Recombinant Human Transmembrane protein 174 (TMEM174)

What is the basic structure of human TMEM174?

TMEM174 is a type III transmembrane protein with 243 amino acids that contains no clear signal peptide. It is characterized by two transmembrane helices with both N and C terminals located inside the cell . The transmembrane domains are crucial for its function, as truncated mutants (TMEM174ΔTM) lacking these regions do not retain the protein's normal functions . The protein's transmembrane structure determines its subcellular localization in the endoplasmic reticulum, which directly influences its biological activities .

How is TMEM174 distributed in human tissues?

TMEM174 exhibits tissue-specific expression patterns with particularly high expression in kidney tissue . RNA transcript analysis has consistently demonstrated this kidney-predominant expression profile . Additionally, expression profiling has revealed that TMEM174 is also present in the lymphadenoma-derived Raji cell line, suggesting a potential role in lymphoid tissues . This distinct tissue distribution pattern provides important clues about its physiological functions in renal development and potential involvement in kidney-related pathologies.

What are the primary cellular functions of TMEM174?

Research has established several key functions of TMEM174:

• Cell proliferation promotion - Overexpression of TMEM174 stimulates cell proliferation, with studies indicating a role in promoting the G2/S phase transition of the cell cycle .

• Signal transduction - TMEM174 is linked with Ras and Raf in the extracellular-signal-regulated kinase (ERK) pathway and enhances AP-1 transcriptional activity .

• Phosphate metabolism - TMEM174 interacts with NaPi2a (sodium-dependent phosphate transporter) in renal proximal tubules and is associated with FGF23 induction in bones, contributing to the regulation of plasma phosphate concentration .

These multiple functions suggest TMEM174 plays integrated roles in normal kidney physiology and potentially in pathological processes.

How is TMEM174 gene expression regulated at the transcriptional level?

The transcriptional regulation of TMEM174 involves several key elements:

• Core promoter region - The core promoter of the human TMEM174 gene is located within the region spanning −186 to +674 bp . Dual luciferase reporter assays have demonstrated that fragments spanning −186 to +674, −700 to +674, −1,000 to +674 bp and −2,500 to +1 bp exhibit higher activity levels than other promoter fragments .

• Transcription factor binding - Electrophoretic mobility shift assays (EMSA) have demonstrated specific binding of cyclic-AMP response element binding (CREB) protein within the TMEM174 gene promoter region . Additionally, activator protein-1 (AP-1) shows non-specific binding to the promoter region .

• Regulatory complexity - The TMEM174 promoter contains regions with negative regulatory elements, particularly spanning −186 to −466 and −700 to −890 bp, indicating complex interactions between different regulatory elements .

This complex regulation suggests that TMEM174 expression can be finely tuned in response to various cellular stimuli.

What transcription factors are involved in TMEM174 regulation?

Several transcription factors have been identified or predicted to bind to the TMEM174 promoter:

• CREB (cyclic-AMP response element binding protein) - EMSA experiments have confirmed specific binding of CREB to the TMEM174 promoter . CREB is known to affect multiple cellular activities including glucose homeostasis, growth factor-dependent survival, proliferation, and differentiation .

• AP-1 (activator protein-1) - Though binding is non-specific, AP-1 is implicated in TMEM174 regulation . AP-1 family members are important upstream activators of the ERK signaling pathway involved in cell proliferation, transformation, and differentiation .

• Other potential regulators - Bioinformatic analysis has predicted additional transcription factor binding sites in the promoter sequence, including NF-κB, P300, and Oct1, though their functional relevance requires further validation .

The involvement of these transcription factors, particularly CREB, suggests that TMEM174 expression may be regulated through multiple signaling pathways associated with cell growth and differentiation.

How does TMEM174 interact with the MAPK signaling pathway?

TMEM174 has significant interactions with the MAPK cascade:

• ERK pathway activation - TMEM174 is linked with Ras and Raf in the extracellular-signal-regulated kinase (ERK) pathway . Overexpression of TMEM174 leads to marked activation of upstream molecules in the ERK pathway, including ERK, ELK-1, and Fos .

• AP-1 transcriptional activation - TMEM174 overexpression enhances the transcriptional activity of AP-1, a key downstream target of MAPK signaling . This enhancement is dependent on the transmembrane domains, as truncated mutants lacking these regions do not activate AP-1 .

