Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na(+)/H(+) exchanger NHE3. We examined the ...mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA(1) receptor agonist alone decreases surface NHE3, simultaneous DA(2) agonist synergistically enhances the effect of DA(1). Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles.
Type IIa and IIb Na+/Pi-cotransporters have different patterns of expression in vivo: IIa is expressed in apical membranes of renal proximal tubules, and IIb in intestinal and lung epithelia. They ...are found in different subcellular locations when transfected in epithelial cells: IIa is apically expressed in renal proximal cells (OK), but mostly intracellularly in intestinal cells (CaCo2); IIb is apical in both cell types. To identify the domains responsible for the different expression of both cotransporters (in CaCo2), as well as those responsible for the apical expression of IIa (in OK), mutated cotransporters were fused to the Enhanced Green Fluorescent Protein (EGFP), and their expression analyzed by confocal microscopy. We conclude that the apical expression information for CaCo2 is contained within the C-terminal tail of IIb, but is not contained within IIa. From analysis of mutated IIa cotransporters we identified residues, within the C-terminal tail, involved in the apical expression of these cotransporters in OK cells: internal PR-residues and terminal TRL-residues. These signals are functional in OK but not in CaCo2-cells, supporting the concept that polarized targeting can be protein and cell specific.
Molecular determinants for apical expression and regulatory membrane retrieval of the type IIa Na/Pi cotransporter. Renal inorganic phosphate (Pi) reabsorption is a key process in Pi homeostasis. ...Type IIa Na/Pi cotransporters, located at the apical membrane of renal proximal tubular cells, guarantee the vectorial transport of Pi. Renal Pi reabsorption can be modulated by controlling the number of cotransporters expressed at the apical membrane. Indeed, factors that increase Pi reabsorption induce the expression of type IIa cotransporters at the apical membrane, whereas factors that decrease Pi reabsorption lead to their retrieval. Therefore, proper sorting of this type of cotransporters is an essential step in Pi homeostasis. The relevance of polarization has been highlighted by the finding that improper sorting of transporters can cause disease. Here we describe the identification of signals involved in apical expression of newly synthesized type IIa cotransporters and in their hormonal-induced endocytosis.
Type II NaPi cotransporters mediate epithelial phosphate (Pi) reabsorption. In mammals the type IIb protein is expressed in the small intestinal apical membrane and other epithelia; it is not ...expressed in the renal proximal tubule where we find the type IIa isoform.
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