Recombinant Pongo abelii Transmembrane protein 174 (TMEM174)

What is the basic structure of TMEM174?

TMEM174 is a type III transmembrane protein that lacks a clear signal peptide, with both N and C terminals located inside the cell. The Pongo abelii (Sumatran orangutan) TMEM174 protein consists of 243 amino acids with a full sequence of: MEQGSGRLEDFPVNVFSVTPYTPSTADIQVSDDDKAGATLLFSGIFLGLVGITFTVMGWIKYKQGVSHFEW TQLLGPVLLSVGVTFILIAVCKFKMLSCQLCKESEERVLDSEQTPGGPSFVFTGINQPITFHGATVVQYIPPPYGSPEPVGINTSYLQSVVSPCSLITSGGAAAAMSSPSQYYTIYPQDNSAFVVDEGCPSFADGGNHRPNPDADQLEETQLEEEACACFSPPPYEEIYSLPR . Transmembrane topology of TMEM174 can be predicted and visualized using tools such as Protter version 1.0, which helps researchers understand the protein's membrane orientation .

How is TMEM174 expressed in normal tissues?

TMEM174 mRNA is easily detectable in human kidney tissues, but its expression in normal renal tissue is relatively weak. RNA in situ hybridization studies have shown that only about 40% of normal renal tissue samples express TMEM174, and those that do show extremely weak expression (scored as 0-1+) . This limited expression in normal tissues contrasts with its higher expression in certain pathological conditions, suggesting potential regulatory mechanisms that govern TMEM174 expression under normal physiological conditions.

What cellular pathways does TMEM174 participate in?

TMEM174 participates in phosphate homeostasis by interacting with NPT2A, a sodium-dependent phosphate transporter in the renal proximal tubule. This interaction affects how NPT2A responds to regulatory hormones like fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) . Additionally, TMEM174 overexpression enhances the transcriptional activity of activator protein 1 (AP-1), which is partly mediated by the ERK pathway. This AP-1 activation contributes to TMEM174's ability to promote cell proliferation .

How does TMEM174 contribute to phosphate homeostasis?

TMEM174 plays a critical role in phosphate homeostasis by interacting with NPT2A, the primary sodium-dependent phosphate transporter in the renal proximal tubule. Research with knockout models demonstrates that TMEM174 is necessary for the proper regulation of NPT2A by FGF23 and PTH. In the absence of TMEM174, the reduction of NPT2A protein levels normally caused by these hormones is attenuated, resulting in hyperphosphatemia . This regulatory mechanism is essential for maintaining appropriate serum phosphate levels, as inorganic phosphate is crucial for numerous biological processes including energy metabolism, bone formation, and signal transduction.

What happens in TMEM174 knockout models?

TMEM174 knockout (KO) mice exhibit significantly increased levels of serum phosphate, FGF23, and PTH. The methodological approach to generating these knockout models involves inserting a NEO cassette onto exons 1 and 2 of the TMEM174 gene . The phenotypic consequences of TMEM174 deletion include:

• Elevated serum phosphate levels

• Compensatory increases in FGF23 and PTH

• Development of vascular calcification

These findings indicate that TMEM174 functions as a negative regulator of phosphate retention, and its absence disrupts the hormonal feedback mechanisms that normally maintain phosphate homeostasis .

How does TMEM174 interact with NPT2A to regulate phosphate transport?

TMEM174 physically interacts with NPT2A in the proximal tubule, affecting its stability and response to hormonal regulators. When TMEM174 is present, NPT2A can be appropriately downregulated by FGF23 and PTH, which is crucial for reducing phosphate reabsorption when serum phosphate levels rise. Knockdown of TMEM174 attenuates the reduction of NPT2A protein by FGF23 and PTH treatments in both human and opossum proximal tubule cells . This interaction can be studied through co-immunoprecipitation experiments and phosphate uptake assays that measure the functional consequences of TMEM174-NPT2A interactions on phosphate transport.

How is TMEM174 expression altered in renal cancers?

TMEM174 exhibits differential expression across various renal cancer types, as demonstrated by RNA in situ hybridization studies. The expression patterns vary significantly by tumor type:

This expression pattern suggests TMEM174 may have specific roles in the development and progression of certain renal carcinomas, particularly squamous cell carcinoma with necrosis, papillary renal cell carcinoma, and transitional cell carcinoma .

Is there a correlation between TMEM174 expression and clinical cancer stages?

Research has examined the relationship between TMEM174 expression and renal pathological clinical stages. The expression rates across different stages are:

Interestingly, Stage III shows the highest TMEM174 expression rate at 71%, while stages II and IV have lower rates at 50% . This non-linear relationship suggests that TMEM174 expression may be influenced by complex factors beyond mere cancer progression, possibly including specific molecular pathways activated at different disease stages.

What is the relationship between TMEM174 and vascular calcification in CKD?

