Cytoplasmic juxtamembrane region of the insulin receptor: a critical role in ATP binding, endogenous substrate phosphorylation, and insulin-stimulated bioeffects in CHO cells.
We have expressed in CHO cells a mutant receptor (IR delta 960) from which 12 amino acids in the juxtamembrane region (A954-D965), including Tyr960, have been deleted. The mutant receptor bound insulin normally but exhibited an increased Km for ATP during autophosphorylation. Upon prolonged incubation in vitro, or at high ATP concentrations such as those observed in vivo, autophosphorylation of IR delta 960 was similar to wild type, and the in vitro phosphotransferase activity of the autophosphorylated IR delta 960 was normal. These results suggest that the deletion did not cause a nonspecific structural disruption of the catalytic domain of IR delta 960. In vivo autophosphorylation of the IR delta 960 receptor was reduced by 30% after 2 min of insulin stimulation and was similar to the wild-type receptor after 30 min of insulin stimulation. However, the mutant receptor was defective in insulin-stimulated tyrosyl phosphorylation of the endogenous substrate pp185. In addition, IR delta 960 was deficient in mediating insulin stimulation of glycogen and DNA synthesis. Thus, autophosphorylation of the insulin receptor is necessary but not sufficient for signal transmission. These data extend the hypothesis that the cytoplasmic juxtamembrane region of the insulin receptor is important for its interactions with ATP, intracellular substrates, and other proteins and is broadly necessary for biological signal transmission.
Backer
J M
JM
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Schroeder
G G
GG
Cahill
D A
DA
Ullrich
A
A
Siddle
K
K
White
M F
MF
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D010053:Ovary; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin