Recombinant Mouse Transmembrane protein 174 (Tmem174)

What is the molecular structure of mouse TMEM174?

Mouse TMEM174 is a transmembrane protein consisting of 243 amino acids with a molecular weight of approximately 26.4 kDa . The protein contains transmembrane domains that anchor it within cellular membranes, particularly in proximal tubule cells of the kidney. The amino acid sequence of mouse TMEM174 includes specific motifs that enable protein-protein interactions, particularly with the sodium-phosphate cotransporter SLC34A1 (NPT2A) . This structural configuration allows TMEM174 to participate in regulatory complexes that control membrane protein trafficking and cellular phosphate handling. For recombinant expression, the full-length protein (AA 1-243) is typically produced with a His-tag or other purification tags to facilitate isolation and functional studies .

What is the primary physiological function of TMEM174?

TMEM174 functions as a critical regulator of plasma phosphate homeostasis. Its primary role involves decreasing serum inorganic phosphate (Pi) uptake by regulating the sodium-phosphate cotransporter SLC34A1 (NPT2A) trafficking in response to parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) signaling in the kidney . Experimental evidence demonstrates that TMEM174 physically interacts with NPT2A, facilitating its internalization and subsequent degradation when phosphate levels require adjustment. Knockout studies in mice have shown that absence of TMEM174 results in significantly increased serum Pi, FGF23, and PTH levels, ultimately leading to pathological conditions including vascular calcification . This regulatory function positions TMEM174 as a crucial component in the complex network controlling mineral homeostasis.

How does recombinant mouse TMEM174 differ from the native protein?

Recombinant mouse TMEM174 produced for research applications maintains the primary amino acid sequence of the native protein but typically includes additional elements to facilitate purification and detection. The recombinant version is commonly expressed in heterologous systems like HEK-293 cells and includes affinity tags such as His-tag or Strep-tag at the N- or C-terminus . These modifications enable simplified purification through affinity chromatography but may potentially affect certain protein-protein interactions or tertiary structure. Researchers should note that recombinant TMEM174 produced in mammalian expression systems (such as HEK-293 cells) generally provides better post-translational modifications compared to prokaryotic systems, making it more suitable for functional studies that depend on proper protein folding and glycosylation patterns .

What techniques are most effective for detecting TMEM174 expression in tissue samples?

RNA in situ hybridization has proven highly effective for detecting TMEM174 gene expression in renal tissues, allowing for precise localization and relative quantification across different nephron segments and pathological states . This method has successfully demonstrated differential expression patterns across various renal cancer types and normal kidney tissues. For protein-level detection, immunoblot analysis using specific antibodies against TMEM174 can be employed, though researchers should note that some antibodies (like Flare Biotech TMEM174 antibody CSB-PA023755LA01HU) may recognize only denatured TMEM174 protein and not be suitable for immunohistochemistry . Quantitative PCR represents another valuable approach for measuring TMEM174 mRNA levels in experimental systems. The choice of detection method should be guided by the specific research question, with consideration for whether spatial information, protein-protein interactions, or absolute expression levels are most relevant to the study design.

What expression patterns of TMEM174 are observed in renal tissues?

TMEM174 exhibits highly variable expression across different renal tissue types and pathological conditions. RNA in situ hybridization studies have revealed a distinctive pattern:

These differential expression patterns suggest that TMEM174 may play significant roles in the development and progression of specific renal carcinomas, particularly those with high expression levels . The pronounced difference between certain cancer types and normal renal tissue makes TMEM174 a potential biomarker for diagnostic or prognostic applications in renal oncology.

What cell systems are optimal for expressing recombinant mouse TMEM174?

For producing functional recombinant mouse TMEM174, mammalian expression systems, particularly HEK-293 cells, have proven most effective . These cells provide appropriate post-translational modifications and protein folding machinery for transmembrane proteins. Alternatively, cell-free protein synthesis (CFPS) systems have been employed with moderate success, achieving 70-80% purity as determined by SDS-PAGE, Western Blot, and analytical SEC (HPLC) . When designing expression systems, researchers should consider the intended downstream applications. For structural studies requiring high purity, HEK-293 cell expression followed by affinity chromatography using the protein's purification tag is recommended. For functional studies investigating protein-protein interactions, mammalian cells that naturally express interaction partners (such as proximal tubule cell lines) may be preferable to reconstitute physiologically relevant complexes. Expression optimization should include careful consideration of promoter strength, culture conditions, and purification strategies to maximize yield while maintaining protein functionality.

How does TMEM174 regulate NPT2A trafficking in proximal tubule cells?

