Regulation of Na K-ATPase distribution and function by arrestin and spinophilin

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The Na,K-ATPase is an integral membrane protein responsible for translocating sodium and potassium ions across the cell membrane by utilizing ATP hydrolysis as the driving force. The ionic transport conducted by sodium pumps creates both an electrical and chemical gradient across the plasma membrane that is critical for vectorial transport of fluid and electrolytes across polarized epithelial cells. Various aspects of the Na,K-ATPase function have been extensively studied over the past years, but the mechanism of polarized distribution and trafficking of the Na,K-ATPase has not been fully elucidated. We are interested in the mechanisms through which polarized epithelial cells control the distributions and activities of ion trasporting ATPases. Recently, we have identified interactions between the Na,K-ATPase and spinophilin and arrestin. Spinophilin and arrestin are known to be involved in signaling and trafficking of G protein-coupled receptors (GPCR). Activated GPCRs are down regulated by arrestin induced internalizaion, while spinophilin antagonizes arrestin's binding, resulting in prolonged GPCR signaling. We propose that arrestin and spinophilin are also involved in the regulation of trafficking and function of the Na,K-ATPase. Thus, the objectives of this proposal are as follows : 1) Define the mechanism through which arrestin and spinophilin modulate the function of Na,K-ATPase. 2) Charaterize the physiologic effects of these interacting proteins on the Na,K-ATPase. To accomplish these objectives we will: 1a) map the interacting domains in arrestin and spinophilin which participate in forming a complex with Na,K-ATPase, 1 b) determine the effectsof arrestin and spinophilin binding on the trafficking and internalization of Na,K-ATPase by utilizing dominant-negative or mutant arrestin, spinophilin and Na,K-ATPase alpha subunit constructs and 1c) examine the phosphorylation state of sodium pump upon arrestin binding. Using arrestin and spinophilin knock-out mice we will: 2a) analyze the expression and distribution of Na,K-ATPase in epithelial tissues from knock out mice and 2b) examine the renal function in responseto hormonal stimuli in these mice. The Na,K-ATPase plays a central role in regulating body fluid volume, and alterations in its functionmay lead to hypertensionor heart failure. Thus, the studies outlined in this proposal will allow us to elucidate the role of these new ion pump interacting proteins on Na,K-ATPase distribution and stability, potentially shedding new light on the regulation of epithelial function and in the pathogenesis of sodium pump related diseases.