The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.
The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.
Wilden
P A
PA
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
Siddle
K
K
Haring
E
E
Backer
J M
JM
White
M F
MF
Kahn
C R
CR
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D000906:Antibodies; D007328:Insulin; D014443:Tyrosine; D006003:Glycogen; D004247:DNA; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D005947:Glucose; D013936:Thymidine