TMEM174 serves as a critical molecular regulator of NPT2A trafficking in response to phosphaturic hormones. The mechanism involves direct interaction between TMEM174 and NPT2A (detected at approximately 70-110 kDa depending on species and glycosylation state) . When PTH or FGF23-Klotho signaling is activated, TMEM174 facilitates the internalization and subsequent degradation of NPT2A from the apical membrane of proximal tubule cells. Experimental evidence from siRNA knockdown studies demonstrates that TMEM174 depletion blocks the reduction of NPT2A protein typically observed after FGF23 or PTH treatment, resulting in sustained phosphate reabsorption capacity . Interestingly, while TMEM174 is essential for hormone-induced NPT2A trafficking, it does not appear to affect the upstream signaling cascades, as PTH- and FGF23-mediated protein kinase C and extracellular signal-regulated kinase activation remain intact in TMEM174-depleted cells . This suggests that TMEM174 functions downstream of hormone receptor activation, specifically at the level of transporter trafficking.

What are the methodological considerations for measuring Pi uptake in TMEM174 research?

When investigating TMEM174's role in phosphate handling, researchers commonly employ radioisotope-based Pi uptake assays. The standard protocol includes:

• Preparing renal proximal tubule cells (either primary cultures or established cell lines like OK-P cells)

• Treating cells with relevant hormones (PTH, FGF23) and/or TMEM174 modulation (siRNA, overexpression)

• Incubating cells with uptake solution containing 0.1 mM KH₂³²PO₄ (1 μCi) at room temperature for 6 minutes

• Stopping the reaction by washing cells three times with cold stop solution

• Solubilizing cells with 0.1 N sodium hydroxide

• Measuring ³²P signals using liquid scintillation counting

Critical considerations for this assay include maintaining consistent temperature and timing to ensure reproducibility, establishing appropriate controls (untreated cells, scrambled siRNA), and normalizing uptake values to protein content or cell number. Researchers should also consider the half-life of NPT2A (approximately 0.9-1.9 hours depending on experimental conditions) when designing time-course experiments . Alternative non-radioactive methods using fluorescent phosphate analogs may be employed, though these typically offer lower sensitivity than radioisotope-based approaches.

What is the phenotype of TMEM174 knockout mice regarding phosphate metabolism?

TMEM174 knockout (KO) mice exhibit a distinctive phosphate metabolism phenotype characterized by significantly elevated serum inorganic phosphate (Pi) levels compared to wild-type controls . This hyperphosphatemia is accompanied by compensatory increases in two phosphaturic hormones: fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) . Despite these elevated hormone levels, the absence of TMEM174 appears to create resistance to their phosphaturic effects, as NPT2A internalization and degradation are impaired. The physiological consequence of this dysregulated phosphate homeostasis manifests as pathological vascular calcification , a serious complication also observed in various clinical conditions associated with hyperphosphatemia. These findings establish TMEM174 as an essential component of the hormonal regulation of phosphate balance and suggest that therapeutic strategies targeting TMEM174 might be relevant for treating disorders of mineral metabolism. When designing experiments with TMEM174 KO models, researchers should comprehensively assess multiple parameters, including serum biochemistries, tissue calcification, bone metabolism markers, and renal transporter expression patterns.

What evidence links TMEM174 to renal carcinogenesis?

Multiple lines of evidence connect TMEM174 to renal carcinogenesis. RNA in situ hybridization studies have demonstrated markedly elevated TMEM174 expression in several renal cancer subtypes, particularly squamous cell carcinoma with necrosis, papillary renal cell carcinoma, and transitional cell carcinoma . This differential expression pattern, with high levels in specific cancer types compared to extremely weak expression in normal renal tissue, suggests a potential role in tumor development or progression. Mechanistically, TMEM174 has been shown to activate AP-1 transcription factors and promote cell proliferation in 293T cells , which may contribute to the dysregulated growth characteristic of cancer cells. The specific function of TMEM174 in phosphate metabolism may also be relevant, as altered cellular metabolism is a hallmark of cancer, and phosphate serves critical roles in energy metabolism, signal transduction, and nucleic acid synthesis . These connections indicate that TMEM174 may serve as both a potential biomarker for certain renal cancer subtypes and a target for therapeutic intervention, though further research is needed to fully elucidate its role in oncogenic processes.

How is TMEM174 expression altered in different pathological kidney conditions?

TMEM174 expression varies significantly across different pathological kidney conditions, creating a distinctive expression profile that may have diagnostic implications. In inflammatory conditions such as interstitial nephritis, TMEM174 shows a low positive expression rate, while in acute and chronic pyelonephritis, expression is extremely weak . Among malignancies, expression levels create a clear stratification pattern:

This expression pattern does not appear to be a simple function of malignancy, as some cancer types (like collecting duct carcinoma) show extremely weak expression similar to normal tissue . Rather, TMEM174 upregulation seems to be associated with specific oncogenic processes occurring in particular renal cancer subtypes. Understanding these expression patterns could contribute to improved molecular classification of renal pathologies and potentially inform targeted therapeutic approaches.

