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J Am Soc Nephrol 11:1961-1968, 2000
© 2000 American Society of Nephrology

PTH-Induced Downregulation of the Type IIa Na/Pi-Cotransporter Is Independent of Known Endocytic Motifs

NATI HERNANDO, JUTKA FORGO, JÜRG BIBER and HEINI MURER

Institute of Physiology, University of Zürich, Switzerland.

Correspondence to Dr. Heini Murer, Physiologisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Phone: 41-1-635-50-30; Fax: 41-1-635-57-15; E-mail: hmurer{at}access.unizh.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Parathyroid hormone (PTH)-induced inhibition of renal proximal tubular Na/Pi cotransport involves two consecutive steps: endocytosis followed by lysosomal degradation of the type IIa Na/Pi cotransporter. Tyrosine-, dileucine-, and diacidic-based motifs are suggested to be involved in endocytosis and/or lysosomal targeting of different plasma membrane proteins. The rat type IIa cotransporter (NaPi2) contains two cytoplasmic tyrosine residues (Y) within sequences highly homologous to tyrosine-based motifs (GY402FAM and Y509RWF), three cytoplasmic dileucine (LL101, LL374, and LI591) and two cytoplasmic diacidic motifs (EE81 and EE616). We studied the role of these motifs on the PTH-induced retrieval and lysosomal degradation of the NaPi2 cotransporter. To follow its trafficking in vivo, the NaPi2 protein was fused to the carboxyl-terminal end of the enhanced green fluorescence protein. This fusion did not impair the apical targeting or the PTH-induced endocytosis of the wild-type cotransporter when transfected in opossum kidney cells. Single and multiple Y and LL mutants retained the apical targeting and the PTH-induced degradation. Mutations of the diacidic motifs were also without effect. These data suggest that the above three motifs are not required for the PTH-induced internalization and/or degradation of the cotransporter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Renal phosphate (Pi) reabsorption is a physiologically controlled process that is largely mediated by the renal-specific, brush border membrane-associated, type IIa Na/Pi cotransporter (NaPi2) (1,2). Confirming this view, it has been reported that brush border membranes from mice that are deficient in the type IIa cotransporter gene (Npt2) show only 20 to 30% of the Na/Pi cotransport activity observed in wild-type animals (3). The NaPi2 cotransporter is the main target for physiologic regulation of renal Pi reabsorption. We have documented that parathyroid hormone (PTH) treatment leads to the downregulation of the cotransporter, as a result of a retrieval from the apical membrane and ultimate lysosomal targeting and degradation of the protein (4,5,6,7). In addition, after PTH treatment, type IIa cotransporters colocalized with AP2, a component of the clathrin-coated endocytic vesicles (8).

Retrieval of membrane proteins from the cell surface and/or targeting to lysosomes can involve several motifs located within cytoplasmic domains: tyrosine (Y)-, dileucine (LL), and diacidic (EE)-based motifs (9,10,11). Tyrosine-based motifs are sequences of four to six amino acids that contain a critical Y residue (12,13,14,15,16,17). The sequence context of the critical Y and its relative position to the transmembrane domain are important for activity (18). Three tyrosine-based motifs seem to play a role in the internalization of endocytic receptors as well as in the biosynthetic targeting of lysosomal integral proteins: NPXY, GYXX{psi}, and YXX{phi}, where X can be any amino acid, {psi} is M/L/V/F/I, and {phi} is a bulky residue. The dileucine-based motifs consist of an invariant L residue in the first position and a hydrophobic residue (L, I, V, or M) in the second position (19,20,21,22). The mechanisms by which these signals mediate sorting to distinct compartments are poorly understood, although they seem to require the interaction of the critical Y or LL residue with the medium µ (for Y) or ß (for LL) chains of the clathrin-associated adaptor complexes AP-1, AP-2, and AP-3 (23,24,25,26,27,28). In an in vitro binding assay, the rates of AP-1 and AP-2 association with peptides that contain Y motifs correlated with the rates of internalization (29). Interactions of Y- and LL-based motifs with the adaptor complex are differently regulated process (10,21,23,24, 27,28,29,30).

In some cases, the endocytic step can be uncoupled from the lysosomal targeting. The independent nature of these membrane traffic events has been recently shown for Nef: an LL motif is responsible for the clathrin-coated—mediated internalization, whereas an EE diacidic sequence is involved in the binding of Nef to ß-COP in endosomes, thereby controlling the lysosomal targeting (11).

