Signal-mediated sorting of neuropeptides and prohormones: secretory granule biogenesis revisited.

Neuropeptides and hormones, in contrast to constitutive secretory proteins, are sorted to and stored in secretory granules and released upon a stimulus. During the last two decades, signals and mechanisms involved in their sorting to the regulated pathway of protein secretion have been addressed in numerous studies. Taken together these studies revealed three important features of regulated secretory proteins: aggregation, sorting signal motifs and membrane binding. Here we try to dissect the sorting process with regard to these features and discuss their relevance in the context of current sorting models. We especially address the question where in the secretory pathway sorting takes place and discuss a possible role of sorting receptors.

Signal-mediated sorting to the regulated pathway of protein secretion.

The existence of specific sorting signals which direct regulated secretory proteins to secretory granules (SGs) was hypothesized two decades ago and since then has been addressed in numerous studies. The discovery that aggregation of regulated secretory proteins is involved in their sorting to SGs questioned the existence of specific sorting signals. In this short review we summarize the identification of a specific sorting signal for chromogranin B (CgB), a regulated secretory protein which undergoes Ca2+/pH-dependent aggregation. This signal is represented by the N-terminal disulfide-bonded loop of CgB encoded by exon 3 and is necessary to direct CgB to SGs. Its essential role was revealed only by the expression of a loopless deletion mutant in the absence of endogenous protein synthesis to preclude aggregative sorting of the former with the latter. The signal is also sufficient to direct a reporter protein to SGs, but only its multiple presence on the reporter leads to high sorting efficiency. Importantly, the identified signal functions at the level of the TGN by binding to membrane components that give rise to SGs. Furthermore, these studies lead to further insights into the mechanism of sorting. First, conclusive evidence is provided that regulated secretory proteins lacking a specific signal, can be sorted via coaggregation with proteins containing a specific sorting signal. Second, the data support an additional function of aggregation in the TGN which is multimerization of sorting signals per sorting unit leading to highly efficient sorting to SGs.

The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules.

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.

Essential role of the disulfide-bonded loop of chromogranin B for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis.

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide- bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Deltacys-hCgB) lacking the 22-amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Deltacys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Deltacys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus-mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.

Targeting of green fluorescent protein to neuroendocrine secretory granules: a new tool for real time studies of regulated protein secretion.

Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.