TMEM174 plays a crucial role in preventing vascular calcification, particularly in the context of chronic kidney disease (CKD). TMEM174 knockout mice develop significantly increased levels of serum phosphate, FGF23, and PTH, which consequently results in vascular calcification . The mechanism appears to involve TMEM174's regulation of NPT2A, the primary phosphate transporter in the proximal tubule. When TMEM174 is absent, the normal downregulation of NPT2A by FGF23 and PTH is impaired, leading to hyperphosphatemia. This elevated phosphate level is a major driver of vascular calcification, a serious complication in later stages of CKD that contributes significantly to cardiovascular morbidity and mortality.

What techniques are available for detecting TMEM174 expression?

Several complementary techniques can be employed to detect and quantify TMEM174 expression:

The choice of detection method should be based on the specific research question and available materials, with consideration for species-specific antibody reactivity.

How can researchers generate TMEM174 knockout models?

The generation of TMEM174 knockout models involves several methodological steps:

• Design a targeting vector with a NEO cassette inserted into exons 1 and 2 of TMEM174.

• Amplify DNA fragments encompassing parts of exons 1 and 2 using PCR with appropriate bacterial artificial chromosome (BAC) clone as template.

• Clone a left homologous arm encompassing the DNA sequence upstream of exon 1 at the appropriate restriction site downstream of the Neo cassette.

• Linearize the targeting vector and introduce it by electroporation into embryonic stem cells (e.g., murine B6/129 hybrid EC7.1).

• Select karyotypically normal embryonic stem cell clones and microinject them into blastocysts to produce chimeric founders.

• Backcross the chimeric mice with desired strains (e.g., C57Bl6 or DBA/2J) to establish the knockout line.

• Confirm knockout by PCR genotyping using appropriate primers designed to detect the wild-type and modified alleles .

This approach creates a valuable model for studying the physiological and pathological roles of TMEM174 in vivo.

What assays can measure the functional impact of TMEM174?

Several functional assays can be employed to assess TMEM174's biological activities:

• Phosphate uptake assays: Measure the transport of radioactive phosphate (32P) in proximal tubule cells with manipulated TMEM174 expression. The methodology includes incubating cells with uptake solution containing 0.1 mM KH₂³²PO₄ (1 μCi) for 6 minutes at room temperature, followed by quantification using liquid scintillation .

• AP-1 transcriptional activity assays: Since TMEM174 activates AP-1 and promotes cell proliferation, luciferase reporter assays with AP-1 response elements can be used to measure the functional impact of TMEM174 expression or mutations.

• Co-immunoprecipitation: To detect and study the physical interaction between TMEM174 and other proteins like NPT2A.

• Cell proliferation assays: Measure the effect of TMEM174 expression on cell growth, which is particularly relevant given its role in promoting cell proliferation and potential involvement in cancer development .

• Serum biochemistry: In animal models, measuring serum phosphate, calcium, FGF23, and PTH levels provides critical information about TMEM174's role in mineral homeostasis .

How do post-translational modifications affect TMEM174 function?

The function of transmembrane proteins is often regulated by post-translational modifications such as phosphorylation, glycosylation, and ubiquitination. For TMEM174, examining these modifications would provide insights into how its activity is dynamically regulated. Researchers should consider:

• Identifying potential phosphorylation sites in the cytoplasmic domains of TMEM174 and determining which kinases target these sites

• Investigating whether TMEM174 undergoes ubiquitination that might regulate its degradation or trafficking

• Determining if glycosylation affects TMEM174's stability or interaction with partners like NPT2A

Methodological approaches should include mass spectrometry to identify modifications, site-directed mutagenesis to test their functional significance, and phospho-specific antibodies to monitor regulatory events in response to various stimuli. These studies would help elucidate how TMEM174's activity is fine-tuned in response to changing physiological conditions.

What is the evolutionary conservation of TMEM174 across species?

Understanding the evolutionary conservation of TMEM174 could provide valuable insights into its fundamental biological importance. Current research has primarily focused on human, mouse, and Pongo abelii TMEM174, but comprehensive phylogenetic analysis is lacking. Researchers should:

• Compare TMEM174 sequences across vertebrate species to identify conserved domains that likely serve critical functions

• Determine when TMEM174 emerged during evolution and whether it correlates with specific physiological adaptations

• Assess whether TMEM174's role in phosphate regulation is conserved across species with different physiological demands

Methodologically, this would involve bioinformatic analysis of sequence databases, expression analysis in diverse species, and functional studies in evolutionary distant model organisms. The findings could reveal whether TMEM174's role in phosphate homeostasis represents an ancient regulatory mechanism or a more recent evolutionary adaptation.

How does the interactome of TMEM174 change in disease states?

TMEM174 interacts with NPT2A to regulate phosphate transport, but its complete interactome, particularly under pathological conditions, remains largely unexplored. Advanced research should:

• Perform comprehensive protein-protein interaction studies using proximity labeling techniques (BioID or APEX) coupled with mass spectrometry to identify the complete TMEM174 interactome

• Compare TMEM174's interaction partners in normal versus cancerous renal tissues

• Investigate how these interactions change in response to altered phosphate levels or hormonal signals

This research would require developing tools for proximity labeling of TMEM174 in appropriate cell models, followed by proteomic analysis and validation of key interactions through orthogonal methods. Understanding the dynamic interactome would provide mechanistic insights into how TMEM174 contributes to both normal physiology and disease pathogenesis.