PEPTIDE-BASED RADIOIMMUNOASSAY FOR THE 2 ISOFORMS OF THE HUMAN INSULIN-RECEPTOR

The insulin receptor exists in two isoforms differing by the absence ( HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of t he alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms em ploying antibodies raised against two peptides, one (Pep-12) correspon ding to residues encoded by exon 11, and the other (Pep-13) correspond ing to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproduci ble, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-1 2 or Pep-13 as standards. The radioimmunoassay is applicable to the me asurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to m easure the relative abundance of the two isoforms in fat and muscle fr om normal, obese non-diabetic and non-insulin-dependent diabetic (NIDD M) subjects. Results demonstrate that expression of the low-affinity H IR-B form is significantly increased in obese and NIDDM subjects compa red with control subjects. In addition, the increased expression of th e HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001 ).