Untersuchungen zum Insulinbindungsbereich des Insulin-IGF-I-Rezeptorsystems mittels kovalenter Markierung

Goal of this thesis was to provide information about the insulin binding region und stoichiometry of the insulin and the IGF-I receptor as well as of two chimeric insulin-IGF-I receptors. To achieve this two photoreactive insulin analogues, B29-Azidotetrafluorobenzoyl-biocytinyl-Insulin (B29-Atf-Bct-Insulin) and B29-Azidosalicyloyl-Insulin (B29-Asa-Insulin), were synthesized and covalently linked to the receptors. After digestion of the hormon-receptor-complexes it was possible to isolate and N-terminally sequence labelled insulin binding fragments. Via orthogonal photoaffinity labelling it was possible to demonstrate the receptors' bivalence for insulin. 1. Two different B29-substituted photoinsulin analogues were synthesized starting from B29-Msc2-Insulin: B29-Asa-Insulin and B29-Atf-Bct-Insulin. The resulting derivatives were chemically and biologically characterized. 2. Radioiodination of B29-Asa-Insulin with Chloramin T succeded in a fast reaction and led to high iodine incorporation. B29-Atf-Bct-Insulin was iodinated with Chloramin T as well as with Lactoperoxidase/H2O2. Incorporation rates were 80-90% for B29-Asa-Insulin and 60-70% for B29-Atf-Bct-Insulin. 3. Culturing NIH-3T3 Cells, which permanently over expressed the studied receptors, was performed according to standard procedures. Prior to photoaffinity labelling the receptors were purified via two different routes. Sequential centrifugation steps let to purified plasma membranes whereas WGA-affinity chromatography let to solubilized membrane receptors. 4. Polyclonal anti-insulin receptor-peptide antisera have the potential to serve as highly specific tools for purification and detection of the insulin receptor. Four polyclonal anti-insulin receptor-peptide antisera were purified by affinity chromatography and analysed by biomolcular interaction analysis. Though none of the sera was able to specifically bind to the insulin receptor ectodomain. 5. B29-Asa-Insulin and B29-Atf-Bct-Insulin bound specifically to the insulin receptor. Both ligands could be displaced from the receptor with a 1000fold excess of the native hormone. As demonstrated with radioiodinated B29-Asa-Insulin photoinsulin could also be displaced by an excess of native IGF-I. Maximal hormone binding rates were 35,6% for the native insulin receptor, 31,5% for the chimeric receptor IR/IGF-IR C2, 29,6% for the chimeric receptor IGF-I/IR C1,3 and 19,4 % for the native IGF-I receptor. 6. Receptors covalently crosslinked to B29-Atf-Bct-Insulin could be detected highly specific by Enhanced Chemiluminescence (ECL). Sensitivity of ECL was comparable to that of radioactivity. Prerequisite was separation of the receptors from endogenous biotinylated proteins via short lysis and WGA-affinity chromatography. 7. Insulin and IGF-I receptors as well as the two chimeric receptors covalently crosslinked with 125I-B29-Asa-Insulin were tryptically digested. The resulting fragment pattern showed partly coincidencing main fragments. This could be interpreted as a first proof of homologous insulin binding regions of the insulin and the IGF-I receptor. 8. The native insulin receptor and the chimeric receptor IGF-I/IRC1,3 were labelled with B29-Asa-Insulin in a larger scale. After tryptic digestion insulin binding fragments were separated via HPLC. Sequencing of the binding fragment of the native insulin receptor let to sequence (84-102 of the (-subunit of the insulin receptor. Sequencing of the insulin binding region of the chimeric recpetor IGF-IR/IR C1,3 let to exactly the same receptor region. 9. The demonstration of the bivalence for insulin of the four receptors succeded via photoaffinity labelling with two different photoreactive insulin derivatives. After labelling the receptors with both insulin analogues the receptors were purified via affinity chromatography with immobilized streptavidin and detected via autoradiography. The biotin residue could be additionally detected via ECL.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2002

Author dc:creator

• Engels-Lange, Ulrike

Contributors dc:contributor

• Höcker, Hartwig

• info:eu-repo/classification/ddc/610
• Medizin
• Insulin
• IGF-I
• Rezeptor

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123180%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61522