Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (∆6) and 16+295+337+418+730+743+881 (∆7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (∆7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of ∆7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of ∆6 and ∆7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The ∆6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.
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May 2000
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Research Article|
April 25 2000
Mutational analysis of the N-linked glycosylation sites of the human insulin receptor
Thomas C. ELLEMAN;
Thomas C. ELLEMAN
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Maurice J. FRENKEL;
Maurice J. FRENKEL
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Peter A. HOYNE;
Peter A. HOYNE
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Neil M. MCKERN;
Neil M. MCKERN
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Leah COSGROVE;
Leah COSGROVE
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Dean R. HEWISH;
Dean R. HEWISH
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Kim M. JACHNO;
Kim M. JACHNO
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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John D. BENTLEY;
John D. BENTLEY
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Sonia E. SANKOVICH;
Sonia E. SANKOVICH
1Commonwealth Scientific and Industrial Research Organisation, Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Colin W. WARD
Colin W. WARD
1
1To whom correspondence should be addressed (e-mail colin.ward@;hsn.csiro.au).
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Biochem J (2000) 347 (3): 771–779.
Article history
Received:
December 06 1999
Revision Received:
February 07 2000
Accepted:
February 29 2000
Citation
Thomas C. ELLEMAN, Maurice J. FRENKEL, Peter A. HOYNE, Neil M. MCKERN, Leah COSGROVE, Dean R. HEWISH, Kim M. JACHNO, John D. BENTLEY, Sonia E. SANKOVICH, Colin W. WARD; Mutational analysis of the N-linked glycosylation sites of the human insulin receptor. Biochem J 1 May 2000; 347 (3): 771–779. doi: https://doi.org/10.1042/bj3470771
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