Temporal and spatial control of intracellular Ca2+ fluctuations are of critical importance for eukaryotic cell function. The long-term research goal underlying this proposal is to understand the regulation of Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA) as the primary high affinity calcium efflux system of eukaryotic cells. Over twenty PMCA isoforms are generated from a multigene family and via alternative RNA splicing. The complex pattern of tissue and cellular distribution of these isoforms suggests their functional adaptation to the specific needs of a given cell. This project will explore the hypothesis that specific plasma membrane Ca2+ ATPase isoforms are targeted to multiprotein complexes where they associate with other receptors, transporters, channels, and signaling molecules to form """"""""units of local Ca2+ regulation"""""""" at specific membrane sites. This concept has recently gained experimental support by the demonstration that some PMCA isoforms interact via their C-terminal tails with PDZ (PSD-95/Dlg/ZO-1) protein-protein interaction domains present in a growing number of multi-modular proteins thought to cluster and anchor membrane transporters and signaling proteins at the plasma membrane.
The specific aims are (1) to determine the effect of PDZ domain interaction on the function of PMCAs; (2) to identify and characterize bona fide PDZ domain proteins that interact with different PMCA isoforms; and (3) to determine the cellular localization and possible co-localization of PMCA isoforms with candidate interacting PDZ domain proteins. Methods will include functional Ca2+ uptake assays, yeast two-hybrid screenings and biochemical, molecular and immunological techniques to identify and characterize specific protein-protein interactions; as well as confocal light microscopy combined with protein overexpression and fluorescent labeling methods to determine specific protein localizations. These experiments will open a new area for investigations of the physiological role of Ca2+ ATPase isoforms in the local control of Ca2+ regulation at the plasma membrane. For the plasma membrane Ca2+ ATPases, this represents a paradigm shift, changing their static image as housekeeping system responsible for the global maintenance of intracellular Ca2+ levels to one of a dynamic component involved in local Ca2+ control and Ca2+ signal transduction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058710-02
Application #
6151241
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Chin, Jean
Project Start
1999-02-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$220,619
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Kip, Sertac N; Strehler, Emanuel E (2004) Vitamin D3 upregulates plasma membrane Ca2+-ATPase expression and potentiates apico-basal Ca2+ flux in MDCK cells. Am J Physiol Renal Physiol 286:F363-9
Strehler, Emanuel E; Treiman, Marek (2004) Calcium pumps of plasma membrane and cell interior. Curr Mol Med 4:323-35
Chicka, Michael C; Strehler, Emanuel E (2003) Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting. J Biol Chem 278:18464-70
Goellner, Geoffrey M; DeMarco, Steven J; Strehler, Emanuel E (2003) Characterization of PISP, a novel single-PDZ protein that binds to all plasma membrane Ca2+-ATPase b-splice variants. Ann N Y Acad Sci 986:461-71
Burette, Alain; Rockwood, Julia M; Strehler, Emanuel E et al. (2003) Isoform-specific distribution of the plasma membrane Ca2+ ATPase in the rat brain. J Comp Neurol 467:464-76
Kip, Sertac N; Strehler, Emanuel E (2003) Characterization of PMCA isoforms and their contribution to transcellular Ca2+ flux in MDCK cells. Am J Physiol Renal Physiol 284:F122-32
DeMarco, Steven J; Chicka, Michael C; Strehler, Emanuel E (2002) Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes. J Biol Chem 277:10506-11
Usachev, Yuriy M; DeMarco, Steven J; Campbell, Colin et al. (2002) Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4. Neuron 33:113-22
Strehler, E E; Zacharias, D A (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81:21-50
Garcia, M L; Usachev, Y M; Thayer, S A et al. (2001) Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells. J Neurosci Res 64:661-9

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