This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Neuronal rafts are plasma membrane compartments with a unique lipid and protein composition; they are enriched in cholesterol, sphingolipids, and proteins involved in signal transduction. There is compelling evidence to implicate neuronal rafts as being local sites for the organization of Ca2+ signaling events but no information exists on specific Ca2+ regulatory proteins that mediate the Ca2+ signal. We have recently discovered that the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ transporter is enriched in rafts isolated from rat brain synaptic plasma membranes (SPMs). Confocal microscopy of intact hippocampal neurons further confirmed the co-localization of PMCA in rafts domains, suggesting the possibility of this association occurring in vivo. Immunoblot analysis showed that all 4 isoforms of PMCA, including their 'a' and 'b' splice variants, were localized in rafts. The presence of both 'a' and 'b' variants and not exclusively PMCA 2b, as proposed in our original hypothesis suggests that raft domains contain the full complement of PMCA subtypes necessary for fine-tuning of the Ca2+ signal. Measurement of PMCA activity in raft vs non-raft domains showed that the raft-associated PMCA had higher specific activity suggesting that this pool of PMCA may be a more significant contributor to overall PMCA function in neurons. To determine the nature of the association of PMCA with rafts, we depleted cellular cholesterol, the major lipid known to maintain raft structure in vivo. Both chronic and acute depletion of cellular cholesterol did not disrupt the association of PMCA with rafts suggesting a very tight interaction. In efforts to determine the potential protein partners that may anchor PMCA to rafts, co-immunoprecipitation studies were performed. The results showed that raft PMCA was associated with calmodulin, NAP-22, and GAP-43, proteins known to organize neuronal raft domains. Future goals are to determine molecular mechanisms that recruit PMCA into and out of raft domains.
| Garabedian, Alyssa; Baird, Matthew A; Porter, Jacob et al. (2018) Linear and Differential Ion Mobility Separations of Middle-Down Proteoforms. Anal Chem 90:2918-2925 |
| Jeanne Dit Fouque, Kevin; Garabedian, Alyssa; Porter, Jacob et al. (2017) Fast and Effective Ion Mobility-Mass Spectrometry Separation of d-Amino-Acid-Containing Peptides. Anal Chem 89:11787-11794 |
| Alaofi, Ahmed; Farokhi, Elinaz; Prasasty, Vivitri D et al. (2017) Probing the interaction between cHAVc3 peptide and the EC1 domain of E-cadherin using NMR and molecular dynamics simulations. J Biomol Struct Dyn 35:92-104 |
| Pang, Xiao-Yan; Wang, Suya; Jurczak, Michael J et al. (2017) Retinol saturase modulates lipid metabolism and the production of reactive oxygen species. Arch Biochem Biophys 633:93-102 |
| McNiff, Michaela L; Chadwick, Jennifer S (2017) Metal-bound claMP Tag inhibits proteolytic cleavage. Protein Eng Des Sel 30:467-475 |
| Johnson, Troy A; Mcleod, Matthew J; Holyoak, Todd (2016) Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase. Biochemistry 55:575-87 |
| Tucker, Jenifer K; McNiff, Michaela L; Ulapane, Sasanka B et al. (2016) Mechanistic investigations of matrix metalloproteinase-8 inhibition by metal abstraction peptide. Biointerphases 11:021006 |
| Yadav, Rahul; Vattepu, Ravi; Beck, Moriah R (2016) Phosphoinositide Binding Inhibits Actin Crosslinking and Polymerization by Palladin. J Mol Biol 428:4031-4047 |
| Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G et al. (2016) Actin polymerization is stimulated by actin cross-linking protein palladin. Biochem J 473:383-96 |
| Budiardjo, S Jimmy; Licknack, Timothy J; Cory, Michael B et al. (2016) Full and Partial Agonism of a Designed Enzyme Switch. ACS Synth Biol 5:1475-1484 |
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