Functional Analysis of PRAF1 and Its Effect on Corticotrophic ACTH Secretion
Type of DegreeDissertation
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Prenylated Rab acceptor domain family member 1 (PRAF1), a transmembrane Golgi apparatus protein, interacts with Dexras1, a corticotrophic protein that may play a role in glucocorticoid-mediated inhibition of ACTH secretion. The purpose of this study was to evaluate the effect of mutating PRAF1 on Dexras1 interaction, PRAF1 and ACTH localization, cell morphology, ACTH secretion, and processing and transcription of POMC, the precursor of ACTH. PCR and site-directed mutagenesis were used to generate the PRAF1 mutations PRAF(54-175), PRAF(54-112), PRAF(N70T), PRAF(Y73A), and PRAF(V161A). Yeast two-hybrid assay was used to assess Dexras1/PRAF1 mutant interaction. PRAF1(54-112) did not interact with Dexras1, however PRAF(54-175) did.1 The Dexras1 interaction was enhanced in the 3 point mutated proteins, PRAF(N70T), PRAF(Y73A), and PRAF(V161A). AtT-20 cells were stably transfected with either empty vector (3X) or an expression vector containing mutated or full-length (FL) PRAF1. Basal secretion, CRH-stimulated secretion and the effect of dexamethasone (Dex) on stimulated secretion were studied in wild type (WT) and transfected cells. In WT, and cells stably transfected with 3X- or FL-containing vectors, CRH increased ACTH secretion compared to basal, and Dex inhibited this response. The response to CRH and Dex was significantly altered in all cell lines stably transfected with a PRAF1 mutant construct. Confocal microscopy revealed that PRAF1 and ACTH localization was altered in all cell lines stably transfected with a PRAF1 mutant construct. Further, the Golgi complex morphology was altered in 2 cell lines. Stable transfection of AtT-20 cells with FL or a PRAF1 mutant increased POMC expression, which was not reflected by an increase in ACTH-containing peptide. However, the ratio of ACTH/pre-ACTH and ACTH/POMC was decreased in all cells stably transfected with a PRAF1 construct except for PRAF(Y73A). Our results demonstrate that PRAF1 mutations affect PRAF1/Dexras1 interaction, PRAF1 and ACTH localization, Golgi complex morphology, ACTH secretion, and POMC expression and processing. In addition, a PRAF1/Rab3A/VAMP2 interaction may be vital for stimulated secretion of ACTH. Thus, the PRAF1/Dexras1 interaction may play a role in glucocorticoid-mediated inhibition of ACTH secretion in the intermediate time frame.