Sequence requirements for prohormone processing in mouse pituitary AtT-20 cells. Analysis using prorenins as model substrates.
Although cleavage of peptides at sites marked by paired basic amino acids is a common feature of prohormone processing, little is known about the properties of endoprotease(s) responsible for cleavage of the precursor. To examine the cleavage specificity of a processing endoprotease, we have altered the Lys-Arg cleavage site of human prorenin to Arg-Arg, Lys-Lys and Arg-Lys by site-directed mutagenesis, and expressed the native and mutated precursors in mouse pituitary AtT-20 cells which are known to process foreign prohormones, including prorenin, at paired basic sites during the regulated secretory process. All native and mutated human prorenins were sorted into the regulated secretory pathway. The mutated precursor with Arg-Arg instead of the Lys-Arg native pair was processed at about half the efficiency of the native one, while the Lys-Lys and Arg-Lys mutants were not processed. Rat prorenin, which naturally has a Lys-Lys pair, was not processed in the cells. In addition, mouse Ren2 prorenin, which has a Ser residue next to the Lys-Arg pair, but not mouse Ren1 prorenin, which has a Pro residue next to the pair, was processed. These results suggest that the Arg residue at the COOH side of the basic pair is essential for cleavage of prorenins by a processing enzyme during the regulated secretory process in AtT-20 cells, although the NH2-side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg-Pro peptide bond.
Nagahama
M
M
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Nakayama
K
K
Murakami
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010911:Pituitary Neoplasms; D011499:Protein Processing, Post-Translational; D051381:Rats; D012083:Renin / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014162:Transfection
D004792:Enzyme Precursors; D010450:Endopeptidases; D012083:Renin