Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter.

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.

Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation.

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.

A dibasic motif involved in parathyroid hormone-induced down-regulation of the type IIa NaPi cotransporter.

Type II NaPi cotransporters are expressed in the apical membrane of P(i)-(re)absorbing epithelia: the type IIa in renal proximal tubule and the type IIb in small intestine. Parathyroid hormone (PTH) leads to a retrieval from the apical membrane of the type IIa NaPi cotransporter. The type IIa cotransporter is also expressed in opossum kidney (OK) cells, and its expression is under the control of PTH. In the present study, we identified the molecular "domains" involved in the PTH-induced retrieval of the type IIa NaPi cotransporter. Wild-type mouse type IIa (mIIa) and type IIb (mIIb) as well as several mIIa-mIIb chimeras and site-directed mutants were fused to the enhanced green fluorescent protein and transfected into OK cells. We found that mIIa but not mIIb was internalized and degraded after incubation with 1-34 (or 3-34) PTH. Using chimeras, we found that the N and C termini were not required in this effect, whereas a "domain" located between residues 216 and 658 seemed to be necessary. This region contains two putative intracellular loops with highly conserved sequences between mIIa and mIIb; in the last intracellular loop, two charged amino acids of type IIa (K(503)R(504)) are replaced by uncharged residues in type IIb (N(520)I(521)). We generated two mutants in which these residues were interchanged: mIIaNI and mIIbKR. Similarly to mIIa, the mIIbKR mutant was endocytosed in response to 1-34 PTH; in contrast, mIIaNI behaved as mIIb and was not internalized. In conclusion, a dibasic amino acid motif (K(503)R(504)) located in the last intracellular loop of the type IIa NaPi cotransporter is essential for its PTH-induced retrieval.

Asymmetrical targeting of type II Na-P(i) cotransporters in renal and intestinal epithelial cell lines.

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK(1)), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.

Requirement of a leucine residue for (apical) membrane expression of type IIb NaPi cotransporters.

Type II NaPi cotransporters mediate epithelial phosphate (P(i)) 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. To look for molecular determinant(s) involved in apical expression of type IIb cotransporters, we have made deletion mutations within the C-terminal tails of mouse IIb (mIIb) and human IIb (hIIb) transporter proteins. The constructs were fused to the enhanced green fluorescent protein and transiently transfected into intestinal CaCo2-cells. Both mIIb and hIIb were located exclusively in the apical membrane of the cells. For mIIb, the removal of a cysteine cluster or the last three amino acids (TVF) had no effect on the location of the protein. However, truncation at the level of the conserved L691/689 prevented the apical membrane expression of both mIIb and hIIb, respectively, and the mutated proteins were located in endosomal and lysosomal structures. A similar expression pattern of the mIIb and hIIb constructs was found in renal proximal tubular opossum kidney cells. Our data suggest that L691/689 is involved in mechanisms leading to an apical expression of type IIb NaPi cotransporters.