INTRACELLULAR SITES OF PROTHYROTROPIN-RELEASING HORMONE PROCESSING

Post-translational processing of proteins plays a key role in regulati ng their subcellular localization, enzymatic activity, and protein-pro tein interactions by such diverse mechanisms as phosphorylation, glyco sylation, and proteolytic cleavage, The prothyrotropin-releasing hormo ne (pro-TRH) precursor (26 kDa) undergoes proteolytic cleavage at eith er 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 prepr o-TRH cDNA, we have previously reported that this initial set of cleav ages 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 compartm ent where this initial cleavage takes place as well as to determine th e sites of processing of the intermediates produced, Our strategy was to block the transport of pro-TRH or its intermediates from one subcel lular compartment to the next and to assay for the accumulation of int ermediates, presumably because their processing occurs in a post-block ade compartment. Radiolabeling experiments in AtT20 cells in the prese nce of the drug brefeldin A, which blocks transport from the endoplasm ic reticulum to the Golgi complex, led to an accumulation of the 26-kD a precursor, suggesting a post-endoplasmic reticulum site of processin g, When Golgi complex-to-secretory granule transport was blocked at 20 degrees C, the processing of the 26-kDa precursor was not affected, s uggesting a Golgi complex site of processing, At this temperature, the 15-kDa N-terminal intermediate accumulated, suggesting a post-Golgi c omplex processing site, while the 16.5-kDa C-terminal intermediate was processed in the Golgi complex to produce a 5.4-kDa peptide.