Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides

Abstract

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the transGolgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.All of the peptides derived from pro-TRH 2 are targeted to the regulated secretory pathway of neuroendocrine cells (1, 2), but it is not completely clear how pro-TRH and other prohormones are targeted to secretory granules from the trans-Golgi network (TGN). Proteins and peptides targeted to the regulated secretory pathway are ultimately stored in secretory granules until the cell receives a stimulus signaling for granule content release (3). Proteins that are secreted without storage in a cellular compartment are said to transit through the constitutive or constitutive-like secretory pathway (4, 5). Scientific debate continues as to what targets prohormones and other proteins into the regulated secretory pathway with some favoring a receptor-mediated model (6 -8), self-aggregation of regulated secretory pathway cargo (9 -11), or binding of the prohormone to lipid rafts (12-14), but it is generally agreed that there are unique structural elements of prohormones, not necessarily universal, that result in their targeting to the regulated secretory pathway (15)(16)(17)(18)(19)(20)(21)(22). It has yet to be reported which elements of pro-TRH might be important in the targeting of this important prohormone to the regulated secretory pathway.Rat pro-TRH is a prohormone composed of 255 amino acids, and when fully processed...

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

“…Pulse-Chase Experiments-The experimental conditions for pulse-chase experiments in transfected AtT20 cells with preproTRH cDNA was fully described in our earlier studies (20). In this study, to analyze the prohormone (26 kDa) maturation, AtT20 cells transfected with either wild type preproTRH or ⌬PGL and PGL/GGG mutants were preincubated 30 min with media lacking leucine.…”

“…Cell Culture and Transient Transfection-AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 mM sodium bicarbonate, 1 mM sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37°C and 5% CO 2 as previously described (20). Rat pituitary GH4C1 cell line was grown in the same media as AtT20 cells.…”

“…The tubes were centrifuged for 30 s, and the supernatants were fractionated with SDS-PAGE gels as described in the pulse-chase experiments section below. Levels of in vitro protein translated were determined by radioactivity counting (cpm) in a ␤ counter (Beckman Counter, Fullerton, CA) and corrected as previously described (20).…”

“…Processing of proTRH generates several peptides, including the N-terminal peptide pEH24, the C-terminal 5.4-kDa fragment, and TRH, which can be readily quantified by radioimmunoassay (20). We measured the amount of these peptides for the mutants listed in Table 1 in three different fractions: basal media, stimulated (with BaCl 2 ) media, and cell content.…”

“…Extensive research has been done during the last two decades to understand the post-translational processing of proTRH (11)(12)(13), the convertases responsible for pro-TRH cleavage (14 -17) the intracellular sites of proTRH processing (18,19). More recently the importance of the initial cleavage for proTRH sorting (20), and the implications of the C-terminal disulfide bond in this process (21) has been uncovered. However, the maturation process of the proTRH precursor in ER, and the structural features involved in intracellular traffic from ER to the Golgi has yet to be elucidated.…”