Trafficking of Mutant Carboxypeptidase E to Secretory Granules in a β-Cell Line Derived from Cpefat/Cpefat Mice

Abstract

We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic beta-cell line derived from the Cpe(fat)/Cpe(fat) mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules

“…CPE S202P remained at a low level as a proform because its position is a little higher than that of WT CPE, as reported previously (Berman et al, 2001;Cawley et al, 2003) (Fig. 6A).…”

“…a 100% increase) from NIT-2 and NIT-3 (Varlamov et al, 1997). Although Loh's group further demonstrated a constitutive secretion of prohormones, including proinsulin and proenkephalin, using CPE-deleted Neuro-2a cells (Normant and Loh, 1998), they showed a regulated secretion of CPE S202P and PC2 from NIT-3 cells by glucagon-like polypeptide-1 (Cawley et al, 2003). Further, the gonadotropins LH and FSH were secreted upon stimulation with GnRH from primary-cultured Cpe fat pituitary cells (Srinivasan et al, 2004).…”

Binding of the Golgi Sorting Receptor Muclin to Pancreatic Zymogens through Sulfated O-linked Oligosaccharides

“…2D). To prepare a sensitive probe for these studies, pancreatic acini were incubated with carrier-free [ 35 Fig. 2A).…”

“…Unlabeled Muclin in PrepCell fractions was detected by immuno-dot blot (16) with a Muclin-specific antiserum (14). 35 S-labeled Muclin was detected in the fractions by liquid scintillation counting. The specific activity of […”

“…In brief, 50 g of ZGC in 250 l of the above buffer was adjusted to pH 6.0 or left at pH 8.0, incubated for 15 min at room temperature, and pelleted at 16,000 ϫ g for 15 min. The pellets were analyzed by SDS-PAGE or liquid scintillation counting when 35 S-labeled Muclin was used. For the Muclin overlay assay, isolated ZGC (10 g/lane) were separated on SDS-PAGE under reducing conditions, and transferred to PVDF.…”