätsmarkierung des Insulinrezeptors aus menschlichen Plazentamembranen eingesetzt. Die bei der Radioiodierung des Insulinderivates entstehenden 5 Monoiodisomere (Bl, B16, B26, A14 und A19) ergaben in der SDS-Polyacrylamidgelelektrophorese ein unterschiedliches Markierungsmuster. Aus dem 125I-Asa[BctB1,LysB2]insulin-Rezeptor-Kom-
plex entstand nach vollständigem tryptischen Abbau ein stabiles Fragment von 18 kDa, das durch HPLC gereinigt wurde. Dieses tryptische Fragment des intakten Rezeptors korrespondiert in HPLC, TricinSDS-PAGE und 2D-Elektrophorese mit dem sequenzierten Fragment der Ektodomäne des Insulinrezeptors^. Wir schließen auf gleiche Kontaktbereiche des Liganden mit dem nativen Insulinrezeptor und der verkürzten Ektodomäne.
Key terms: Insulin, insulin receptor, photoaffinity labelling, peptide mapping.
The insulin receptor is a heterotetrameric integral glycoprotein, ß-a-a-ß, comprised of the extracellular, insulin-binding -subunit and the ß-subunit, a
tyrosine-kinase, with extracellular, transmembraneous and cytosolic domains (for reviews, see ref.[1]).
Abbreviations: ABL-insulin, 4-azidosalicyloyl[Bl-biocytinyl-B2-lysine]insulin; Asa, 4-azidosalicyloyl; Bet, biocytinyl, biocytin, -biotinyl-L-lysine; Boc, te/t.-butyloxycarbonyl; BSA, bovine serum albumin; DTE, dithioerythritol; EDTA, ethylenediamine tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid; hIR, human insulin receptor; IEF, isoelectric focusing; KRH, KrebsRinger-Henseleit; Msc, methylsulfonylethoxy-carbonyl; Nap, 4-azido-2-nitrophenyl; Napa, 4-azido-2-nitrophenylacetyl; PAGE, poly-acrylamide gel electrophoresis; PAMAC, photoaffinity-mediated avidin complexing; Ptc, phenylthiocarbamoyl; PVDF, polyvinylidene fluoride; RP-HPLC, reversed phase high performance liquid chromatography; SDS, sodium dodecyl sulfate;TCA, trichloroacetic acid;TEAP, triethylammoniumphosphate;TFA, trifluoroaceticacid;TPCK, L-(7V-tosyl-phenylalanyl)chloromethane; WGA, wheat germ agglutinine.
Brought to you by | Brown University Rockefeller Library Copyright © by WalterAuthenticated de Gruyter & Co · Berlin · New York Download Date | 5/19/15 11:31 PM
916
M. Fabry and D. Brandenburg
Since binding of the hormone to, and its recognition by, the receptor is the first step in insulin's action, detailed knowledge of the interacting surfaces is pivotal to our understanding of the molecular mechanism(s) of signal generation and transduction. X-ray or NMR data still pending, information on the hormone binding site is being gathered via mutated receptors, antiinsulin antibodies, and photoaffinity labelling (see, for instance, refsJ 2 " 6 ^). Only the latter technique gives direct information on contact sites of both hormone and receptor. Applying a novel technique, which we termed PAMAC (photoaffinity-mediated avidin complexing)16'"^, we have recently identified the sequence 20121 of the purified human placental receptor as an insulin binding domain^ 7 '. The availability of recombinant soluble receptor ectodomain in milligram amounts^8' greatly facilitates further work. With IR 921 (ßi-a-a-ßi), comprising the complete a-subunit and the extracellular domain of the /3-subunit (except for the 8 C-terminal residues) a second contact site could be discovered[9]. In order to test whether these receptors of different structure and origin bind insulin similarly and form comparable complexes, we have now analysed the human placental receptor with the aid of the same novel photoreactive insulin, 125I-Asa[BctB1,LysB2]insulin*'10^. Following similar strategies as before^, we find covalent binding to a domain, which, according to the analyses performed, corresponds to that identified with the receptor ectodomain.