We mutated two Y located at positions 402 (GY402FAM) and 509 (Y509RWF) of the rat NaPi2 cotransporter. On the basis of hydropathy plot predictions (31) and epitope tagging studies (32), these residues are suggested to lie within the third and fourth intracellular loops of the cotransporter. Both sequences are conserved in all of the type IIa cotransporters cloned so far and show a high homology with the GYXX{psi} and YXX{phi} consensus motifs, respectively. We also mutated three LL-type sequences located at positions 101 (LL101), 374 (LL374), and 590 (LI590). These three sequences are also conserved in most of the known type IIa cotransporter, and most probably they lie within the cytoplasmic N-terminal tail, third intracellular loop, and the cytoplasmic C-terminal tail, respectively (31,32). Finally, we also mutated two conserved diacidic motifs (EE81 and EE616) predicted to lie within the cytoplasmic N- and C-terminal tails, respectively (31,32).

Because membrane trafficking, i.e., retrieval from plasma membrane and subsequent lysosomal targeting, is involved in the PTH-induced inhibition of the NaPi2 cotransporter, the goal of this work was to study the implication of the Y-, LL-, and EE-based motifs in that process. We fused the NaPi2 constructs to the enhanced green fluorescence protein (EGFP), which enabled us to study in vivo the hormonal regulation on single opossum kidney (OK) cells transfected with the EGFP-fused cotransporter. We provide evidence that none of these motifs is involved in the PTH-induced internalization and/or lysosomal targeting of the NaPi2 cotransporter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
EGFP—NaPi2 Fusion Constructs
A mammalian expression vector containing the EGFP under the control of the cytomegalovirus immediate early promoter (pEGFP-C1; Clontech, Palo Alto, CA) was digested with BglII and SalI. Wild-type (WT) NaPi2 was fused to the carboxyl terminal end of the EGFP through PCR and subcloning. Briefly, the NaPi2 cDNA was amplified with the sense primer Bgl-S that contained a BglII recognition site (underlined) just downstream of the NaPi2 initiator methionine (bold), and the antisense primer Sal-AS that contained a SalI recognition site (underlined) 20 bp downstream of the NaPi2 stop codon. Amplifications were carried out using Pfu DNA polymerase. PCR products were digested with BglII and SalI and ligated to the pEGFP-C1 previously digested with the same enzymes. After the EGFP-NaPi2 plasmid was obtained, the 20-bp recognition site of the T7 RNA polymerase was introduced upstream of the EGFP coding sequence; this allowed us to transcribe in vitro cRNA to test the activity of the EGFP-fused cotransporter in Xenopus laevis oocytes. The RNA polymerase site was introduced with the use of a site-directed mutagenesis kit (Stratagene, La Jolla, CA). Because this system allows only up to 12 bp insertions, the 20-bp recognition site (bold) was split between the sense (T7-S) and antisense (T7-AS) primers. After PCR amplification with Pfu DNA polymerase, the parental DNA was digested with DpnI and the amplified strands were phosphorylated for 1 h at 37°C in the presence of polynucleotide kinase and ligated overnight at 16°C with T4 DNA ligase. The following primers were used:

Site-Directed Mutagenesis
Construction of single Y402 and Y509 mutations to phenylalanine has been reported (33). The double Y mutant (Y402/509) as well as the single and multiple LL and EE mutants were constructed following a similar approach: WT-NaPi2 cDNA fused to EGFP (for the single mutations) or the already mutated cDNA (for multiple mutations) were used as templates for amplification with Pfu DNA polymerase. We used complementary sense (S) and antisense (AS) primers, both of which contained in the middle of their sequence the desired mutated codon (bold). PCR amplification, DpnI digestion, and competent cell transformation were done as previously reported (33). The following primers were used:

For the construction of the mutant in which all of the Y and LL sequences were mutated, a 300-bp SmaI-BamHI fragment containing the LI590 pair mutated was first cut from the LI590 mutant and pasted into the Y402/509 plasmid previously digested with the same enzymes. Then an EcoRI site (bold) was introduced by PCR between residues D390 and F391 of the Y402/509LI590 and LL101/374 mutants, using the primers RI-S and RI-AS. After digestion of both plasmids with BglII and EcoRI, a 1.1-Kb fragment containing the LL 101 and LL374 pairs mutated was cut from the LL101/374 mutant and pasted into the Y402/509LI590 plasmid. Finally, the Y402/509 + LL 101/374/590 construct was used as a template to generate the plasmid in which all of the Y-, LL-, and EE-based motifs were mutated. The correctness of all mutations was confirmed by sequencing.

