ABSTRACT
Since many receptors apparently contain only one or two membrane-spanning segments, their transmembrane topology should be similar. This feature suggests that these receptors share common mechanisms of transmembrane signaling. To test the degree of conservation of signaling properties, a chimeric receptor containing the ligand-binding extracellular domain of the Escherichia coli aspartate chemoreceptor and the cytosolic portion of the human insulin receptor was constructed. This chimeric receptor is active as a tyrosine kinase, and aspartate stimulates its activity. Some interesting differences are noted in the target proteins phosphorylated by the chimera compared to the wild-type insulin receptor. These results indicate that features of the signaling mechanisms used by these diverse receptors are conserved, but that interesting changes in the protein properties are caused by differences in the neighboring domains.
Subject(s)
Escherichia coli/metabolism , Receptor, Insulin/metabolism , Receptors, Amino Acid , Receptors, Cell Surface/metabolism , Signal Transduction , Amino Acid Sequence , Aspartic Acid/metabolism , Cell Membrane/metabolism , Chimera , Escherichia coli/genetics , Humans , Kinetics , Molecular Sequence Data , Phosphorylation , Plasmids , Receptor, Insulin/genetics , Receptors, Cell Surface/geneticsABSTRACT
The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.
Subject(s)
Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor, Insulin/metabolism , Animals , Enzyme Activation , Insulin/analogs & derivatives , Insulin/metabolism , Kinetics , Macromolecular Substances , PhosphorylationABSTRACT
The cheR methyltransferase, known to be necessary for the methyl esterification of receptors involved in chemotaxis, is shown to be essential to the synthesis of S-methyl glutathione from glutathione and S-adenosylmethionine in intact Escherichia coli. S-Methyl glutathione is not, however, found to be essential for chemotaxis. It is suggested that the synthesis of S-methyl glutathione may be due to a "parasitic" reaction of glutathione with S-adenosylmethionine bound to the methyltransferase.
Subject(s)
Escherichia coli/metabolism , Glutathione/analogs & derivatives , Methyltransferases/metabolism , Chemotaxis , Escherichia coli/enzymology , Escherichia coli/physiology , Glutathione/analysis , Glutathione/biosynthesis , Glutathione/metabolism , Methylation , S-Adenosylmethionine/metabolismABSTRACT
The formation of tyrosine from phenylalanine catalyzed by rat liver phenylalanine hydroxylase is coupled to the generation of a 4a-hydroxy adduct from the requisite tetrahydropterin cofactor. As indicated by its circular dichroism (CD) spectrum, the optical activity of the adduct generated from racemic 6-methyltetrahydropterin requires stereoselectivity of the oxygenation. The absolute configuration of this new stereocenter is 4a(S)-hydroxy-6(RS)-methyltetrahydropterin by analogy to the CD spectrum of one of the four stereoisomers of 5-deaza-4a-hydroxy-6-methyltetrahydropterin. The source of the 4a-hydroxy oxygen is O2, as demonstrated by the observation of a 18O-induced 13C shift in the 13C NMR spectrum of the adduct when generated from [4a-13C]-6-methyltetrahydropterin and 18O2.
