Substitution of the insulin receptor transmembrane domain with the c-neu/erbB2 transmembrane domain constitutively activates the insulin receptor kinase in vitro.
To examine the role of the transmembrane domain (TM) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a Val938----Asp substitution (IR/TMv----D), 2) insertion of a 3-amino acid repeat (Val938-Phe939-Leu940) (IR/TM+3), or the entire TM was replaced by the corresponding domain of either the 3) platelet-derived growth factor (PDGF) receptor (IR/TMPDGFR) or 4) c-neu/erbB2 proto-oncogene product (IR/TMc-neu). Each mutant receptor was stably expressed in Chinese hamster ovary cells, assessed by fluorescence-activated cell sorting, insulin binding, and biosynthetic labeling. All mutant receptors exhibited normal affinity for insulin. Pulse-chase experiments showed that each proreceptor was processed into alpha- and beta-subunits, although the rate of IR/TMV----D conversion was reduced approximately 3-fold. With IR/TMPDGFR, IR/TMV----D, and IR/TM+3 basal and insulin-stimulated levels of autophosphorylation and tyrosine kinase activation were normal, both in wheat germ agglutinin (WGA)-purified receptor preparations and intact cells. By contrast, following WGA purification or isolation of crude membranes, IR/TMc-neu was a constitutively active autokinase and substrate kinase in vitro. However, in intact cells insulin-stimulated autophosphorylation and kinase activity appeared normal. We conclude that although there is considerable latitude in acceptable structure, residues within the insulin receptor transmembrane domain can play a functional role in regulation of insulin receptor tyrosine kinase activity.
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215.
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K
Yamada
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Goncalves
C R
CR
Kahn
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SE
Shoelson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D004247:DNA; D007328:Insulin; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011972:Receptor, Insulin