• Sequential signaling - Studies using sequential blockade of upstream factors in the ERK pathway have demonstrated that TMEM174 functions within this signaling cascade to promote cellular responses .

This integration with the MAPK pathway provides a molecular mechanism for TMEM174's observed effects on cell proliferation and other cellular functions.

What is the relationship between TMEM174 and phosphate metabolism?

TMEM174 plays important roles in phosphate homeostasis:

• Interaction with phosphate transporters - Tmem174 is localized in the renal proximal tubules and directly interacts with NaPi2a (sodium-dependent phosphate transporter), but not with NaPi2c .

• FGF23 regulation - In Tmem174-knockout mice, serum FGF23 (fibroblast growth factor 23) concentration is markedly increased, suggesting that Tmem174 influences the production or clearance of this phosphaturic hormone .

• Response to phosphate load - Tmem174-knockout mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration, and a dietary phosphate load causes marked hyperphosphatemia and abnormal NaPi2a regulation in these animals .

These findings suggest that TMEM174 is an important regulator in the complex network controlling phosphate balance, potentially functioning as a protective factor against hyperphosphatemia during high phosphate loading conditions.

What evidence suggests a role for TMEM174 in cancer?

Several lines of evidence implicate TMEM174 in cancer development:

• Expression in renal cancer - RNA in situ hybridization analyses have shown high expression of TMEM174 in several types of renal cancer, including squamous cell carcinoma with necrosis, papillary renal cell carcinoma, and transitional cell carcinoma .

• Proliferation promotion - TMEM174 stimulates cell proliferation and influences the G2/S phase of the cell cycle, suggesting a potential oncogenic function when dysregulated .

• Association with transcription factors - TMEM174 is regulated by transcription factors like CREB, which has been implicated in tumor development . CREB overexpression confers oncogenic characteristics on cells in various tissues and abnormal CREB expression is associated with tumor development in humans .

• Pathway involvement - TMEM174 activates pathways associated with cell proliferation, transformation, and survival, including the ERK signaling pathway and AP-1 transcription factor activity .

These observations collectively suggest that TMEM174 may contribute to renal cancer development through its effects on cell proliferation and survival pathways.

What developmental roles does TMEM174 play in renal physiology?

TMEM174 appears to have significant roles in renal development and function:

• Kidney-specific expression - The high expression of TMEM174 in kidney tissue suggests tissue-specific functions related to renal development or physiology .

• Phosphate homeostasis regulation - TMEM174 interacts with NaPi2a in renal proximal tubules and influences the body's response to phosphate loading, suggesting a role in maintaining mineral balance critical for proper kidney function .

• FGF23-bone-kidney axis - TMEM174 is thought to be associated with FGF23 induction in bones and regulation of phosphate transporters in the kidney, indicating its involvement in the bone-kidney endocrine axis that regulates mineral metabolism .

• Protection against kidney injury - TMEM174 appears to help prevent increases in plasma phosphate concentration due to high phosphate loads and kidney injury, suggesting a protective role in renal physiology .

These developmental and physiological roles highlight TMEM174's importance beyond simple cellular functions, positioning it as a significant player in kidney development and homeostasis.

What are the optimal methods for studying TMEM174 expression and localization?

For comprehensive analysis of TMEM174 expression and localization, researchers should consider these methodological approaches:

• RNA expression analysis:

  • RT-PCR and qPCR for quantitative expression analysis in tissues and cell lines

  • RNA in situ hybridization for spatial localization in tissue sections, particularly effective for detecting TMEM174 in kidney tissues and renal cancer samples

  • RNA-seq for genome-wide expression profiling in different contexts

• Protein detection and localization:

  • Western blotting using specific antibodies against TMEM174

  • Immunohistochemistry and immunofluorescence for tissue and cellular localization

  • Subcellular fractionation followed by immunoblotting to confirm endoplasmic reticulum localization

• Reporter systems:

  • GFP-tagged TMEM174 constructs for live cell imaging and localization studies

  • Dual luciferase reporter assays for analyzing promoter activity, as successfully used to characterize the TMEM174 promoter regions

These complementary approaches provide a comprehensive understanding of TMEM174 expression patterns across tissues, cells, and subcellular compartments.

What techniques are effective for studying TMEM174 function?