Vol. 373 (1992)
Purification of insulin receptors Insulin receptor was purified from human placenta membranes by wheat germ agglutinin and insulin-Sepharose affinity chromatography as described116'17'. The purification of 5 placentae resulted in ca. 400 ^g receptor, which was calculated from the amount from one placenta.The insulin-binding activity was 18 -g per mg of protein. Preparative photoaffinitv labelling, tryptic digestion and fragment isolation Purified receptors from 5 placentae were incubated in a total volume of 10 m/Tris/HCl buffer (50mM, pH 8.0) with 4.8 x KT7M ABL-insulin (30 ^g) and 3 x 10"'°M !25 I-ABL-insulin (19 ng, 1()7 cpm) for 16 h at 4°C (molar ratio of receptor to insulin of 1.0:1.5). Irradiation (6 flashes) was carried out in a Petri dish under helium gas. Non-covalently bound insulin derivative was removed by Centricon-30 (Amicon) centrifugation by washing 3 times with 2 m/ Tris/HCl buffer (50mM. pH 8.0). Digestions were performed with 30 A
Analysis of the Human Insulin Receptor Marlies FABRY and Dietrich BRANDENBURG Deutsches Wollforschungsinstitut an derTechnischen Hochschule Aachen
(Received 5 June 1992)
Summary: The insulin derivative 4-azidosalicyloyl[Bl-biocytin-B2-lysine]insulin was used to photoaffinity-label the highly purified insulin receptor from human placenta. As shown by SDS-polyacrylamide gel electrophoresis, the 5 monoiodo isomers, with iodine in positions B l, B16, B26, A14 orA19, gave different labelling patterns. After complete tryptic digestion of the covalent receptor complex with 12:>I-Asa[BctB1,LysB2]insulin, a stable fragment of 18 kDa was
isolated, which was further purified by HPLC. This tryptic fragment of the intact receptor corresponds, according to HPLC,Tricin-SDS-PAGE and 2D-electrophoresis, to the similarly labelled sequenced domain of the receptor ectodomain (Fabry, M. et al. (1992) J. Biol. Chem. 267, 8950-8956). We thus conclude that insulin is bound to identical contact sites of native receptor and truncated ectodomain.
Analyse des humanen Insulinrezeptors Zusammenfassung: Das Insulinderivat 4-Azidosalicyloyl[Bl-biocytinyl-B2-lysin]insulin wurde zur Photoaffinitätsmarkierung des Insulinrezeptors aus menschlichen Plazentamembranen eingesetzt. Die bei der Radioiodierung des Insulinderivates entstehenden 5 Monoiodisomere (Bl, B16, B26, A14 und A19) ergaben in der SDS-Polyacrylamidgelelektrophorese ein unterschiedliches Markierungsmuster. Aus dem 125I-Asa[BctB1,LysB2]insulin-Rezeptor-Kom-
plex entstand nach vollständigem tryptischen Abbau ein stabiles Fragment von 18 kDa, das durch HPLC gereinigt wurde. Dieses tryptische Fragment des intakten Rezeptors korrespondiert in HPLC, TricinSDS-PAGE und 2D-Elektrophorese mit dem sequenzierten Fragment der Ektodomäne des Insulinrezeptors^. Wir schließen auf gleiche Kontaktbereiche des Liganden mit dem nativen Insulinrezeptor und der verkürzten Ektodomäne.
Key terms: Insulin, insulin receptor, photoaffinity labelling, peptide mapping.
The insulin receptor is a heterotetrameric integral glycoprotein, ß-a-a-ß, comprised of the extracellular, insulin-binding -subunit and the ß-subunit, a
tyrosine-kinase, with extracellular, transmembraneous and cytosolic domains (for reviews, see ref.[1]).
