Sequence requirements for prohormone processing in mouse pituitary AtT-20 cells. Analysis using prorenins as model...

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Prohormone processing involves peptide cleavage at paired basic amino acid sites by endoproteases.
• The specific properties and cleavage specificities of these processing endoproteases remain largely unknown.

Purpose of the Study:

• To investigate the cleavage specificity of endoproteases involved in prohormone processing.
• To determine the role of specific amino acid residues within paired basic sites in prorenin cleavage.

Main Methods:

• Site-directed mutagenesis was used to alter the Lys-Arg cleavage site of human prorenin to Arg-Arg, Lys-Lys, and Arg-Lys.
• Native and mutated human prorenins were expressed in mouse pituitary AtT-20 cells, known for processing foreign prohormones.
• Processing efficiency of native and mutated prorenins was assessed within the regulated secretory pathway.

Main Results:

• All prorenin variants were correctly sorted into the regulated secretory pathway.
• The Arg-Arg mutant showed reduced processing efficiency compared to native Lys-Arg.
• Lys-Lys and Arg-Lys mutants, as well as native rat prorenin (Lys-Lys), were not processed.
• Mouse Ren2 prorenin (with Ser next to Lys-Arg) was processed, but mouse Ren1 prorenin (with Pro next to Lys-Arg) was not.

Conclusions:

• The arginine residue at the COOH side of the basic pair is essential for prorenin cleavage by processing enzymes in AtT-20 cells.
• The NH2-side lysine residue also contributes to efficient cleavage.
• The processing enzyme is unable to cleave the Arg-Pro peptide bond, highlighting sequence-specific constraints.