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

Abstract

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.

Two Activation States of the Prohormone Convertase PC1 in the Secretory Pathway

“…Using AtT-20 cells transfected with human prorenin, we have previously shown that the processing site for a granulespecific endoprotease between the profragment and the renin molecule seems necessary for appropriate prorenin sorting to the regulated secretory pathway (24). This result suggests that prorenin can be driven in the regulated secretory pathway by binding to the active site of the processing enzyme prior to sorting.…”

“…Following a 2-h pulselabeling period with radioactive methionine, the cells were chased by incubation in complete medium for 15, 30, or 60 min. Cell supernatants were immunoprecipitated with an anti-renin antibody and submitted to SDS-PAGE (24). As shown in Fig.…”

“…Sixty h after transfection, the cells were depleted of methionine for 1 h, labeled with 300 Ci of [ 35 S]methionine/well (pulse), and incubated in complete medium for 15, 30, or 60 min (chase). Culture supernatants were immunoprecipitated with protein G-agarose-coupled anti-human renin antibody as described previously (24). Immunoprecipitated proteins were fractionated by SDS-PAGE (10% acrylamide gel) and gels were subjected to fluorography.…”

“…In all cases, the control vector consisted of an analogous expression vector, which coded for an inert portion of the mouse immunoglobulin heavy chain constant region (24). Twenty-four h after transfection, the cells were glycerol shocked.…”

The Role of Dibasic Residues in Prohormone Sorting to the Regulated Secretory Pathway

“…Thus, although initial studies suggested that the N-terminal domain of prosomatostatin may play a role in precursor targeting to the RSP (24), further work indicated that the somatostatin 28-containing C-terminal region might also be involved in sorting (25) and that a single mutation of the Arg-Lys dibasic that is normally processed to yield somatostatin 14 prevented prosomatostatin from entering the RSP (26). Similarly, following initial studies suggesting that in prorenin no signal was present in the prodomain that may be involved in its routing to the RSP (27-30), a more recent work showed that mutation of the dibasic that is normally processed in prorenin to produce renin prevented sorting of the protein to the RSP (31).…”