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

Abstract

Type II NaPi cotransporters are expressed in the apical membrane of Pi-(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 503R504) are replaced by uncharged residues in type IIb (N520I521). 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 503R504) located in the last intracellular loop of the type IIa NaPi cotransporter is essential for its PTH-induced retrieval.PTH ͉ endocytosis R egulated P i reabsorption in the renal proximal tubule is an important element in overall P i homeostasis. Its rate is mainly controlled by the amount of type IIa NaPi cotransporters expressed in the brush-border membrane of proximal tubular cells (1-4).Parathyroid hormone (PTH) is the main phosphaturic hormone (5). PTH exerts its effect by an inhibition of proximal tubular brush-border NaPi cotransport associated with a membrane retrieval and lysosomal degradation of type IIa NaPi cotransporters (4, 6-9). These PTH-induced regulations are retained in cultured opossum kidney (OK) cells for both the endogenous (NaPi-4) and the transfected rat (NaPi-2) type IIa NaPi cotransporter (10)(11)(12)(13)(14). In proximal tubules and in OK cells, two signaling pathways are involved in PTH-induced downregulation of P i transport and of type IIa NaPi cotransporter: protein kinase A and protein kinase C (15-19). The mechanisms by which these kinase activities affect the cotransporter are not known.The small intestinal brush-border membrane contains the type IIb NaPi cotransporter, identified in a procedure based on homology to IIa (20). Type IIa and type IIb NaPi cotransporters differ in several properties such as tissue distribution (20-22), pH sensitivity (1, 20, 23), and Na ϩ and P i kinetics (1,20,24). Type IIa and type IIb NaPi cotransporters are differently regulated; the type I...

Regulation of phosphate transport in proximal tubules

“…Although putative endocytic motifs such as tyrosine-or di-leucine-based motifs are contained within the amino acid sequence of NaPi-lla, mutations of these motifs did not affect the retrieval of NaPi-lla [50]. Instead, a dibasic amino acid motif (KR), most likely localized within an intracellular loop, was found to be required for internalization and degradation of NaPi-lla [56]. The absence of this KR motif in the NaPi-llc protein could explain the different endocytic rates of the NaPi-lla and NaPi-llc cotransporters after PTH stimulation.…”

Physiological Regulation of Renal Sodium-Dependent Phosphate Cotransporters

“…The two charged amino acids in the type IIa cotransporter (K 503 R 504 ) are replaced by uncharged residues in type IIb (N 520 I 521 ), which is not regulated by PTH. The results of chimera experiments with type IIb indicate that this motif is important for PTH-induced endocytosis [29].…”

“…By the yeast two-hybrid approach, several proteins were identified that interact with type IIa transporter protein and are localized either in the brush border or in the subapical compartment of proximal tubular cells [25][26][27][28]. Karim-Jimenez et al [29] indicated that the N-and C-terminals are not required for the PTHinduced internalization, whereas the region between residues 216 and 658 seems to be necessary. This region contains two putative intracellular loops with sequences in the last intracellular loop that are highly conserved between the types IIa and IIb proteins.…”

Phosphate transporters of the SLC20 and SLC34 families

“…This model incorporates predictions from biochemical and biophysical studies including epitope labeling, cysteine scanning mutagenesis and in vitro glycosylation assays (for review see: ( ). The C-terminal region is important for targeting, hormonal regulation and proteinprotein interactions, for example the TRL motif in the C-terminal plays a role as a PDZ binding motif Karim-Jimenez et al, 2001), and a KR motif located in an intracellular linker region (Fig 2A) is critical for PTH sensitivity (Karim-Jimenez et al, 2000). Sequence analysis reveals an inverted repeat motif that is conserved amongst SLC34 isoforms and homologs in all phyla (Werner and Kinne, 2001).…”