Effects of amino acid replacements within the tetrabasic cleavage site on the processing of the human insulin receptor precursor expressed in Chinese hamster ovary cells.
We have studied the specificity requirements for processing of the human insulin proreceptor by successively replacing each basic amino acid in the tetrabasic cleavage site with alanine. These mutated receptor cDNAs have then been overexpressed in Chinese hamster ovary cells, using vectors containing the mouse dihydrofolate reductase gene to amplify the transfected cDNAs in the presence of increasing concentrations of methotrexate. High levels of expression, ranging up to 6 x 10(7) receptors/cell were achieved in these experiments. Replacement of the P1 arginine with alanine led to the complete suppression of processing, as occurs also in a naturally occurring serine mutation at this site (Yoshimasa, Y., Seino, S., Whittaker, J., Kakehi, T., Kosaki, A., Kuzuya, H., Imura, H., Bell, G. I., and Steiner, D. F. (1988) Science 240, 783-787). A small amount of cleavage at alternative sites was detected. Replacement of the P2 arginine or P3 lysine with alanine did not in either case affect conversion to mature alpha and beta subunits, while replacement of the P4 arginine significantly inhibited processing. The binding isotherms for the processed versions of the receptor were comparable to previously published normal values. The unprocessed proreceptor bound insulin normally but was autophosphorylated less efficiently than processed versions of the receptor expressed in the same cells. These results suggest that a single processing protease with trypsin-like specificity may be involved in processing both insulin and insulin-like growth factor-I receptor precursors as well as a variety of viral envelope glycoprotein precursors.
Yoshimasa
Y
Y
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Paul
J I
JI
Whittaker
J
J
Steiner
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010053:Ovary; D010766:Phosphorylation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011972:Receptor, Insulin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
D007328:Insulin; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D013762:Tetrahydrofolate Dehydrogenase; D011972:Receptor, Insulin