Expression in X. Laevis Oocytes and Transport Assay
The procedures for oocyte isolation, cRNA injection, and 32P-uptake assay have been described (34). Briefly, in vitro synthesis and capping of cRNA were done by incubating previously linearized WT and EGFP-fused NaPi2 plasmids with SP6 and T7 RNA polymerase, respectively. 32P uptakes were done 3 d after cRNA injection.

Cell Culture and Transfections
Opossum kidney cells (OK cells, clone 3B/2) were maintained in DMEM/Ham's F-12 medium (1:1) as described previously (35). Cells were plated in 35-mm dishes (Nunc; Life Technologies, Switzerland), and subconfluent cultures were transfected with either the empty pEGFP plasmid or the different EGFP-fused NaPi2 constructs. Transfections were carried out overnight, using 1 µg of DNA and 3 µl of FuGENE (Boehringer Mannheim, Mannheim, Germany) per dish, according to the manufacturer's procedures. Subcellular location and PTH effect were studied in confluent cultures of transiently transfected cells. We have made several attempts to generate stable transfected cell lines containing the EGFP-fused transporter proteins but have not succeed (see below).

Subcellular Location: Confocal Microscopy
OK cells grown on coverslips were fixed for 10 min in 3% paraformaldehyde in PBS, washed with PBS and incubated for 10 additional min with 20 mM L-glycine in PBS. After 30 min of permeabilization with 0.1% saponine, cells were incubated either with a polyclonal antibody against NaPi2 (2) followed by incubation with a rhodamine-conjugated IgG, or with phalloidine-rhodamine (Calbiochem, San Diego, CA) for actin detection. After extensive washings in PBS, the coverslips were mounted using Dako-Glycergel (Dakopatts, Carpinteria, CA) containing 2.5% 1,4-diazabicyclo-[2.2.2]octane (Sigma, St. Louis, MO) as a fading retardant. Confocal images were taken using a Leica laser scan microscope (TCSSP, Wetzlar, Germany) equipped with a 63x oil immersion objective.

PTH Effect: Fluorescence Microscopy
To study the PTH effect on the NaPi2 constructs in vivo, we first identified OK cells that expressed the typical patches known to reflect the presence of the cotransporter in the apical membrane (5). Pictures of these cells were taken using the 20x objective of an inverted microscope (Eclipse TE3OO/200; Nikon, Germany) equipped with a TE-FM Epi-Fluorescence Attachment (Nikon) to detect the EGFP fluorescence. Then, cells were incubated at 37°C with either 10 -8 M PTH or vehicle, as it has been reported (35). After different time intervals, the same cells were identified again and pictures were taken using the same fluorescence intensity setup that was used before the PTH addition. To inhibit lysosomal degradation, cells were incubated with leupeptine (100 µg/ml) for 30 min before addition of the hormone. Pictures of the culture monolayer were taken by phase contrast microscopy. The images were processed using the Lucia analysis software (Laboratory Imaging Ltd., Czech Republic). For each set of data, at least four independent experiments were performed, and from each of them at least six cells were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGFP Fluorescence Reflects the Presence of the NaPi2 Cotransporter
Confocal microscopy of confluent OK cultures transfected with the empty plasmid showed that the native EGFP exhibited a diffuse distribution throughout the cell (Figure 1A). In cross sections, the EGFP fluorescence was homogeneously detected in the cytoplasm and nucleus (Figure 1a). By contrast, in cells that were transfected with the EGFP-fused WT cotransporter, the EGFP fluorescence was specifically located at the plasma membrane (Figure 1, B and b). After staining with an antibody against an N-terminal peptide of the cotransporter, both the EGFP (Figure 2A) and the antibody (Figure 2B) signals revealed a "patchy" pattern of expression that reflected the presence of the cotransporter at the apical cell surface (Figure 2, a and b). As shown in Figure 2C, there was a total overlapping between the intrinsic fluorescence of the fused cotransporter (EGFP signal) and the NaPi2 immunostaining.



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  		Figure 1. Confocal microscopy of opossum kidney (OK) cells transfected with the empty pEGFP plasmid or the pEGFP-NaPi2 construct. OK cells transfected with either the empty pEGFP-CI plasmid (A) or with the pEGFP-fused NaPi2 (B) were processed for immunofluorescence as described in the Materials and Methods section. The intrinsic EGFP fluorescence is shown in green, and the actin staining is shown in red. Apical focal planes (A, B) and cross sections along the planes represented by white lanes (a, b) are depicted.

