Inorganic phosphate (P_i) homeostasis is achieved by modulating urinary excretion (renal reabsorption) according to the dietary intake (intestinal reabsorption). Renal P_i reabsorption is largely dependent on the activity of the type IIa Na⁺/P_i-cotransporter. The precise nature of the intestinal transporter remains unknown, although type IIb Na⁺/P_i-cotransporter is a good candidate. Type IIa and type IIb cotransporters are highly conserved proteins, but they show specific expression and regulation properties. Type IIa Na⁺/P_i-cotransporters are located at the apical membrane of renal proximal tubular cells, and are responsible for up to 80 % of renal P_i reabsorption. Therefore, reabsorption of P_i in the kidney can be modulated by controlling the number of type IIa cotransporters expressed at the apical membrane. Indeed, factors that increase P_i reabsorption (as low dietary P_i) induce the expression cotransporters at the apical membrane, whereas factors that decrease P_i reabsorption (as parathyroid hormone, PTH) lead to their retrieval. In transfection experiments, apical expression of type IIa requires a cell line from renal proximal origin (OK). On the other hand, type IIb Na⁺/P_i-cotransporters are expressed at the apical membrane of lung and intestinal epithelia, but are not detected in kidney. They are not regulated by PTH. In transfection experiments type IIb is apically expressed in cells of either renal proximal (OK) or intestinal (CaCo-2) origin.

Since regulation of type IIa cotransporters involves changes in apical expression, we have studied two processes that control the amount of cotransporters apically expressed: insertion of newly synthesized cotransporters, and hormonally induced retrieval. For that we exploited the differences between type IIa and type IIb cotransporters and constructed several type IIa-IIb chimeras as well as type IIa truncations and point mutations. Our studies suggest that:

(1) The C-terminal cytoplasmic tail of type IIb contains a conserved leucine required for apical expression both in OK and CaCo-2 cells. Removal of this residue yielded a cotransporter able to leave the ER and Golgi but retained in an endosomal/lysosomal compartment, suggesting impaired targeting and/or stability at the plasma membrane.

(2) The C-terminal cytoplasmic tail of type IIa contains two signals involved in apical expression: a terminal sequence matching a PDZ-binding motif (TRL), and an internal PR-based sequence. Removal of any of these signals partially prevented or destabilized apical expression of the cotransporter. Both signals are properly recognized in OK but not in CaCo-2 cells. The TRL-signal mediates the interaction between the type IIa C-terminal cytoplasmatic tail and several PDZ-containing proteins, among them Cap-1 (mouse homologue of the human PDZK1) and NHERF1.

(3) The type IIa-specific PTH-induced retrieval is independent of known endocytic signals such as tyrosine- or dileucine-based motifs. Instead, it depends on the presence in the third intracellular loop of a dibasic motif (K₅₀₃R₅₀₄), substituted by uncharged residues in type IIb (N₅₂₀I₅₂₁). Replacement of these residues reverted the phenotype of the constructs: type IIb containing the KR motif was internalized after PTH treatment, whereas type IIa containing the NI sequence was not regulated. The specific amino acids are located just upstream of a PKC consensus sequence present at an equivalent position in type IIa and IIb (T₅₀₅AK for IIa, S₅₂₂AK for IIb); however, mutations of the putative phosphorylated residues did not affect the PTH response. PTH-induced regulation of the type IIa may involve interacting proteins, and the dibasic motif could mediate this interaction.