Karim-Jimenez Z, Hernando N, Biber J, Murer H
Institute of Physiology, University of Zürich, Zürich CH-8057, Switzerland.
Proc Natl Acad Sci U S A. 2000 Nov 7;97(23):12896-901. doi: 10.1073/pnas.220394197.
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.
II型钠-磷共转运体表达于磷重吸收上皮细胞的顶端膜：IIa型存在于近端肾小管，IIb型存在于小肠。甲状旁腺激素（PTH）可使IIa型钠-磷共转运体从顶端膜回收。IIa型共转运体也在负鼠肾（OK）细胞中表达，其表达受PTH调控。在本研究中，我们确定了参与PTH诱导的IIa型钠-磷共转运体回收的分子“结构域”。将野生型小鼠IIa型（mIIa）和IIb型（mIIb）以及几种mIIa - mIIb嵌合体和定点突变体与增强型绿色荧光蛋白融合，并转染到OK细胞中。我们发现，与1 - 34（或3 - 34）PTH孵育后，mIIa而非mIIb被内化并降解。利用嵌合体，我们发现此效应不需要N端和C端，而位于216至658位残基之间的一个“结构域”似乎是必需的。该区域包含两个假定的细胞内环，mIIa和mIIb之间具有高度保守的序列；在最后一个细胞内环中，IIa型的两个带电荷氨基酸（K(503)R(504)）在IIb型中被不带电荷的残基（N(520)I(521)）取代。我们构建了两个这些残基互换的突变体：mIIaNI和mIIbKR。与mIIa类似，mIIbKR突变体在1 - 34 PTH作用下被内吞；相反，mIIaNI表现得与mIIb一样，未被内化。总之，位于IIa型钠-磷共转运体最后一个细胞内环中的一个双碱性氨基酸基序（K(503)R(504)）对其PTH诱导的回收至关重要。
