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Deletion of V335 from the L2 Domain of the Insulin Receptor Results in a Conformationally Abnormal Receptor That Is Unable to Bind Insulin and Causes Donohue’s Syndrome in a Human Subject

Department of Clinical Biochemistry, Addenbrooke’s Hospital (S.G., M.A.S., K.S., S.O.), Cambridge, United Kingdom CB2 2QQ; Departments of Pediatrics (A.J., H.M.) and Clinical Biochemistry (S.G.), Glostrup University Hospital, DK-2600 Copenhagen, Denmark; Incyte Genomics Ltd. (V.S.), Cambridge, United Kingdom CB2 1FF; and Clinical Genetics, Novo Nordisk A/S (L.H.), DK-2880 Bagsvaerd, Denmark

Address all correspondence and requests for reprints to: Prof. S. O’Rahilly, Department of Clinical Biochemistry, Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom CB2 2QQ. E-mail: sorahill{at}hgmp.mrc.ac.uk.

An infant with Donohue’s syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the V335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the V335 receptor was capable of being immunoprecipitated with antibodies directed against the ß-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the -subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor -subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two ß-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.