What are the implications of TMEM174 in vascular calcification models?

Studies with TMEM174 knockout mice have revealed crucial insights into its role in vascular calcification, a pathological process associated with cardiovascular morbidity and mortality. TMEM174 KO mice develop significant vascular calcification as a consequence of disrupted phosphate homeostasis . The mechanism involves elevated serum phosphate levels (hyperphosphatemia) resulting from impaired ability of PTH and FGF23 to downregulate NPT2A in proximal tubules. This creates a physiological environment conducive to ectopic mineralization in vascular tissues. This model provides valuable experimental evidence linking renal phosphate handling directly to vascular health, a connection relevant to multiple clinical conditions including chronic kidney disease and diabetes. For researchers investigating vascular calcification, the TMEM174 KO mouse represents a useful model that develops pathology through an endogenous mechanism rather than requiring exogenous mineral loading. Importantly, these findings suggest that TMEM174 function might be relevant to human conditions characterized by vascular calcification, though translational studies will be needed to confirm this potential clinical significance.

What protein-protein interaction methods are most suitable for studying TMEM174 binding partners?

Given TMEM174's transmembrane nature and role in protein trafficking, specialized approaches for membrane protein interactions are recommended. Co-immunoprecipitation (Co-IP) using antibodies against TMEM174 or its binding partners (particularly NPT2A) has successfully demonstrated their physical interaction . When designing Co-IP experiments, careful selection of detergents is crucial—mild non-ionic detergents like digitonin or DDM better preserve membrane protein interactions compared to harsher ionic detergents. For more sensitive detection, proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) techniques can visualize interactions in intact cells, providing spatial information about where in the cell these proteins associate. Yeast two-hybrid systems, while powerful for soluble proteins, are generally less suitable for transmembrane proteins like TMEM174. For high-throughput identification of novel interaction partners, BioID or APEX2 proximity labeling coupled with mass spectrometry offers advantages for membrane proteins, as these methods can capture transient or weak interactions in the native cellular environment. When using recombinant TMEM174, researchers should consider whether affinity tags might interfere with certain protein-protein interactions.

How can researchers differentiate between direct and indirect effects of TMEM174 on phosphate transport?

Distinguishing direct from indirect effects of TMEM174 on phosphate transport requires a multi-faceted experimental approach. Direct effects typically involve physical interaction with transport machinery (like NPT2A), while indirect effects might operate through signaling pathways or metabolic changes. Research has shown that TMEM174 siRNA treatment does not affect mRNA levels of phosphate transporters (NPT2A, NPT2C, or PiT2) and does not alter PTH or FGF23 signaling cascades, suggesting direct involvement in protein trafficking rather than transcriptional regulation or signal transduction .

To systematically differentiate these mechanisms, researchers should implement:

• Temporal analysis: Monitoring the kinetics of NPT2A protein levels, membrane localization, and phosphate uptake after TMEM174 manipulation

• Domain mapping: Creating truncation or point mutants of TMEM174 to identify regions required for NPT2A interaction versus other functions

• Reconstitution experiments: Testing whether purified recombinant TMEM174 can directly bind NPT2A in cell-free systems

• Live cell imaging: Using fluorescently tagged proteins to track the real-time dynamics of NPT2A trafficking in response to TMEM174 manipulation

• Structure-function studies: Identifying critical residues in both proteins required for their interaction

Additionally, researchers should control for compensatory mechanisms that might mask direct effects, particularly in long-term knockdown or knockout studies where cellular adaptation can occur.

What are the key quality control parameters for recombinant mouse TMEM174 protein preparations?

Ensuring high-quality recombinant mouse TMEM174 preparations is essential for reliable experimental outcomes. Key quality control parameters include:

• Purity assessment: Recombinant TMEM174 should achieve >90% purity when expressed in HEK-293 cells, as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC (HPLC) . For cell-free protein synthesis systems, expected purity is slightly lower (70-80%) .

• Structural integrity: Circular dichroism spectroscopy can verify proper secondary structure formation, particularly important for transmembrane proteins.

• Functional validation: Activity assays demonstrating TMEM174's ability to interact with known binding partners (e.g., NPT2A) or influence phosphate uptake in reconstituted systems.

• Aggregation analysis: Dynamic light scattering or size exclusion chromatography to confirm monodispersity and absence of protein aggregates.

• Endotoxin testing: Particularly important for preparations intended for in vivo studies, as endotoxin contamination can confound experimental results.

• Storage stability: Monitoring protein integrity after freeze-thaw cycles, as membrane proteins are often sensitive to these procedures .