A protease processing site is essential for prorenin sorting to the regulated secretory pathway.

Transfected mouse pituitary AtT-20 cells were used to examine the sorting of human prorenin to dense core secretory granules and the regulated secretory pathway. These cells secrete prorenin constitutively and sort a portion of the prorenin to secretory granules, where it is converted to active renin by proteolytic processing. Pulse-chase labeling of transfected AtT-20 cells demonstrated that regulated secretion of prorenin was prevented by: 1) the mutagenic deletion of the prosegment, 2) the premature proteolytic removal of the prosegment by a Golgi-resident processing protease, or 3) the mutation of the native cleavage site so as to prevent removal of the prosegment. In addition, expression of fusion proteins containing portions of the prorenin prosegment demonstrated that exposure of potential proteolytic cleavage sites was sufficient to confer cleavage-dependent regulated secretion of the corresponding protein. These data implicate the protease cleavage event in the regulated secretion of prorenin and are consistent with the involvement of a subclass of processing proteases in the sorting of certain proteins to secretory granules in AtT-20 cells.

Molecular determinants of human prorenin processing.

In humans, active renin is generated by the removal of a 43-amino acid prosegment from the zymogen prorenin. This cleavage event is highly specific, occurring at only one of the seven pairs of basic amino acids in the body of preprorenin. This cleavage site selectivity is also displayed by a number of other proteases in vitro and in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector, suggesting that specificity of cleavage is directed in part by the primary sequence, the higher order structure, or both of prorenin itself. To test this hypothesis, single amino acid mutations were introduced in the region of human preprorenin surrounding the natural cleavage site, and the resultant recombinant proteins were expressed in cultured Chinese hamster ovary and AtT-20 cells. The results suggest that amino acids in addition to the pair of basic amino acids surrounding the cleavage site affect the ability of both trypsin and the endogenous AtT-20 processing enzyme to cleave prorenin. Notably, although a proline at position -4 is essential for processing of prorenin in AtT-20 cells and is correlated with predicted formation of a beta-turn at this position, site-directed mutations suggest that this structural feature in addition to a pair of basic amino acids is not sufficient to lead to proteolytic activation of prorenin. Displacement of sequences surrounding the cleavage site to a position 10 amino acids toward the amino terminus led to partial processing of a mutated prorenin.(ABSTRACT TRUNCATED AT 250 WORDS)

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin.

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.

The molecular biology of human renin and its gene.

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.