Protein kinase C activators induce membrane retrieval of type II Na<sup>+</sup>‐phosphate cotransporters expressed in <i>Xenopus</i> oocytes

Forster, Ian C.; Traebert, Martin; Jankowski, Maciej; Stange, Gerti; Biber, Jürg; Murer, Heini

doi:10.1111/j.1469-7793.1999.0327t.x

Cited by 43 publications

(50 citation statements)

References 41 publications

(93 reference statements)

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“…7, none of these mutations had an effect on the PTH response. In agreement with this observation, removal of all protein kinase C sites on the rat IIa was unable to prevent the protein kinase C-dependent effect when tested in the Xenopus laevis oocytes system (48,49).…”

Section: K503r504 Residues Aresupporting

confidence: 65%

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

Karim

Hernando

Biber

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: K503r504 Residues Aresupporting

confidence: 65%

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

Karim

Hernando

Biber

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…The PMA effect in oocytes could not be mimicked by the inactive phorbol ester 4␣-PDD. However, only partial suppression of DOG inhibition was produced by staurosporine, in both oocytes and HEK293 cells, corresponding to previous findings for NaPi-II (30). This might be partly attributable to the opposing (i.e., inhibitory) effect observed with staurosporine alone.…”

Section: Discussionsupporting

confidence: 53%

“…This investigation was designed to determine whether transport inhibition occurs at the cell membrane or is achieved via carrier internalization and whether any of the classic PKC consensus sites of hOAT1 are involved. As our main expression system, we used X. laevis oocytes, which have been used for a variety of studies on the regulation of carriers and ion channels, such as the sodium/dicarboxylate cotransporter NaDC-1 (32), the sodium/phosphate cotransporter NaPi-II (30,34), the sodium/ glucose cotransporter SGLT1 (35), and the epithelial Na ϩ channel ENaC (36). In some cases, this system reflects regulation in the original cellular environment more accurately than does the HEK293 cell system (37).…”

Section: Discussionmentioning

confidence: 99%

“…This effect could not be mimicked by the inactive phorbol ester 4␣-PDD, which slightly increased rather than decreased PAH uptake in hOAT1-expressing oocytes (122.7 Ϯ 10.2% of uptake in the absence of 4␣-PDD, three independent experiments). Similar to PMA, the more PKC-specific agent DOG, which is thought to lack the artifactual effects on Xenopus oocytes observed for phorbol esters (29,30), strongly re-duced hOAT1-mediated PAH uptake, by an average of 82.7 Ϯ 2.9% (n ϭ 17). The time course of DOG inhibition was similar to that observed for PMA ( Figure 3A), whereas the IC 50 was higher for DOG (average of 1 M) ( Figure 3B).…”

Section: Downregulation Of Hoat1-mediated Transport By Agents Stimulamentioning

confidence: 99%

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Protein Kinase C Activation Downregulates Human Organic Anion Transporter 1-Mediated Transport through Carrier Internalization

