Date of Completion

11-25-2014

Embargo Period

11-19-2015

Keywords

AP-1, trafficking, secretory granules, endosomes, PAM, amidation, ATP7A, copper

Major Advisor

Dr. Betty A. Eipper

Associate Advisor

Dr. Richard E. Mains

Associate Advisor

Dr. Stephen M. King

Associate Advisor

Dr. Ann E. Cowan

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Peptides act as chemical messengers to regulate a many physiological functions. First synthesized as inactive precursors, peptides undergo a set of post-translational modifications to gain bioactivity en route through the secretory pathway. Peptides exit the Golgi apparatus in secretory granules (SGs) which are stored until a stimulus triggers their exocytosis. The adaptor protein 1A (AP-1A) is required for SG maturation in neuroendocrine cells and for the formation of specialized SGs such as the glue granules in Drosophila, Weibel-Palade bodies in endothelial cells and rhoptries in Toxoplasma gondii. AP-1A is a heterotetrameric complex (γ/β1/µ1A/σ1) which interacts with cargo proteins and clathrin to transport proteins between the trans-Golgi network (TGN) and endosomes. Lack of any AP-1A subunit is sufficient to impair AP-1A function.

A decrease in expression of μ1A of AP-1A in AtT-20 cells, a corticotrope tumor cell line (sh-μ1A), resulted in vacuolization of the TGN, formation of non-condensing SGs and impaired responsiveness to secretagogue. Impaired AP-1A function in AtT-20 cells also dramatically altered the endocytic pathway. Early and recycling endosomes accumulated above the Golgi complex instead of peripherally and endosome maturation was altered in sh-μ1A cells.

Clinically, patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of σ1A, exhibit clinical and biochemical signs of impaired copper homeostasis. Peptidylglycine α-amidating monooxygenase 1 (PAM-1), a secretory granule membrane protein which catalyzes peptide amidation, requires copper for activity. Atp7a brings copper from the cytosol to the lumen of the secretory pathway for delivery to cuproproteins such as PAM. The endosomal trafficking of both Atp7a and PAM was altered when AP-1 function was diminished, resulting in increased sensitivity of peptide amidation to copper chelation. Neuroendocrine cells respond to high copper levels by increasing the protein expression level of Atp7a rather than moving Atp7a from the Golgi to the cell periphery. Although the pituitary contains moderate levels of copper, the brain is rich in PAM and copper.

PAM expression profile in the developing cortex and hippocampus indicated that PAM-1, the major isoform, was distributed in perikaryon, dendrites and axons in the hippocampus opening future directions to investigate PAM-1 trafficking in hippocampal neurons.

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