Sequence requirements for prohormone processing in mouse pituitary AtT‐20 cells

Abstract

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 Renl 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 NH,-side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg-Pro peptide bond.In endocrine and neuronal cells, many peptide hormones and neuropeptides are produced from larger, biologically inactive precursors through endoproteolytic cleavage, usually, at paired basic amino acids during intracellular transport [l -41. These cells have two secretory pathways. One is the regulated pathway by which mature hormones are produced by prohormone processing and are selectively targeted to secretory granules where they are stored until release is stimulated. The other is the constitutive pathway by which other proteins are secreted continuously without storage [5 -71. Lines of genetic evidence [8 -121 and studies using synthetic analogs [13 -151 and site-directed mutants [16 -201 of prohormones have suggested that paired basic amino acids are essential for prohormone processing. However, little is known about the specificity of endoprotease responsible for the precursor cleavage; precursor proteins often contain basic pairs which are never cleaved and tissue-specific use of certain pairs is often observed [2,4]. It has been recently reported by surveying various prohormone sequences that there is a hierarchy for the use of certain pairs of basic amino acids at the cleavage sites [17,21] : Lys-Arg (70%); Arg-Arg (1 5 % ) ; Lys-Lys (10%); Arg-Lys (5%). The data suggest that processing enzymes may have a preference for cleavage on the COOH side of Arg residues.Mouse pituitary AtT-20 cells, which endogenously produce adrenocorticotropin, P-endorphin, etc., through cleavage of a precursor, pro-opiomelanocortin, during the regulated secretor...

Role of Amino Acid Sequences Flanking Dibasic Cleavage Sites in Precursor Proteolytic Processing

“…Firstly, both endoproteases did not tolerate, at their subsite S:, the p-carbon branched amino acid residues in accord with the low occurrence frequencies observed in the database for these excluded amino acids. Similar results were obtained by site-directed mutagenesis on some of these excluded amino acids occupying the Pi position at the cleavage sites of pro-albumin [35], pro-renin [37] and pro-factor IX [39] in different cell systems. Secondly, the other [Pro'l, Leu151 analogs, which were best hydrolyzed by the Kex2 enzyme, were cleaved by both dibasic proteases and comparable kinetic values were obtained.…”

“…Both the integrity [16,511 and the accessibility [28][29][30]32, 331 of basic doublets were also shown to play a critical role in these processes. However, a series of observations using site-directed mutagenesis of various prohormones had underlined the particular importance of other amino acid residues around the scissile bond in processing [25,30,34,[36][37][38][39]. Therefore, the aim of the present studies was an attempt to define other parameters which might also participate in the specificity of processing endoproteases.…”

“…They were characterized by one-dimensional and two-dimensional NMR spectroscopy in the case of the processing sequences around the LysllArgl2 doublet of the common precursor for ocytocin and neurophysin [27,281 and around the dibasic sites of human proinsulin [32]. Although both the functional role of these structures in the substrate-binding site for processing enzyme recognition and their implicatiion in processing of many prohormones were only recently shovvn [29,30,331, a series of observations using site-directed mutagenesis of various prohormones had also underlined the particular importance of some amino acid residues other than the dibasic ones in specifying correct processing at the cleavage sites 125, 30,[34][35][36][37][38][39].…”

A Protease Processing Site Is Essential for Prorenin Sorting to the Regulated Secretory Pathway

“…The replacement of Arg by Lys does not change the predicted secondary structure of the putative sorting peptide (data not shown) but should abolish cleavage by potential processing enzymes present in AtT-20 cells (23). As shown in Fig.…”

Renin-1 Is Essential for Normal Renal Juxtaglomerular Cell Granulation and Macula Densa Morphology

“…Thus signals required for sorting renin to the regulated secretory pathway of mouse JG cells in vivo reside exclusively in the renin-1 d protein and not in renin-2. This finding suggests that the trafficking and maturation of renin-2 protein within secretory granules in transfected AtT-20 cells (24,25) may not mirror the situation in the intact mouse, especially given that this cell line displays a different range of prorenin processing activities compared with the JG cells of the kidney (see Ref. 26).…”