Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.
Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.
Tsai
A Y
AY
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Itoh
M
M
Streuli
M
M
Thai
T
T
Saito
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006358:Hot Temperature; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D017027:Protein Tyrosine Phosphatases
D006898:Hydroxylamines; D008769:Methylnitronitrosoguanidine; D019811:Hydroxylamine; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases