The human insulin receptor cDNA: a new tool to study the function of this receptor.
The human insulin receptor (hIR) is an integral transmembrane glycoprotein comprised of two alpha and two beta subunits. An immediate consequence of insulin binding to the extracellular alpha subunit is the autophosphorylation of tyrosine residues on the intracellular domain of the beta subunit. The placental hIR cDNA has been cloned and sequenced, providing the primary structural features of the protein. In order to investigate the functions of the beta subunit and particularly the role of autophosphorylation and tyrosine phosphokinase (TPK) activity (a feature shared by other receptors and oncogene proteins) in transmembrane signalling, we designed an expression system of the hIR cDNA in eucaryotic cells. Superexpressing CHO cell lines that contain about 10(6) functional hIR/cell have been developed. In these cells half maximum stimulation of glucose uptake occurs at 5 X 10(-10)M insulin, whereas normal CHO cells require 5 X 10(-12)M insulin. In this expression system we have carried out site-directed mutagenesis experiments in which domains of the molecule have been deleted or particular amino acids have been replaced by others. The replacement of either or both the tyrosine residues 1162 and 1163 compromise an autophosphorylated site that is important for kinase function and the insulin response. Expression of an isolated membrane-bound form of the beta-subunit produces a 6 fold increase in glucose uptake. This insulin-independent effect disappears if the twin tyrosines are mutated or if the beta subunit is expressed in the cytoplasm. These studies also show that the C terminal 112 amino acid portion of the beta subunit is important for the stability of this protein.
Clauser
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Ellis
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Morgan
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D
Edery
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Roth
R A
RA
Rutter
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D010053:Ovary; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D011994:Recombinant Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin