The Characterization of Menkes Copper Transporter and Dopamine ß-monooxygenase Carboxy-Terminus in Neuroendocrine Cells
Dopamine ?-monooxygenase (DBM) requires ascorbate, copper, and molecular oxygen to synthesize norepinephrine. Because little is known about the mechanism by which DBM is routed to catecholamine-containing secretory vesicles, we investigated the role of the DBM carboxy-terminus (DBMCT) by attaching it to the hydroxylating (PHM) domain of peptidyl-glycine ?-amidating monooxygenase. The active PHMs/DBMCT chimera expressed in corticotrope AtT-20 cells was analyzed by Western blot, metabolic labeling, and immunostaining. The PHMs/DBMCT chimera contained a 48 and 51 kDa species, with the 48 kDa formed by proteolytic processing of DBMCT. Immunostaining showed that although PHMs/DBMCT is mainly localized to secretory vesicles in AtT-20 cells, it is also present in the ER. An EGFP/DBMCT chimera transiently transfected into pheochromocytoma PC12 cells showed ER staining, indicating that DBMCT does not contain sorting information for routing to secretory granules but rather it has a structural role. Although copper is essential for DBM function, excess copper is highly toxic and its homeostasis is tightly regulated by transporters and chaperones. Menkes copper transporter (MNK) is a P-type ATPase essential for an organism's survival due to its role in regulating copper efflux. Mutations in MNK affect copper utilization by cuproenzymes involved in catecholamine and peptide biosynthesis, development and growth, thus causing Menkes syndrome that is characterized by mental retardation, neurodegeneration, connective tissue disorders and early childhood death. Since little is known about MNK in neuroendocrine cells containing sequestered cuproenzymes, we investigated the localization and trafficking of endogenous MNK in chromaffin and PC12 cells where catecholamines are synthesized. Sucrose density gradients identified a bi-modal MNK distribution with enrichment of MNK in lighter fractions that co-localized with syntaxin 6, a trans-Golgi network (TGN) marker, and in denser fractions containing secretory vesicle markers. Immunostaining and cell surface biotinylation showed MNK redistribution to the plasma membrane with increasing copper levels, while both MNK and DBM redistributed to the plasma membrane following stimulation of exocytosis. Our findings showed a unique MNK localization to the TGN and secretory vesicles in endocrine cells, suggesting distinctive roles for MNK in these compartments and indicating the importance of understanding MNK function in the catecholaminergic system in Menkes patients.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:menkes dbm secretory granules chromaffin pc12 neuroendocrine
Date of Publication:01/01/2008