Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells

Abstract

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein ␣ 1 -protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30 -70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas ␣ 1 -protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.

Dominant pro-vasopressin mutants that cause diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic reticulum

“…When wild-type pro-vasopressin and other regulated secretory proteins (e.g. secretogranin II) were co-expressed in COS-1 cells, they colocalized in granule-like structures (Beuret et al, 2004). This was also the case for the P7L mutant (Fig.…”

“…COS cells are particularly suitable for visualizing intracellular organelles and aggregations due to their flat cell body and large cytoplasm. As previously shown (Beuret et al, 2004), expression of regulated secretory proteins such as granins and prohormones, including pro-vasopressin, is sufficient to generate granule-like structures in a variety of nonendocrine cell lines due to aggregation in the trans-Golgi. Accordingly, wild-type precursor was concentrated in granular accumulations in the cell periphery of about half the expressing cells (Fig.…”

“…4C for C28Y). When COS-1 cells expressing pro-vasopressin were co-stained with antibodies against NPII and against BAP31 (B-cell receptorassociated protein 31; an endogenous ER membrane protein), the granule-like structures of P7L [like those of the wild type (Beuret et al, 2004)] were not positive for BAP31 (Fig. 4B).…”

Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

“…As an alternative to specific on/off switches for secretory granule biogenesis, it is possible that a "cargo effect" is involved in granule biogenesis (Beuret et al, 2004). According to this model, high levels of secretory proteins in the trans-Golgi network induce the formation of protein complexes that become membrane-enclosed, thus forming the secretory granules.…”

Modulation of Secretory Granule-targeting Efficiency by Cis and Trans Compounding of Sorting Signals

“…A direct correlation between the ability to aggregate in vitro and to be sorted to secretory granules in transfected cells has been established for chromogranin A (9) and rat pro-atrial natriuretic factor (10). Indeed, expression of a variety of granule cargo proteins including chromogranin A (11,12), provasopressin, prooxytocin, proopiomelanocortin, secretogranin II, and chromogranin B (12) is sufficient to induce aggregatecontaining cytoplasmic vesicles, although these vesicles do not have all of the characteristics of secretory granules (12). Proinsulin, proopiomelanocortin, and engineered proteins containing the C-terminal tail of PC1/3 also form multimers; however, this property alone is insufficient to direct granule sorting (3,13).…”