Expression of pro-Muclin in pancreatic AR42J cells induces functional regulated secretory granules

Abstract

It is not clear how protein cargo is sorted to and retained in forming regulated secretory granules (RSG). Here, the sulfated mucin-type glycoprotein pro-Muclin was tested for its ability to induce RSG in the poorly differentiated rat pancreatic cell line AR42J. AR42J cells express RSG content proteins, but they fail to make granules. Adenovirus-pro-Muclin-infected AR42J cells store amylase, accumulate RSG, and respond to hormonal stimulation by secreting the stored protein. Expression of pro-Muclin combined with the inducing effect of dexamethasone resulted in a significant enhancement of the efficiency of regulated secretion. The effect of pro-Muclin was a strong decrease in constitutive secretion compared with dexamethasone-induction alone. A pro-Muclin construct missing the cytosolic tail domain was less effective at improving the efficiency of regulated secretion compared with the full-length construct. Increased expression of cargo (using adenovirus amylase) also modestly enhanced regulated secretion, indicating that part of pro-Muclin's effect may be due to increased expression of cargo protein. Overall, the data show that pro-Muclin acts as a sorting receptor that can induce RSG, and that its cytosolic tail is important in this process.

Effects of Muclin (Dmbt1) deficiency on the gastrointestinal system

“…We suggested that Muclin is important in protein sorting and packaging in the regulated secretory pathway of the acinar cell (4,9). We also showed that when Muclin was expressed in AR42J cells that lack the regulated pathway, the cells developed granules whose secretion could be stimulated by caerulein (11).…”

“…We have previously shown that ectopic expression of Muclin in the rat pancreatic exocrine cell line AR42J induces functional regulated secretory granules (11). Along with the decrease in stimulated amylase release from Muclin-deficient acini, this indicates an important role of Muclin in the formation of zymogen granules.…”

“…A closer examination of protein trafficking in the regulated secretory pathway shows that the rate at which newly made secretory proteins are transported to a stimulus-releasable pool is significantly retarded. We have proposed a model for the role of Muclin in the regulated secretory pathway (4,9,11). Briefly, Muclin is synthesized on the rough endoplasmic reticulum as a type I membrane protein.…”

“…Among the many proposed functions for the products of the gene, the most investigated functions are as a tumor suppressor (37) and/or a regulator of epithelial functional differentiation (53), as a component of the innate immune defenses (46), and as a Golgi-sorting receptor in the regulated secretory pathway (11).…”

“…The most direct evidence for the role of Muclin in granule formation came from experiments where Muclin was ectopically expressed in the poorly differentiated rat pancreatic exocrine cell line AR42J, which lacks the regulated secretory pathway. When Muclin was expressed in AR42J cells, they acquired regulated secretion: they developed functional granules whose exocytosis could be stimulated by the secretagogue caerulein (a cholecystokinin analog) (11).…”