Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (T-m) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthostericbinding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested Lamino acids, but no D-amino acid, induced T-m shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The T-m shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF

A water channel closely related to rat brain aquaporin 4 is expressed in acid- and pepsinogen-secretory cells of human stomach

AbstractWe isolated a cDNA clone encoding a water channel protein, aquaporin (AQP), from human stomach. The encoded protein consisted of 323 amino acid residues, containing six putative transmembrane domains. The protein was designated human aquaporin 4 (hAQP4) because of its 94% sequence similarity to rat brain AQP4. Expression of hAQP4 cRNA in Xenopus oocytes resulted in a significant increase in osmotic water permeability, indicating that this protein functions as a water channel. Northern blot analysis demonstrated a strong signal of hAQP4 mRNA in brain, lung, and skeletal muscle as well as in stomach. Immunohistochemical experiments with human stomach tissues showed that hAQP4 as a protein is expressed mainly in cells located in the glandular portion of the fundic mucosa. These include chief cells which secrete pepsinogen and parietal cells which secrete hydrochloric acid. These results strongly indicate that hAQP4 is a principal factor involved in the osmotic regulation of pepsinogen and acid secretion in the stomach

How has the Covid-19 pandemic affected wheelchair users? Time-series analysis of the number of railway passengers in Tokyo

Abstract The Coronavirus disease 2019 (COVID-19) has posed ‘new barriers’ to people with disabilities (PwDs) who have already experienced many barriers to using public transportation. However, there is limited quantitative knowledge of how PwDs have been affected by the COVID-19 pandemic. This study investigated the impact of the COVID-19 pandemic on the use of public transportation by PwDs over time. Specifically, we analysed time-series data on wheelchair rail passenger numbers and all rail passenger numbers in Tokyo from April 2012 to December 2021. The impact of COVID-19 was more accurately assessed by excluding seasonal variations in the time-series, and two key findings were obtained. First, the change point for the decline in the number of passengers owing to the COVID-19 pandemic was March 2020, one month earlier than the declaration of the state of emergency. Second, using the time-series model, the actual and estimated values were compared, and we found that wheelchair rail passenger numbers reduced by approximately 20 percentage points on average compared with all rail passengers. Wheelchair rail passengers were more severely affected by the COVID-19 pandemic than all rail passengers. Based on previous studies, these findings demonstrated that opportunities to participate in society were disproportionately reduced for PwDs during the COVID-19 pandemic. This study’s quantitative data and the resulting conclusions on wheelchair users are useful for inclusive planning for mitigating the pandemic’s impact by national administrations and public transport authorities

Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3

<div><p>The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.</p></div

Identification of two α-subunit species of GTP-binding proteins, Gα15 and Gαq, expressed in rat taste buds

AbstractWe cloned cDNAs for two G protein α-subunits belonging to the Gαq family, each capable of activating PLCβ, from rat tongue. One is a Gαq in the narrow sense, and the other, termed rat Gα15, is a rat counterpart of mouse Gα15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Gα15 and Gαq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Gα15, together with the Gαq, was localized mainly in taste buds. These studies suggest the possibility that these two Gα proteins function for taste signal transduction in sensory cells

Surface expression of VFTM-truncated human T1R2/T1R3 mutants.

<p>A. Construction of the truncated mutants of human T1R2 and T1R3. All mutants were tagged with c-Myc or FLAG epitopes under the mGluR1 signal peptide. B. Surface expression of the truncated mutants. HEK293 cells expressing mutants were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). C. Immunoblot analysis of truncated mutant-expressing cells. (Left panel) The sample proteins for truncated hT1R2 (CxHH2) or c-Myc tagged hT1R2 (CH2) were obtained through immunoprecipitation from 7.5×10⁵ cells/well using an anti-c-Myc antibody. (Right panel) The cell lysates from 2.5×10⁴ cells/well were used for truncated hT1R3 (FxHH3) or FLAG tagged hT1R3 (FH3) proteins. Arrowheads indicate molecular weights of approximately 33 kDa for both CxHH2 and FxHH3. D. Flow cytometry quantification of cell surface expression of the truncated mutants. Data are expressed as the MFI ratio of FLAG (MFI [FLAG]) labeling in mutant-expressing cells. Statistical significance was calculated by ANOVA followed by Tukey test (*: P<0.05). Error bars: SEM (n = 3). E. Responses of truncated T1rs to cyclamate. Cells expressing CxHH2/FH3 (open triangles) or CH2/FxHH3 (filled squares) showed little response to cyclamate, whereas cells expressing CH2/FH3 (filled circles) did respond to cyclamate. The intensity of the response was represented as the ratio (ΔF) relative to the baseline (F) and was plotted versus ligand concentration. Error bars: SD (n = 3–6).</p

Surface expression of tagged T1r2 and T1r3.

<p>A. Surface expression of tagged T1r2 and T1r3 solely in HEK293 cells. HEK293 cells stably expressing tagged T1r2 or tagged T1r3 were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under non-permeabilized conditions (scale bar = 50 µm). B. Surface expression of tagged T1r3 in COS-7 and CHO cells. Tagged T1r3 were transiently expressed in COS-7 or CHO cells and were labeled with a rabbit anti-FLAG antibody under non-permeabilized conditions (scale bars = 50 µm) C. Tagged T1rs expression in HEK293 cells. The cells were labeled with a rabbit anti-c-Myc antibody or rabbit anti-FLAG antibody under permeabilized conditions (scale bar = 50 µm). D. Surface expression of FH3 with transient CH2 expression. FH3 cells were labeled with a rabbit anti-FLAG antibody under non-permeabilized conditions after 24-h lipofection with CH2 (scale bar = 50 µm). E. Representative flow cytometry histogram data. HEK293 cells were used as a control. F. Flow cytometry quantification of cell surface expression of tagged T1r3 in intact cells. Data are expressed as the mean fluorescence intensity (MFI) ratio of FLAG labeling (MFI [FLAG]) in cells expressing tagged mouse T1r2, T1r3, T1r2/T1r3, human T1R2, T1R3, or T1R2/T1R3 to that in control HEK293 cells. Statistical significance was calculated by ANOVA followed by Tukey test (*: P<0.05). Error bars: SEM (n = 3).</p