Insulin receptor autophosphorylation occurs asymmetrically.
The unoccupied insulin receptor is a structurally symmetric, disulfide-linked dimer, comprising two alpha beta halves, each with a potential insulin binding alpha subunit and a kinase active beta subunit. In the accompanying paper (Shoelson, S. E., Lee, J., Lynch, C. S., Backer, J. M., and Pilch, P. F. (1993) J. Biol. Chem. 268, 4085-4091), we described the utility of a novel insulin analogue, L-benzoylphenylalanineB25,B29 epsilon-biotin insulin (BBpa insulin)1 as a probe for receptor behavior, and we determined that binding and cross-linking of one BBpa insulin molecule could fully stimulate insulin receptor autophosphorylation. Here we use this analogue to determine the symmetry of the autophosphorylation reaction. The alpha beta half-receptor that does not covalently couple to BBpa insulin incorporates 50% more orthophosphate than the alpha beta half that becomes coupled to the insulin analogue. Phosphopeptide mapping of each receptor half shows minimal differences in the phosphorylation sites or their relative contribution to the phosphate content of each half. The kinetics of 32P incorporation into each receptor half are essentially identical over a 10-20-min time course. Phosphopeptide mapping analysis reveals that the phosphate incorporation patterns do not change between the two alpha beta half-receptor forms (BBpa insulin-linked and unlinked, respectively) at different time points or concentrations of ATP ranging from 12 to 200 microM. Based on these and other data, we propose a model of insulin receptor activation whereby binding of one insulin molecule can trigger autophosphorylation in an asymmetric fashion.
Lee
J
J
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
O'Hare
T
T
Pilch
P F
PF
Shoelson
S E
SE
D016475:3T3 Cells; D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D007328:Insulin / Q000031:analogs & derivatives; D051379:Mice; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*
D000345:Affinity Labels; C079861:B29-biotin insulin; D007328:Insulin; D010748:Phosphopeptides; D001710:Biotin; D011972:Receptor, Insulin