Hello, I copy and paste the article to save image space. Can you please summarize what this paper is about and elaborate the result? The discussion should explain what this article is about. Please summarize the paper and its conclusion as if your explaing step by step to the class. movement of the three cytoplasmic domains, N (nucleotide binding), P (phosphorylation), and A(actuator) (37). A recent study (28) suggests that the NH2 terminus may act as an auto-regulatorydomain, modulating E1/E2 conformational transition. In El conformation, the NH2 terminus is inassociation with the first cytoplasmic loop of Na,K-ATPase. Transition from E1 to E2 conformationmay release the NH2 terminus from its interaction with the first cytoplasmic loop of Na,KATPase,thus making the NH2 terminus available for interaction either with the InsP3R directly or with aprotein bridging between Na,K-ATPase and InsP3R. Such an effect could explain why truncation ofthe first 32 amino acids of the NH2 terminus will prevent ouabain-induced Ca2 oscillations. Analternative explanation is that NH2-terminal truncation has displaced the E1 3 E2 conformation ofNa,K-ATPase in favor of E1 (8, 38). The concept that Na,K-ATPase may, in addition to its functionas an ion pump, also act as a signal transducer, is now rapidly evolving (2-5). Most previous studieson the signaling role of Na,K-ATPase have been carried out on cardiac myocytes (for a review, seeRef. 39). In these cells, activation of Src kinase was found to be the primary event in the signalingcascade initiated by the ouabain/Na,K-ATPase complex, and downstream effects includedphosphorylation of epidermal growth factor receptor and activation of the mitogen-activated proteinkinase pathway. Since cardiac myocytes exhibit spontaneous Ca2 sparks of high frequency, it has notyet been possible to establish whether Na,K-ATPase/InsP3R-triggered Ca2 oscillations also occur inthese cells. Another model where Na,K-ATPase-mediated Ca2 signaling is dependent on a closeproximity between the plasma membrane and ER Ca2 stores has been presented previously (40). Inthis model, Na,KATPase 2- and 3-subunits modulate Ca2 release from ER via local changes inintracellular Na concentration. Our study was performed on cells expressing only the 1-subunit ofNa,KATPase, and the results from the protocols with the truncated NKA1.M32 imply that increasedintracellular Na concentration was, in our model, not the main cause of ouabain-induced Ca2oscillations. Furthermore, we previously established that increasing intracellular Na, by loweringextracellular K to the same extent as 250 M ouabain, is not sufficient to elicit a Ca2 oscillatoryresponse (2). Ca2 oscillations trigger such universal processes as fertilization, cell division, celldifferentiation, and apoptosis (41). The specificity of this signal is encoded by the frequency and/orthe amplitude of the Ca2 oscillations (42). In the case of G-proteincoupled receptors, Ca2 oscillationsgenerally have a periodicity of seconds (30, 43). The highly regular ouabain/Na,KATPase-inducedCa2 oscillations displayed a longer periodicity in the minute range and should thereby provide amechanism to ensure a different kind of physiological readout. Endogenous ouabain levels are highduring pregnancy and in the perinatal period (44, 45). Thus, it is of great physiological interest thatthe ouabain-induced slow Ca2 oscillations were found to activate NF-B. This pluripotenttranscription factor activates genes modulating cell proliferation, apoptosis, and development as wellas responses of the immune system (46). Cells expressing GFP-NKA1.M32 did not respond toouabain with Ca2 oscillations. In these cells, ouabain did not activate NF-B. These findings supportthe physiological significance of a signaling microdomain containing Na,K-ATPase 1-subunit andInsP3R as a trigger mechanism for NF-B activation. In conclusion, we have demonstrated theexistence of a cell signaling microdomain containing Na,K-ATPase and InsP3R that acts as asignaling pathway for Ca2 oscillations and activation of NF-B. Our findings represent, in manyaspects, a novel mechanism for generation of Ca2 signaling where an ion pump, Na,K-ATPase, alsofunctions as a signal transducer. The physiological importance of slow Ca2 oscillations is generallyrecognized, yet few biological generators of this signal have been identified previously. Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium OscillationsIt is now generally agreed that many, if not most, important processes in the cell arecontrolled by proteins aggregated together in complexes (21, 22, 31-33). The assembly of complexesthat contain a receptor and components of signal machinery provides the cell with a highly selectivemeans to engage a specific signaling pathway. The finding that ligandbound Na,K-ATPase assembleswith InsP3R and that this assembly can give rise to intracellular Ca2 oscillations with a constantperiodicity in the minute range represents a novel principle for such a protein complex. Based on ourcombined results, we suggest that the ouabaininduced Ca2 oscillation and signal-transducingfunction of Na,K-ATPase is made possible by the local organization of Na,K-ATPase and InsP3Rinto a spatially organized functional microdomain that links the plasma membrane to intracellular ERCa2 stores. A signaling microdomain can function without a diffusible messenger provided that thetransducer and the effector are in such proximity that they can communicate via protein-proteininteraction, either directly or via one or more interacting/scaffolding proteins. Molecular strategiesusing the InsP3 sponge and pharmacological studies using the PLC inhibitor, U73122, indicate thatthe ouabain-induced Ca2 oscillatory response may be elicited via an InsP3-independent mechanismof InsP3R activation. Significant energy transfer between the donor and acceptor complexes, GFP-NKA1 and anti-InsP3R-anti-mouse IgG-Cy3, indicate a distance of less than 12 nm (16). It is wellestablished that the ER is juxtaposed to the plasma membrane (34). FRET was recorded in a regionof the plasma membrane, and the result is clearly compatible with previous electron microscopystudies showing that the distance between the plasma membrane, where Na,K-ATPase is located, andthe membrane of ER, where the InsP3R is located, can be as short as 10 nm (35). It should be noted,however, that FRET is recorded between the GFP-labeled Na,K-ATPase and Cy3-labeled goat-anti-mouse IgG antibody that binds to the InsP3R antibody. Taking the size of the antibodies and the GFPmolecule into account, the maximal distance between Na,K-ATPase and InsP3R could be somewhatlarger than the working distance for FRET fluorophores, GFP and Cy3. Hence, the FRET results donot rule out the possibility that Na,K-ATPase and InsP3R may interact via a scaffolding protein. Theobserved increase in FRET between Na,KATPase and InsP3R following ouabain exposure and theloss of FRET upon CytD treatment indicate that ouabain-induced Ca2 oscillations were dependent ona dynamic physical association between Na,K-ATPase and InsP3R and therefore add furtherevidence to the concept that the Ca2 signal arises from a signaling microdomain containing Na,K-ATPase and InsP3R. The question remains as to how the localization of Na,KATPase and InsP3R iscontrolled. As evidenced by our FRET and co-immunoprecipitation studies using CytD, an intactcytoskeleton is required for the physical association between Na,K-ATPase and InsP3R and ouabain-induced Ca2 signaling. This suggests that actin, or cytoskeletal proteins associated with actin, willstabilize the Na,K-ATPase/InsP3R complex by physical tether cross-linking. Ankyrins, a family ofadaptor proteins believed to participate in the organization of proteins into specialized regions in theplasma membrane and ER Ca2 stores, represent an interesting putative mechanistic partner fororchestrating Na,K-ATPase and InsP3R proximity (25). The complete framework of adaptor andscaffolding proteins that may be involved in facilitating the structure and signal-transducing functionof this microdomain is still uncertain, but our results also do not preclude the necessity of a directphysical interaction between Na,K-ATPase and InsP3R in this event. The activation of InsP3R islikely due to an allosteric effect of ouabain on Na,K-ATPase. Na,K-ATPase is a P-type ATPase thatcan exist in distinct E1 and E2 conformational states that are at least partially determined byintramolecular interactions between the NH2 terminus and cytoplasmic loops of the enzyme.Ouabain binds to the E2 conformation of the Na,K-ATPase and causes a robust shift in the E1/E2poise toward E2 forms (36). Information derived from the crystal structure of another P-typeATPase, the SERCA pump, suggests that the E1 3 E2 state transition is accompanied by significant

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Various proteins form an aggregate inside the cells and regulate different functions inside the…