Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form.
Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form.
Le Marchand-Brustel
Y
Y
Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, Nice, France.
Ballotti
R
R
Grémeaux
T
T
Tanti
J F
JF
Brandenburg
D
D
Van Obberghen
E
E
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System; D006801:Humans; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D010766:Phosphorylation; D011865:Radioisotope Dilution Technique; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D011972:Receptor, Insulin