To investigate the multifaceted functions of TMEM174, these methodological approaches are recommended:

• Gain- and loss-of-function studies:

  • Overexpression systems using full-length TMEM174 and domain-deleted variants (such as TMEM174ΔTM)

  • RNA interference (siRNA or shRNA) for knockdown studies

  • CRISPR/Cas9-mediated knockout in cell lines

  • Transgenic or knockout mouse models, as demonstrated with Tmem174-KO mice

• Protein-protein interaction analyses:

  • Co-immunoprecipitation to identify binding partners such as NaPi2a

  • Proximity labeling methods (BioID or APEX)

  • Yeast two-hybrid screening for novel interactors

  • Protein fragment complementation assays

• Signaling pathway analysis:

  • Phosphorylation-specific antibodies to track ERK pathway activation

  • Transcription factor reporter assays, particularly for AP-1 activity

  • Pharmacological inhibitors of specific pathway components for epistasis experiments

  • Phosphoproteomic analysis following TMEM174 manipulation

• Functional assays:

  • Cell proliferation assays (MTT, BrdU incorporation, cell cycle analysis)

  • Phosphate transport assays when studying interactions with NaPi2a

  • Serum biochemistry in animal models to assess effects on phosphate metabolism and FGF23 levels

These diverse experimental approaches enable comprehensive investigation of TMEM174's cellular and physiological functions.

What expression systems are most suitable for producing recombinant TMEM174?

For the production of functional recombinant TMEM174, consider these methodological approaches:

• Mammalian expression systems:

  • HEK293T cells have been successfully used for TMEM174 expression studies

  • Stable cell lines with inducible expression systems (Tet-On/Tet-Off)

  • Optimization considerations: codon optimization, inclusion of affinity tags (His, FLAG, GST) for purification, and signal sequences if secretion is desired

• Specialized expression vectors:

  • Vectors containing strong promoters (CMV, EF1α)

  • Vectors that facilitate proper membrane protein folding and trafficking

  • Bicistronic vectors allowing co-expression with chaperones or folding factors

• Post-translational processing considerations:

  • Since TMEM174 is a transmembrane protein localized to the endoplasmic reticulum , expression systems should support proper membrane insertion

  • Detergent screening for solubilization while maintaining native conformation

  • Lipid nanodisc or liposome reconstitution for functional studies

• Purification strategies:

  • Affinity chromatography using engineered tags

  • Size exclusion chromatography to ensure homogeneity

  • Validation of proper folding through circular dichroism or limited proteolysis

The choice of expression system should be guided by the intended application of the recombinant protein, whether for structural studies, functional assays, or antibody production.

What are the key considerations for designing TMEM174 constructs for research applications?

When designing TMEM174 constructs for various research applications, consider these methodological details:

• Domain-specific constructs:

  • Full-length constructs (1-243 amino acids) for complete functional studies

  • Transmembrane domain mutants (such as TMEM174ΔTM) to study the importance of membrane localization

  • N-terminal and C-terminal domain-specific constructs to dissect function

• Fusion protein design:

  • Reporter protein fusions (GFP, RFP) for localization studies

  • Split protein complementation constructs for interaction studies

  • Proximity labeling fusions (BioID, APEX) to identify the proximal proteome

• Mutation strategies:

  • Site-directed mutagenesis of predicted phosphorylation sites within TMEM174

  • Mutation of residues at the interface with binding partners like NaPi2a

  • Creation of constitutively active or dominant-negative variants

• Regulatory elements:

  • Incorporation of tissue-specific promoters for in vivo studies

  • Inducible expression systems for temporal control

  • Viral vectors for efficient delivery to difficult-to-transfect cells

These design considerations ensure that the recombinant TMEM174 constructs are optimally suited for addressing specific research questions while maintaining relevant biological properties.

What are the most promising areas for future TMEM174 research?

Based on current knowledge, these research directions represent significant opportunities for advancing TMEM174 understanding:

• Structural biology:

  • Determination of TMEM174's three-dimensional structure through X-ray crystallography or cryo-electron microscopy

  • Structural analysis of TMEM174 interaction with binding partners, particularly NaPi2a

  • Investigation of conformational changes associated with activation states

• Physiological roles:

  • Further characterization of TMEM174's role in the kidney-bone axis regarding phosphate homeostasis

  • Investigation of TMEM174 function in additional tissues beyond kidney

  • Exploration of potential roles in other physiological processes beyond known functions

• Disease associations:

  • Comprehensive analysis of TMEM174 expression and mutations in various renal cancers

  • Evaluation of TMEM174 as a biomarker or therapeutic target in kidney diseases

  • Investigation of potential roles in metabolic disorders related to phosphate handling

• Regulatory networks:

  • Comprehensive mapping of TMEM174's position in signaling networks

  • Identification of additional transcription factors beyond CREB and AP-1 that regulate TMEM174

  • Systems biology approaches to understand TMEM174's role in broader cellular and physiological networks

These research directions would significantly expand our understanding of TMEM174 and potentially reveal new therapeutic opportunities.