Abbreviations: ABL-insulin, 4-azidosalicyloyl[Bl-biocytinyl-B2-lysine]insulin; Asa, 4-azidosalicyloyl; Bet, biocytinyl, biocytin, -biotinyl-L-lysine; Boc, te/t.-butyloxycarbonyl; BSA, bovine serum albumin; DTE, dithioerythritol; EDTA, ethylenediamine tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid; hIR, human insulin receptor; IEF, isoelectric focusing; KRH, KrebsRinger-Henseleit; Msc, methylsulfonylethoxy-carbonyl; Nap, 4-azido-2-nitrophenyl; Napa, 4-azido-2-nitrophenylacetyl; PAGE, poly-acrylamide gel electrophoresis; PAMAC, photoaffinity-mediated avidin complexing; Ptc, phenylthiocarbamoyl; PVDF, polyvinylidene fluoride; RP-HPLC, reversed phase high performance liquid chromatography; SDS, sodium dodecyl sulfate;TCA, trichloroacetic acid;TEAP, triethylammoniumphosphate;TFA, trifluoroaceticacid;TPCK, L-(7V-tosyl-phenylalanyl)chloromethane; WGA, wheat germ agglutinine.
Brought to you by | Brown University Rockefeller Library Copyright © by WalterAuthenticated de Gruyter & Co · Berlin · New York Download Date | 5/19/15 11:31 PM
916
M. Fabry and D. Brandenburg
Since binding of the hormone to, and its recognition by, the receptor is the first step in insulin's action, detailed knowledge of the interacting surfaces is pivotal to our understanding of the molecular mechanism(s) of signal generation and transduction. X-ray or NMR data still pending, information on the hormone binding site is being gathered via mutated receptors, antiinsulin antibodies, and photoaffinity labelling (see, for instance, refsJ 2 " 6 ^). Only the latter technique gives direct information on contact sites of both hormone and receptor. Applying a novel technique, which we termed PAMAC (photoaffinity-mediated avidin complexing)16'"^, we have recently identified the sequence 20121 of the purified human placental receptor as an insulin binding domain^ 7 '. The availability of recombinant soluble receptor ectodomain in milligram amounts^8' greatly facilitates further work. With IR 921 (ßi-a-a-ßi), comprising the complete a-subunit and the extracellular domain of the /3-subunit (except for the 8 C-terminal residues) a second contact site could be discovered[9]. In order to test whether these receptors of different structure and origin bind insulin similarly and form comparable complexes, we have now analysed the human placental receptor with the aid of the same novel photoreactive insulin, 125I-Asa[BctB1,LysB2]insulin*'10^. Following similar strategies as before^, we find covalent binding to a domain, which, according to the analyses performed, corresponds to that identified with the receptor ectodomain.
Vol. 373 (1992)
Purification of insulin receptors Insulin receptor was purified from human placenta membranes by wheat germ agglutinin and insulin-Sepharose affinity chromatography as described116'17'. The purification of 5 placentae resulted in ca. 400 ^g receptor, which was calculated from the amount from one placenta.The insulin-binding activity was 18 -g per mg of protein. Preparative photoaffinitv labelling, tryptic digestion and fragment isolation Purified receptors from 5 placentae were incubated in a total volume of 10 m/Tris/HCl buffer (50mM, pH 8.0) with 4.8 x KT7M ABL-insulin (30 ^g) and 3 x 10"'°M !25 I-ABL-insulin (19 ng, 1()7 cpm) for 16 h at 4°C (molar ratio of receptor to insulin of 1.0:1.5). Irradiation (6 flashes) was carried out in a Petri dish under helium gas. Non-covalently bound insulin derivative was removed by Centricon-30 (Amicon) centrifugation by washing 3 times with 2 m/ Tris/HCl buffer (50mM. pH 8.0). Digestions were performed with 30 A
