Secretory Pathway Calcium-ATPases (SPCA) are a newly defined family of ion pumps that transport calcium and manganese into the lumen of the Golgi apparatus where they are essential for sorting, processing, and glycosylation of proteins. The first member of this family, named PMR1, was described in Saccharomyces cerevisae, and more recently, 2 mammalian homologues of PMR1, SPCA1 and SPCA2, have been identified. Mutations in hSPCAl cause Hailey Hailey disease, a debilitating disorder characterized by severe ulceration of the skin, thought to result from dysregulation of cellular calcium. Excess manganese is deposited in the brain and leads to Parkinsonism. The SPCA have been implicated in diverse physiological processes, ranging from manganese detoxification in the liver, calcium transport across intestinal epithelia, and milk production by the mammary glands, although there is little molecular evidence for their specific roles. This proposal combines 3 parallel approaches to investigate the SPCA: biochemical studies using purified proteins or Golgi membranes, cell biological studies in polarized cultured mammalian cells, and large scale phenomic analysis of ion homeostasis in a model organism. In previous studies, we have defined the transmembrane helices and residues critical for ion transport and have identified a role for helix packing in determining ion selectivity.
In Aim 1 of this proposal, we will shift our focus to understanding the ion binding and modulatory role of EF motifs in the cytoplasmic N-terminal domain. We have new evidence for trafficking of the pumps between the Golgi stacks and a novel, vesicular compartment in polarized cell models of hepatocytes and enterocytes.
In Aim 2, we will determine if trafficking is ion-dependent and related to transcellular transport, and whether specific retrieval or PDZ-binding motifs at the C-terminus are important for localization. Gene knockdown approaches will be used to evaluate the isoform-specific contributions of the 2 SPCA pumps in the enterocyte model. Finally, we propose to use the yeast model organism for high resolution phenomic analysis that will identify new genes and pathways associated with the cellular function of the SPCA pumps (Aim 3). Taken together, this proposal lays the foundation for understanding the role of this novel family of transporters at the molecular, cellular and physiological level. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM062142-05A1
Application #
7150782
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Chin, Jean
Project Start
2001-06-01
Project End
2010-05-31
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
5
Fiscal Year
2006
Total Cost
$318,744
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Dang, Donna; Prasad, Hari; Rao, Rajini (2017) Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells. Mol Carcinog 56:2474-2485
Dang, Donna; Rao, Rajini (2016) Calcium-ATPases: Gene disorders and dysregulation in cancer. Biochim Biophys Acta 1863:1344-50
Kondapalli, Kalyan C; Llongueras, Jose P; Capilla-González, Vivian et al. (2015) A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma. Nat Commun 6:6289
Prasad, Hari; Rao, Rajini (2015) Applying knowledge of autism to brain cancer management: what do we know? Future Oncol 11:1847-50
Prasad, Hari; Rao, Rajini (2015) The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease. J Biol Chem 290:5311-27
Cross, Brandie M; Breitwieser, Gerda E; Reinhardt, Timothy A et al. (2014) Cellular calcium dynamics in lactation and breast cancer: from physiology to pathology. Am J Physiol Cell Physiol 306:C515-26
Feng, Ming-Ye; Rao, Rajini (2013) New insights into store-independent Ca(2+) entry: secretory pathway calcium ATPase 2 in normal physiology and cancer. Int J Oral Sci 5:71-4
Cross, Brandie M; Hack, Anniesha; Reinhardt, Timothy A et al. (2013) SPCA2 regulates Orai1 trafficking and store independent Ca2+ entry in a model of lactation. PLoS One 8:e67348
Patenaude, Cassandra; Zhang, Yongqiang; Cormack, Brendan et al. (2013) Essential role for vacuolar acidification in Candida albicans virulence. J Biol Chem 288:26256-64
Leitch, Sharon; Feng, Mingye; Muend, Sabina et al. (2011) Vesicular distribution of Secretory Pathway Ca²+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells. Biometals 24:159-70

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