What methodological advances would benefit TMEM174 research?

Advanced methodological approaches that would enhance TMEM174 research include:

• Advanced imaging techniques:

  • Super-resolution microscopy to better visualize TMEM174 localization in subcellular compartments

  • Live-cell imaging with FRET-based sensors to monitor TMEM174 activity in real-time

  • Correlative light and electron microscopy to link function with ultrastructure

• Proteomics and interactomics:

  • Proximity-dependent biotinylation (BioID, APEX) to identify the TMEM174 proximal proteome

  • Cross-linking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to analyze conformational dynamics

• Genomic and transcriptomic approaches:

  • CRISPR screens to identify genes that modify TMEM174 function

  • Single-cell transcriptomics to understand cell-specific TMEM174 expression patterns

  • ChIP-seq for comprehensive mapping of transcription factor binding to the TMEM174 promoter

• Translational research methods:

  • Patient-derived organoids to study TMEM174 in human disease contexts

  • Tissue-specific conditional knockout models to overcome potential developmental effects

  • Development of specific small molecule modulators of TMEM174 activity

These methodological advances would provide deeper insights into TMEM174 function and regulation, potentially revealing new therapeutic opportunities for diseases involving dysregulation of cell proliferation or phosphate metabolism.

What are common challenges in TMEM174 expression studies and how can they be addressed?

Researchers working with TMEM174 may encounter these experimental challenges:

• Low expression levels:

  • Solution: Optimize codon usage for the expression system

  • Try different promoters (CMV, EF1α) for increased expression

  • Use expression-enhancing elements such as WPRE or Kozak sequences

  • Consider stable cell line generation for consistent expression

• Membrane protein solubilization:

  • Solution: Screen different detergents (DDM, CHAPS, digitonin) for effective solubilization

  • Use mild solubilization conditions to maintain protein folding

  • Consider nanodiscs or amphipols for maintaining native conformation

  • Utilize membrane fractionation approaches for enrichment

• Antibody specificity issues:

  • Solution: Validate antibodies using overexpression and knockout controls

  • Consider epitope-tagged versions of TMEM174 when specific antibodies are unavailable

  • Use multiple antibodies targeting different epitopes for confirmation

  • Perform peptide competition assays to verify specificity

• Functional redundancy in knockout models:

  • Solution: Consider double knockout approaches if redundant proteins are identified

  • Use acute knockdown (siRNA, shRNA) alongside chronic knockout models

  • Employ dominant-negative constructs as alternative approaches

  • Consider tissue-specific or inducible knockout strategies

These troubleshooting approaches address common technical challenges in TMEM174 research and provide practical solutions for researchers in the field.

How can researchers resolve conflicting data regarding TMEM174 function?

When faced with contradictory results in TMEM174 research, consider these methodological approaches:

• Cell type and context specificity:

  • Solution: Compare results across multiple cell lines and primary cells

  • Analyze tissue-specific effects using appropriate model systems

  • Consider microenvironmental factors that might influence TMEM174 function

  • Document experimental conditions in detail to identify variables affecting outcomes

• Isoform and post-translational modification differences:

  • Solution: Verify which TMEM174 isoform is being studied

  • Analyze post-translational modifications in different experimental systems

  • Use site-directed mutagenesis to determine the importance of specific residues

  • Consider the effects of tags or fusion proteins on function

• Temporal aspects of signaling:

  • Solution: Perform detailed time-course experiments

  • Consider acute versus chronic effects of TMEM174 manipulation

  • Analyze feedback mechanisms that might compensate for TMEM174 alterations

  • Use synchronized cell populations for cell cycle-dependent effects

• Methodological validation:

  • Solution: Use orthogonal approaches to validate key findings

  • Implement rigorous controls for all experimental systems

  • Consider replication studies with slight methodological variations

  • Collaborate with other labs to independently verify results

By systematically addressing these potential sources of conflicting data, researchers can develop a more coherent understanding of TMEM174 function across different biological contexts.