 


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  		Figure 2. The EGFP fluorescence reflects the presence of the NaPi2 cotransporter. OK cells transfected with the wild-type (WT) cotransporter fused to the EGFP were stained with an antibody against an N-terminal peptide, as described in the Materials and Methods section. The EGFP fluorescence is shown in green (A), the antibody staining is shown in red (B), and the merge of both signals is shown in yellow (C). Apical focal planes (A through C) and cross sections (a through c) are shown.

 

Fusion of NaPi2 to EGFP Yields an Active Cotransporter
X. laevis oocytes were injected with cRNA transcribed from WT and EGFP-fused WT plasmids, and Na+-dependent 32P uptake was measured. The 32P uptake of oocytes that expressed the EGFP-fused cotransporter was approximately 20% of that of the eggs that expressed the WT NaPi2. This reduction in the activity of the EGFP-fused cotransporter correlated with a reduced amount of the EGFP-NaPi2 protein detected in Western blot (data not shown).

Fusion of NaPi2 to EGFP Enables Study In Vivo of the Regulation of the Cotransporter
To study the PTH-induced endocytosis of the WT cotransporter, we followed the fate of the EGFP patches on single living OK cells, after addition of PTH. Incubation with PTH induced the retrieval of the EGFP-fused WT cotransporter from the apical membrane, as reflected by the disappearance of apical patches (Figure 3). Preincubation with leupeptin, a lysosomal inhibitor, led to an increase in the fluorescence signal (Figure 3). When we decreased the fluorescence contrast, it became apparent that the patches were removed from the apical membrane after PTH treatment. Therefore, as previously reported for the native cotransporter (6), leupeptine did not interfere with the endocytic step but prevented the degradation of the endocytosed cotransporters. This indicates that fusion of the cotransporter to EGFP did not interfere with its lysosomal degradation. The loss of the apical signal was time dependent: it was clearly detected after 90 min of incubation with PTH and it was completed after 3 h of treatment (Figures 1 and 5).



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  		Figure 3. Fusion of NaPi2 to the EGFP enables the study in vivo of the regulation of the cotransporter. OK cells were transfected with the WT cotransporter fused to the EGFP. Pictures of the same cells were taken at time 0 and 3 h after incubation with 10-8 M parathyroid hormone (PTH), 100 µg/ml leupeptine (LP) plus 10-8 M PTH, or with vehicle (VH) as described in the Materials and Methods section. In the cell that was pretreated with leupeptine and exposed to PTH, two different intensity setups are shown: the first one was similar to the original one at time 0, and the second corresponds to half the intensity. A picture of the culture monolayer, taken by phase contrast microscopy, is also show.

 


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  		Figure 5. Time course of the PTH-induced endocytosis. OK cells were transfected with plasmids encoding the WT cotransporter (A), the double tyrosine plus triple leucine mutant (B), and the cotransporter containing all of the tyrosine, dileucine, and diacidic signals mutated (C). Picture s of the same cells were taken at time 0 and at the indicated times after incubation with 10-8 M PTH. For each group, two cells from different experiments are shown.

 

Role of Known Endocytic Motifs in the PTH-Induced Downregulation of NaPi2
We studied the effect of single (data not shown) or multiple mutations of the tyrosine-, dileucine-, and diacidic- motifs on the regulation of the NaPi2 cotransporter. The double Y402/509 mutant (Figure 4A), the triple LL101/374LI590 mutant (Figure 4B), the Y402/509 + LL101/374LI590 mutant (Figure 4C), and the Y402/509 + LL101/374LI590 + EE81/616 mutant (Figure 4D) yielded cotransporters that retained the fluorescence patches when transfected in OK cells, indicating their proper apical expression. The apical location of all of the mutants was further confirmed by confocal microscopy (data not shown). In all cases, the apical patches disappeared after 3 h of treatment with PTH, whereas incubation with vehicle did not induce any change in fluorescence (Figure 4). Retrieval of the mutated cotransporters was also time dependent: Figure 5 shows the time course of PTH-induced endocytosis in two cells transfected with the WT cotransporter (A) as well as with the Y402/509 + LL101/374LI590 (B) and Y402/509 + LL101/374LI590 + EE81/616 (C) mutants. No differences between the two mutants and the WT cotransporter were detected. In all three groups, the patches were clearly disturbed 90 min after addition of PTH, and the process was completed after 3 h of treatment.



