Interaction between PKD1L3 and PKD2L1 through their transmembrane domains is required for localization of PKD2L1 at taste pores in taste cells of circumvallate and foliate papillae

Abstract

The polycystic kidney disease 1-like 3 (PKD1L3) and polycystic kidney disease 2-like 1 (PKD2L1) proteins have been proposed to form heteromers that function as sour taste receptors in mammals. Here, we show that PKD1L3 and PKD2L1 interact through their transmembrane domains, and not through the coiled-coil domain, by coimmunoprecipitation experiments using a series of deletion mutants. Deletion mutants lacking the critical interaction region were not transported to the cell surface and remained in the cytoplasm, whereas PKD1L3 and PKD2L1 proteins were expressed at the cell surface when both are transfected. Calcium imaging analysis revealed that neither the coiled-coil domain nor the EF-hand domain located in the C-terminal cytoplasmic tail of PKD2L1 was required for response on stimulation with an acidic solution. Finally, PKD2L1 did not localize to the taste pore but was distributed throughout the cytoplasm in taste cells of circumvallate and foliate papillae in PKD1L3(-/-) mice, whereas it localized to the taste pore in wild-type mice. Collectively, these results suggest that the interaction between PKD1L3 and PKD2L1 through their transmembrane domains is essential for proper trafficking of the channels to the cell surface in taste cells of circumvallate and foliate papillae and in cultured cells.

Figure 1.

Schematic representation of the domain organization of PKD1L3, PKD2L1 and their deletion constructs. Schematic representations show the PKD1L3 (A) and PKD2L1 (B) deletion constructs tested in this study. Black boxes indicate predicted transmembrane (TM) domains. EF, Ca²⁺-binding EF-hand domain; CC, coiled-coil domain.

Figure 2.

Regions required for PKD1L3 and PKD2L1 to interact. Coimmunoprecipitation assays were performed using PKD1L3, PKD2L1, and their deletion mutants. A) Control Western blot analysis indicating expression of PKD1L3, PKD2L1, and their deletion mutants. i) C-terminally truncated PKD1L3 mutants and full-length PKD2L1. ii) PKD1L3 deletion mutants and full-length PKD2L1. iii) PKD1L3TM6-CT and C- or N-terminally truncated PKD2L1 mutants. iv) Full-length PKD2L1 and PKD2L1ΔCT. B) i) Interaction between C-terminally truncated PKD1L3 mutants and PKD2L1. When HA-tagged PKD1L3 or C-terminally truncated PKD1L3 mutants were precipitated, FLAG-tagged PKD2L1 proteins coprecipitated (lanes 1–3). Less FLAG-tagged PKD2L1 protein coprecipitated with the PKD1L3NT-TM6 protein (lane 4). We could not detect any signals for HA-tagged PKD1L3FL in either the lysis (A) or the immunoprecipitation, likely because of low transfer efficiency of high-molecular-mass protein (∼234 kDa) from gels to PVDF membranes. Immunostaining analysis under permeabilized conditions demonstrated that protein expression levels were comparable among PKD1L3FL, PKD1L3NT-TM11, PKD1L3NT-TM10, and PKD1L3NT-TM6 (Fig. 3). ii) Region of PKD1L3 that is required for interaction with PKD2L1. When HA-tagged PKD1L3 deletion mutants were precipitated, FLAG-tagged PKD2L1 proteins were coprecipitated (lanes 5–10). Little FLAG-tagged PKD2L1 protein coprecipitated with the PKD1L3TM11-CT protein (lane 11). iii) Region of PKD2L1 that is required for heteromeric interaction with PKD1L3. When HA-tagged PKD1L3TM6-CT was precipitated, FLAG-tagged PKD2L1 N-terminal or C-terminal truncated proteins coprecipitated (lanes 12–14, 16); in contrast, little PKD2L1CT protein coprecipitated (lane 15). iv) Region of PKD2L1 that is required for homomeric interaction. When HA-tagged PKD2L1 or PKD2L1ΔCT was precipitated, FLAG-tagged PKD2L1 or PKD2L1ΔCT protein coprecipitated (lanes 17–19). Solid arrowheads indicate PKD1L3 deletion mutants; open arrowheads indicate PKD2L1 or PKD2L1 deletion mutants; brackets indicate high-molecular-mass oligomers. Data are representative of ≥3 independent experiments.

Figure 3.

Association of PKD1L3 and PKD2L1 proteins is required for their cell surface localization. HEK293T cells expressing HA-tagged PKD1L3 together with PKD2L1 or PKD2L1 deletion mutants (A) and those expressing HA-tagged C-terminal truncated PKD1L3 mutants in the presence of PKD2L1 (B) were stained with anti-HA antibodies under nonpermeabilized (top panels) or permeabilized conditions (bottom panels). Scale bar = 20 μm. Data are representative of ≥3 independent experiments.

Figure 4.

Regions required for responding to acid stimuli. A) Representative ratiometric images of Fura-2-loaded HEK293T cells. HEK293T cells expressing PKD1L3 together with PKD2L1 or PKD2L1 deletion mutants were stimulated with control buffer (top panels) or buffer containing 25 mM citric acid (pH 2.7) (bottom panels). Color scale bar indicates the ratiometric value of Fura-2 340/380, from 0.5 (cyan) to 1.5 (red) as pseudocolors. B) HEK293T cells expressing PKD2L1 together with truncated PKD1L3 mutants were stimulated with control buffer (top panels) or buffer containing 25 mM citric acid (bottom panels). C) Percentage of responding cells of 100 DsRed2-positive cells. Bars represent means ± se (n=3). *P < 0.05 vs. control; Student's t test.

Figure 5.

Localization of PKD2L1 protein in PKD1L3-knockout mice. A) Schematic representation showing the structure of the PKD1L3 gene and the strategy for generating knockout mice. Targeting construct deleted predicted transmembrane (TM) motifs 7 to 11. Ex, exon; Cre, Cre recombinase gene; Neo, neomycin resistant gene; loxP, loxP site; DT-A, diphtheria toxin A-chain gene; EGF, enhanced green fluorescent protein. B) In situ hybridization experiments demonstrating complete loss of PKD1L3 expression in the taste buds of the circumvallate papillae of PKD1L3^−/− mice and robust expression in wild-type mice. C, D) Sections of circumvallate (C) or foliate (D) papillae in PKD1L3^−/− mice or wild-type littermates were incubated with anti-PKD2L1 (red) and ZO-1 (green) antibodies. PKD2L1 was mainly distributed in the upper half of taste cells, with intensely accumulated signals at the taste pore in circumvallate papillae of wild-type mice, while much stronger PKD2L1 immunoreactivity was observed throughout the cytoplasm in PKD1L3^−/− mice. Dotted lines on differential interference contrast (DIC) images indicate approximate area of the taste buds. Stained images were obtained with a confocal laser-scanning microscope at high magnification. E) Signal intensity in each third of taste bud was quantified using the ImageJ program in circumvallate (a) and foliate (b) papillae. Percentage of PKD2L1 protein distributed in the top third of taste cells in circumvallate and foliate papillae was larger in wild-type mice than in PKD1L3^−/− mice. Bars represent means ± se (n=3). *P < 0.05; Student's t test. Scale bars = 20 μm.