Intracellular sites of prothyrotropin-releasing hormone processing

Post-translational processing of proteins plays a key role in regulating their subcellular localization, enzymatic activity, and protein-protein interactions by such diverse mechanisms as phosphorylation, glycosylation, and proteolytic cleavage, The prothyrotropin-releasing hormone (pro-TRH) precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating a 15/10-kDa or a 9.5/16.5-kDa N/C-terminal pair of intermediates, Using transfected AtT20 cells encoding a prepro-TRH cDNA, we have previously reported that this initial set of cleavages occurs prior to entry into the secretory granules (Nillni, E. A., Sevarino, K. A., and Jackson, I. M. D. (1993) Endocrinology 132, 1271-1277). In this study, we set out to identify the subcellular compartment where this initial cleavage takes place as well as to determine the sites of processing of the intermediates produced, Our strategy was to block the transport of pro-TRH or its intermediates from one subcellular compartment to the next and to assay for the accumulation of intermediates, presumably because their processing occurs in a post-blockade compartment. Radiolabeling experiments in AtT20 cells in the presence of the drug brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi complex, led to an accumulation of the 26-kDa precursor, suggesting a post-endoplasmic reticulum site of processing, When Golgi complex-to-secretory granule transport was blocked at 20 degrees C, the processing of the 26-kDa precursor was not affected, suggesting a Golgi complex site of processing, At this temperature, the 15-kDa N-terminal intermediate accumulated, suggesting a post-Golgi complex processing site, while the 16.5-kDa C-terminal intermediate was processed in the Golgi complex to produce a 5.4-kDa peptide.