Wolff¹,

Thies²,

Kuhnke³

et al. 2003

Journal of the American Society of Nephrology

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Abstract. Organic anion transport in intact renal proximal tubule cells in animal model systems is downregulated by treatments that activate protein kinase C (PKC). How this downregulation is achieved is not yet known. Stimulation of PKC with sn-1,2-dioctanoylglycerol resulted in strong inhibition of p-aminohippurate transport mediated by the cloned human organic anion transporter 1 (hOAT1) expressed in Xenopus oocytes and HEK293 cells, as well as hOAT1 internalization in both expression systems. The sn-1,2-dioctanoylglycerol-induced transport inhibition was partially prevented by staurosporine. It was independent of the conserved canonical PKC consensus sites in hOAT1, however, and was unaffected by agents that destabilize actin filaments or microtubules, which altered baseline hOAT1-mediated p-aminohippurate uptake activity in oocytes. It is concluded that PKC-induced hOAT1 downregulation is achieved through carrier retrieval from the cell membrane and does not involve phosphorylation of the predicted classic hOAT1 PKC consensus sites.The renal secretory organic anion transport pathway plays an important role in the elimination of a variety of potentially toxic environmental chemicals, such as phenoxyacetate herbicides and fungicides, as well as anionic drugs, including ␤-lactam antibiotics, cytostatic agents, diuretics, and antiviral drugs (1,2). The importance of this pathway is exemplified by the increased toxicity of methotrexate when it is coadministered with nonsteroidal anti-inflammatory drugs (3), which compete for the same elimination pathway (4) and thus lead to higher serum methotrexate concentrations (3). In a number of nonmammalian and mammalian renal proximal tubule model systems, peritubular uptake and/or transepithelial secretion of prototypical organic anions have been demonstrated to be inhibited by agents that stimulate protein kinase C (PKC) activity. The nonselective PKC activator phorbol-12-myristate-13-acetate (PMA) inhibited cellular and luminal fluorescein accumulation in isolated nonperfused killifish renal tubules (5), net secretion of 2,4-dichlorophenoxyacetic acid across winter flounder proximal tubule monolayer cultures (6), peritubular uptake and transepithelial fluorescein flux in isolated nonperfused (7) and perfused (8) rabbit proximal tubule S2 segments, respectively, and basolateral uptake and transcellular movement of p-aminohippurate (PAH) in opossum kidney cells (9,10).Because a number of agents, such as parathyroid hormone (PTH) (11), ␣ 1 -adrenergic agonists (12), angiotensin II (13), and bradykinin (14), modulate renal proximal tubule function via activation of PKC, these agents might be expected to impair renal elimination of organic anions. Indeed, bradykinin and phenylephrine were observed to inhibit basolateral uptake (7) and transepithelial secretion (8) of fluorescein in isolated rabbit proximal tubule S2 segments, and these effects were suppressed by the PKC inhibitor bisindolylmaleimide.On the basis of their localization, two of the cloned organic anion transpo...

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“…In a previous study we have shown that, in oocytes, transport activity correlates with the transporter present in the surface membrane but not with the total amount of protein (23). Here, we have measured Na ϩ -dependent P i uptake and its pH dependence for all the different constructs.…”

Section: Resultsmentioning

confidence: 99%

Molecular Determinants of pH Sensitivity of the Type IIa Na/Pi Cotransporter

Horra

Hernando

Lambert

et al. 2000

Journal of Biological Chemistry

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Type II Na/P i cotransporters play key roles in epithelial P i transport and thereby contribute to overall P i homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N-and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P i cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P i cotransporter.The kidney and the small intestine are involved in maintaining overall phosphate (P i ) homeostasis. Renal tubular P i reabsorption contributes to "acute" and "chronic" regulatory control, whereas only slow "adaptive" changes occur in small intestinal P i absorption (1-4). Na/P i cotransporters located in renal proximal tubular and small intestinal brush border membrane are the rate-limiting and physiologically controlled steps (4 -6). Recently they have been structurally identified as type II Na/P i cotransporters (7-11): the proximal tubule expresses the type IIa isoform (7, 12), whereas the type IIb isoform in mammals is preferentially expressed in small intestine and in other tissues such as lung, colon, liver, and testis but not in kidney (11).Mouse IIa and IIb cotransporters show an overall identity of 57%, which increases to 75% in the predicted transmembrane domains (11). Most of the differences are found in the N-and C-terminal regions (11). A topology model of both cotransporters, based on hydropathy analysis and epitope tagging, predicts eight transmembrane segments, with the N-and C-terminal tails facing the cytoplasm and a large hydrophilic loop between the third and fourth transmembrane domains (7,11,13). This large loop is glycosylated (14).The kinetic properties of mouse IIa and IIb cotransporters have been characterized after expression in Xenopus laevis oocytes. There are some differences in the affinities for the substrates, Na ϩ and P i (11). Moreover, IIa and IIb show different pH dependence of both 32 P i uptake and P i -induced electrogenic responses (11,15). For the IIa, a decrease in external pH results in a sign...

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Protein kinase C activators induce membrane retrieval of type II Na⁺‐phosphate cotransporters expressed in Xenopus oocytes

Cited by 43 publications

References 41 publications

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

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

Protein Kinase C Activation Downregulates Human Organic Anion Transporter 1-Mediated Transport through Carrier Internalization

Molecular Determinants of pH Sensitivity of the Type IIa Na/Pi Cotransporter

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Protein kinase C activators induce membrane retrieval of type II Na+‐phosphate cotransporters expressed in Xenopus oocytes

Cited by 43 publications

References 41 publications

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

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

Protein Kinase C Activation Downregulates Human Organic Anion Transporter 1-Mediated Transport through Carrier Internalization

Molecular Determinants of pH Sensitivity of the Type IIa Na/Pi Cotransporter

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Protein kinase C activators induce membrane retrieval of type II Na⁺‐phosphate cotransporters expressed in Xenopus oocytes