Peptide-based radioimmunoassay for the two 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 the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding 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 reproducible, 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-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the 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).
Sesti
G
G
Dipartimento di Medicina Interna, Università di Roma-Tor Vergata, Italy.
D'Alfonso
R
R
Vargas Punti
M D
MD
Frittitta
L
L
Trischitta
V
V
Liu
Y Y
YY
Borboni
P
P
Longhi
R
R
Montemurro
A
A
Lauro
R
R
D016475:3T3 Cells; D000273:Adipose Tissue / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001667:Binding, Competitive; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D005091:Exons; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000378:metabolism*; D009765:Obesity / Q000378:metabolism; D009928:Organ Specificity; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011863:Radioimmunoassay / Q000379:methods; D011972:Receptor, Insulin / Q000032:analysis* / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D012016:Reference Values; D012044:Regression Analysis; D014162:Transfection
D000906:Antibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011972:Receptor, Insulin