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.
Karim-Jimenez
Z
Z
Institute of Physiology, University of Zürich, Zürich CH-8057, Switzerland.
Hernando
N
N
Biber
J
J
Murer
H
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009893:Opossums; D010281:Parathyroid Hormone / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters
D002352:Carrier Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C493554:SLC34A1 protein, human; C493557:SLC34A2 protein, human; C493555:Slc34a1 protein, mouse; C493558:Slc34a2 protein, mouse; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; C031617:parathyroid hormone (3-34)