Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

Abstract

Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.

A Cleavable Propeptide InfluencesToxoplasmaInfection by Facilitating the Trafficking and Secretion of the TgMIC2–M2AP Invasion Complex

“…It is also possible that a subpopulation of the labeled vesicles contains the excess pool of proTgM2AP trafficking through dense granules or in transport vesicles for recycling back to endosomes for redirection into an alternative pathway for constitutive secretion. This would be similar to the constitutive-like secretion of proinsulin, which occurs when a small fraction of the hormone enters clathrin-coated vesicles budding from ISGs (Halban, 1994;Kuliawat et al, 2000;Molinete et al, 2000). This material is subsequently delivered to early endosomes before being secreted in a nonstimulus-dependent manner.…”

Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

“…GH4C1 is a rat pituitary cell line that lacks prohormone convertases PC1/3 and PC2. It has secretory granules but with low efficiency sorts and stores endogenously and exogenously expressed proteins to the regulated secretory pathway (23). These properties make this cell line a good model to study the importance of PGL motif within the N-terminal proregion independently of processing by PC1/3 and PC2.…”

Evidence of a Novel Dipeptidyl Aminopeptidase in Mammalian GH3 Cells: New Insights into the Processing of Peptide Hormone Precursors

“…Protein secretion from mammalian cells can be either constitutive or regulated. It has been generally assumed that while the constitutive secretory pathway is present in all eukaryotic cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine or exocrine cells (Kuliawat et al, 2000). Several characteristics serve to distinguish these two pathways.…”