PDZ Adaptor Protein PDZK2 Stimulates Transport Activity of Organic Cation/Carnitine Transporter OCTN2 by Modulating Cell Surface Expression

2006 ◽  
Vol 34 (11) ◽  
pp. 1927-1934 ◽  
Author(s):  
Chizuru Watanabe ◽  
Yukio Kato ◽  
Tomoko Sugiura ◽  
Yoshiyuki Kubo ◽  
Tomohiko Wakayama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Organic Cation ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Carnitine Transporter

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

scholarly journals Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

scholarly journals Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1

1998 ◽  
Vol 331 (1) ◽  
pp. 257-264 ◽  
Author(s):  
Ana M. PAJOR ◽  
Ning SUN ◽  
Heidi G. VALMONTE
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Cell Surface Biotinylation

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopusoocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

Role ofN-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

2012 ◽  
Vol 302 (5) ◽  
pp. C781-C795 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Yukari Yamashita
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Tryptic Digestion ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Biochemical Modification ◽  
Glycosylation Sites ◽  
Increased Susceptibility

SLC26A3 is a Cl−/HCO3−exchanger that plays a major role in Cl−absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

2021 ◽  
Author(s):  
Osamu Yamazaki ◽  
Maho Yamashita ◽  
Jinping Li ◽  
Fumika Ochiai-Homma ◽  
Tadashi Yoshida ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Transport Activity ◽  
Wild Type ◽  
Single Nucleotide Variants

Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69 % reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39 % of that of wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm. These data demonstrate that I551F-NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention, and suggest that the variant can have a dominant-negative effect by forming complexes with wild-type NBCe1-A.

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