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The identification of Histidine 712 as a critical residue for constitutive TRPV5 internalization.

ABSTRACT: The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura-2 analysis. Removal of amino acid His(712) elevated the [Ca(2+)](i), indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His(712) for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His(712) was confirmed by patch clamp analysis, which demonstrates increased Na(+) and Ca(2+) currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca(2+)-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca(2+)](i). Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His(712) plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.

SUBMITTER: de Groot T

PROVIDER: S-EPMC2937873 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The identification of Histidine 712 as a critical residue for constitutive TRPV5 internalization.

de Groot Theun T Verkaart Sjoerd S Xi Qi Q Bindels René J M RJ Hoenderop Joost G J JG

The Journal of biological chemistry 20100713 37

The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity ...[more]

PMID: 20628046

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Project description:Susceptibility to ecotropic murine leukemia viruses (MLV) is restricted to mice and rats at the level of virus binding to the host cell receptor. Asparagine 232, valine 233, tyrosine 235, and glutamic acid 237 in the third extracellular domain (EL3) of the receptor are critical determinants of the host range difference between mice and humans. However, placing these residues in the human homolog confers only partial binding, indicating that other divergent sequences are involved. We sought to determine if the other sequences lie within or outside EL3. Here we report the identification of lysine 234 as another critical residue that influences virus binding and infection, as well as evidence that the unidentified sequences lie outside EL3. Each of the four basic residues in the third extracellular domain were changed to an acidic residue and initially examined in combination with a change at position 235 or position 237. Substitution of lysine 211, 215, or 222 combined with substitution of the critical tyrosine 235 or glutamic acid 237 did not affect virus infection. However, combined substitution of lysine 234, a conserved residue between mice and humans, and tyrosine 235 resulted in a marked decrease in virus infection and binding. A lysine 234 change alone reduced virus binding, contrary to previous observations that at least two of the other four residues must be changed before binding is reduced. Interestingly, there was no decrease in infection when lysine 234 was replaced in combination with glutamic acid 237. This result suggests that residue 234 may act by influencing the local structure of residues 233 to 235, whereas the presence of a glycine at position 236 may prevent this influence from extending to residue 237. With this report, the involvement of all the residues divergent between mice and humans in the third extracellular domain has been ruled out, suggesting that as yet unidentified determinants lie in other extracellular domains.

Project description:BackgroundAtypical chemokine receptors (ACKRs) play an important role in regulating the availability of chemokines and are responsible for the formation of chemokine gradients required for the directed migration of immune cells in health and disease. ACKR4 shapes gradients of the chemokines CCL19 and CCL21, which are essential for guiding leukocyte homing to lymphoid organs where they initiate an adaptive immune response against invading pathogens. How ACKRs internalize and scavenge chemokines on the molecular level remains poorly understood. Current state-of the art methods to study βarrestin recruitment, signaling and trafficking of ACKRs - and G-protein-coupled receptors in general - rely heavily on C-terminally tagged receptors with unknown consequences for receptor functions.MethodsFluorescently labelled CCL19 was used to quantify chemokine internalization by native and tagged receptors as assessed by flow cytometry and live cell confocal microscopy. Steady-state interaction and chemokine-driven recruitment of βarrestins was determined by NanoBiT bystander assays. βarrestin-dependency for CCL19 internalization was determined in wild-type versus βarrestin1/2-double deficient cell lines. Statistical significance was determined by unpaired t-test or one-way ANOVA with Dunnett's or Tukey's multiple comparison tests.ResultsAddition of a C-terminal tag selectively affected the function of ACKR4, but not other ACKRs. Fusing a short peptide tag or a fluorescent protein to ACKR4 significantly augmented its ability to internalize its cognate ligand CCL19. In comparison to native ACKR4, its C-terminal tagging provoked an elevated pre-association of βarrestins with the plasma membrane, yet a reduction in chemokine-driven βarrestin recruitment. Furthermore, the addition of a C-terminal tag led to a shift from a βarrestin-dependent towards a βarrestin-independent endocytosis pathway. Similar results on chemokine uptake and on βarrestin-dependency were obtained with ACKR4 variants, in which a putative class II PDZ-binding domain located at the C-terminal tip of the receptor was mutated.ConclusionThis study identifies that the integrity of the C-terminus of ACKR4 is critical for receptor function. The addition of a C-terminal tag to ACKR4 enhances chemokine uptake and alters the involvement of βarrestins in receptor trafficking.

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The identification of Histidine 712 as a critical residue for constitutive TRPV5 internalization.

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The identification of Histidine 712 as a critical residue for constitutive TRPV5 internalization.

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