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  		Figure 4. Role of tyrosine-, dileucine-, and diacidic-based motifs on the PTH-induced downregulation of NaPi2. OK cells were transfected with plasmids encoding the double tyrosine Y402/509 mutant (A), the triple dileucine LL101/374 -LI590 mutant (B), the double tyrosine plus triple dileucine mutant (C), and the cotransporter containing all of the tyrosine, dileucine, and diacidic signals mutated (D). As described in Figure 3, pictures of the same cells were taken at time 0 and 3 h after incubation with either 10-8 M PTH or with VH. Pictures of the culture monolayer, taken by phase contrast microscopy, are also shown.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH treatment leads to a reduction in renal Pi reabsorption as a result of a rapid downregulation of the NaPi2 cotransporters (4). This effect can be reproduced in OK cells, a cell line derived from proximal tubules (5,6). PTH-induced downregulation of the NaPi2 cotransporter involves two steps: internalization of the cotransporter into a subapical tubulovesicular compartment (independent of microtubules) and its delivery to lysosomes for degradation (dependent on microtubules) (36). After hormonal treatment, the cotransporters colocalized with the adaptor protein AP2, suggesting that endocytosis could take place in clathrin-coated vesicles (8). Endocytic processes are known to involve at least two types of sorting signals located within cytoplasmic domains of the internalized protein: tyrosine- and dileucine-based motifs, e.g., (9). The aim of this work was to study the implication of tyrosine-, dileucine-, and diacidic-based motifs in the PTH-induced endocytosis of the Na/Pi-cotransporter.

We transfected OK cells with the WT and the different NaPi2 mutants fused to the intrinsically fluorescence protein EGFP. To prove that fusion of EGFP at the cytoplasmic N-terminal tail of the cotransporter did not impair its delivery to the apical surface and that the EGFP-related fluorescence of the fused cotransporter reflected the presence of the NaPi2 protein, we stained transfected OK cells with an antibody against an N-terminal peptide of the cotransporter (2). We found a total overlapping between the intrinsic fluorescence of the fused cotransporter (EGFP signal) and the NaPi2 immunostaining. Both signals revealed a patchy pattern of expression that was reminiscent of the one that can be detected in OK cells after staining with an antibody against the endogenous NaPi4 cotransporter (5). Scanning electron microscopy has shown the presence of clustered microvilli at the apical surface of OK cells corresponding to endogenous NaPi4 patches (5). Cross sections of the transfected cells showed that the EGFP patches also reflected the presence of the cotransporter at the apical cell surface. Therefore, fusion of EGFP at the cytoplasmic N-terminal tail of the NaPi2 did not impair its delivery to the apical surface. Moreover, the intrinsic fluorescence of the EGFP-fused NaPi2 indeed reflected the presence of the cotransporter and mimicked the pattern of expression detected after antibody staining. Furthermore, the transport activity of the WT cotransporter was not disturbed by fusion to EGFP; the reduced activity measured in oocytes was related to a reduced amount of fused protein expressed in the oocyte membrane. Thus, fusion to EGFP may impair the processing and/or the stability of the cotransporter in oocytes rather than the transport function per se.

We have previously reported that PTH treatment of OK cells causes inhibition of Na/Pi-cotransport by increasing the retrieval and lysosomal degradation of the intrinsic (NaPi4) and transfected (NaPi2) type IIa Na/Pi-cotransporters (5,6). The disappearance of patches paralleled the inhibition of the transporter activity. Intracellular accumulation of the endocytosed cotransporters could be observed only if lysosomal degradation was prevented, suggesting that once the cotransporters are internalized they are rapidly degraded (6). Here we report that PTH induces internalization of the EGFP-fused WT cotransporter. Preincubation with leupeptin did not have any effect on the removal of apically targeted cotransporters but prevented the loss of intracellular signal. This observation confirms that after PTH treatment, the EGFP-fused cotransporter has a similar fate than the native protein, i.e., it is targeted for degradation in lysosomes. The PTH-induced endocytosis of the EGFP-fused cotransporter is a time-dependent process that was clearly visible 90 min after addition of PTH and was completed after 3 h of incubation with the hormone. This kinetics of endocytosis of the EGFP-fused WT cotransporter is in agreement with our previous studies in OK cells, in which inhibition of cotransporter activity and removal of apical patches was apparent after 1 h of incubation with PTH and was completed after 4 h of treatment (5). Taken together, these data indicate that fusion of the NaPi2 cotransporter with EGFP does not interfere with the PTH-induced endocytosis and lysosomal degradation of the cotransporter and therefore can be used to study the downregulation of the cotransporter in single living cells. Our approach relies on transiently transfected OK cells expressing NaPi2-related apical patches, which excludes the possibility to perform any type of quantitative analysis. Several attempts to generate stable transfected lines that express the EGFP-fused cotransporters have failed, although we succeeded in the past in generating lines that express the WT cotransporter (5).

