Identification of the intact insulin receptor using a sequence-specific antibody directed against the C-terminus of the beta-subunit.
An antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal amino acids of the human insulin receptor (Anti-R beta C). Immunoprecipitation of the human insulin receptor and immunoblotting to the beta-subunit by Anti-R beta C could be inhibited by competition with the corresponding peptide. However, even at saturating concentrations, anti-R beta C could not completely immunoprecipitate or immunodeplete insulin receptors compared to a human autoantibody (anti-R B2). Using receptor labeled directly by 125I, evidence of multiple forms of the beta-subunit was found. When the receptor could be immunoprecipitated by anti-R beta C, the beta-subunit migrated with an apparent mol wt (MW) of 96,000 (at or above the phosphorylase b MW marker). However, in preparations where anti-R beta C was not able to immunoprecipitate the insulin receptor, the beta-subunit migrated at a significantly lower MW of 91,000 (below phosphorylase b), as detected by immunoprecipitation with Anti-R B2. Intermediate forms could also be detected. Phosphorylation of partially purified insulin receptor did not affect is ability to be immunoprecipitated by anti-R beta C, although insulin-stimulated phosphorylation increased the apparent MW of the beta-subunit. However, insulin receptor that was phosphorylated in solubilized extracts of whole cells had a beta-subunit that migrated at lower MW and was not immunoprecipitated by anti-R beta C. One possible explanation for this is that the beta-subunit may be degraded during preparation. When the MW of insulin receptor that has been purified to homogeneity from human placenta is compared to our data, it is clear that many of these insulin receptor preparations contain lower MW beta-subunits. These results must be taken into account when the sites of phosphorylation and kinase activity of purified insulin receptor preparations are studied.
Grunfeld
C
C
Shigenaga
J K
JK
Huang
B J
BJ
Fujita-Yamaguchi
Y
Y
McFarland
K C
KC
Burnier
J
J
Ramachandran
J
J
D000595:Amino Acid Sequence; D000906:Antibodies; D000918:Antibody Specificity; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000032:analysis*
D000906:Antibodies; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011972:Receptor, Insulin