The effect of single Y substitutions on the activity and regulation of the type IIa cotransporter expressed in X. laevis oocytes has been reported (33). We found that the Y509A mutant was internalized as efficiently as the WT after pharmacologic activation of protein kinase C, whereas the level of expression of the Y402 mutants was too low to perform the experiment. Replacement of each Y by phenylalanine (F) had a less dramatic effect on the amount of cotransporters detected in the plasma membrane than mutations to alanine (A). On the basis of this information, only Y to F substitutions were used in the present work. Here we report that transfection of OK cells with mutants lacking different combinations of the Y-, LL-, and EE-based signals yielded cotransporters that retained the fluorescence patches, indicating their proper apical expression. The apical location of all of the mutants was further confirmed by confocal microscopy (data not shown).

PTH treatment of OK cells led to an unaltered membrane retrieval of the mutated cotransporters that lacked either the Y or the LL motifs. Y- and LL-based motifs bind to different components of the endocytic machinery (Y motifs bind to the µ chain of the AP complex, whereas the binding site of the LL motif is the ß subunit) and are differentially regulated (21,23,25,27,29; therefore, they can work independent of each other. A construct that contained all of the putative Y and LL motifs mutated was also downregulated, ruling out the simultaneous involvement of both endocytic signals in the regulation of the NaPi2 cotransport. Usually, mutations in the Y- or LL-based signals affect both the endocytosis and the lysosomal degradation of the protein. However, several exceptions to this rule have been published. Thus, Nef-induced CD4 degradation depends on the presence of two independent motifs on Nef: an LL motif controls the endocytosis, while an EE sequence mediates the lysosomal targeting of the Nef/CD-4 complex (11). Nef mutants that lack the EE motif can induce CD4 endocytosis, but the receptor rapidly recycles back to the cell surface. The potential implication of two cytoplasmic diacidic sequences (EE81 and EE616) on the PTH-induced lysosomal degradation of the NaPi2 cotransporter was therefore analyzed. Substitution of these residues either in the WT or in the mutant with the two Y and three LL replaced did not alter the degradation of the cotransporter after PTH treatment. Moreover, the kinetics of endocytosis of the mutated cotransporters were similar to that of the WT cotransporter. These data strongly suggest that tyrosine-, dileucine-, and diacidic-based motifs are not required for the PTH-induced internalization and lysosomal degradation of the NaPi2 cotransporter.

Although a large body of evidence links Y and LL motifs to protein endocytosis of single transmembrane domain proteins, only a few reports connect them with the retrieval of multiple transmembrane spanning proteins, such as the NaPi2 cotransporter. Thus, for GLUT4, a double leucine within the C-terminal tail seems to participate in its rapid endocytosis in the absence of insulin (37,38) and efficient endocytosis of the cystic fibrosis transmembrane regulator requires a tyrosine-based signal (39). The observation that after PTH treatment NaPi2 cotransporters and AP2 colocalized in renal proximal tubules led us to postulate the involvement of Y- and/or LL-based motifs in the cotransporter regulation. Because this hypothesis could not be experimentally confirmed, new paths must been taken. Thus, although speculative, colocalization of NaPi2 cotransporters with components of clathrin-coated vesicles could be explained by the presence of an intermediary factor connecting the cotransporter with the AP2 complex. This indirect connection has been described for the Nef-induced CD4 degradation, where two independent motifs in Nef are responsible for the formation of CD4-specific clathrin-coated pits. Additional experiments must be undertaken to identify the sequence domains and the interacting proteins involved in the PTH-induced downregulation of the NaPi2 cotransporters, thereby controlling the apical expression of this protein, a key step in the regulation of proximal tubular Pi handling.


   Acknowledgments
 
We thank C. Gasser for assistance in preparing the figures. This work was supported by Swiss National Science Foundation Grant 31.46523 (to H.M.), Hartmann Müller-Stiftung (Zurich, Switzerland), Olga Mayenfisch Stiftung, Novartis Foundation, and the Schwerzerische Bank-Gesellschaft (Zurich; Bu 70417-1).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 29, 1999. Accepted for publication April 10, 2000.




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