Recombinant Mouse Transmembrane protein 87B (Tmem87b)

What is the basic structure of mouse TMEM87B protein?

Mouse TMEM87B (Transmembrane Protein 87B) is a highly conserved transmembrane protein encoded by the Tmem87b gene. The protein contains a signal peptide followed by six transmembrane domains . The full-length mouse TMEM87B protein corresponds to the sequence in RefSeq NP_082524.2, with the gene located on chromosome 2 . The protein structure includes an N-terminal signal peptide that is crucial for proper protein trafficking and membrane insertion. The six transmembrane domains anchor the protein within cellular membranes, with both cytoplasmic and extracellular regions playing potentially important roles in protein-protein interactions and signaling functions .

What is currently known about the physiological function of TMEM87B?

TMEM87B is a protein of currently unknown precise function, although emerging evidence suggests roles in several biological processes. Recent research indicates TMEM87B may play important roles in cardiac development, as evidenced by its association with restrictive cardiomyopathy (RCM) and congenital heart defects . The high evolutionary conservation of TMEM87B across species suggests fundamental biological importance (demonstrated by conservation scores: GERP = 5.66, phastCons = 1, PhyloP = 8.366) . Studies utilizing model organisms and cell lines suggest TMEM87B may function in membrane trafficking or signaling pathways, though specific mechanisms remain to be fully elucidated. The protein's involvement in gene fusion events in cancer also points to potential roles in regulating cell growth, survival, or differentiation pathways when dysregulated .

How evolutionarily conserved is TMEM87B across species?

TMEM87B demonstrates remarkable evolutionary conservation across species, particularly at functional domains. Comparative analysis shows that specific amino acid residues, such as Asn456 in human TMEM87B, are completely conserved across diverse species . This conservation is evidenced by high conservation scores across multiple algorithms (GERP = 5.66, phastCons = 1, PhyloP = 8.366) . The table below illustrates TMEM87B amino acid conservation at key functional residues:

This remarkable evolutionary conservation strongly suggests that TMEM87B performs essential biological functions that have been maintained throughout evolutionary history, making it an important target for comparative functional studies across model organisms .

What are the optimal expression systems for producing recombinant mouse TMEM87B protein?

For recombinant mouse TMEM87B production, mammalian expression systems consistently yield the highest quality protein with proper folding and post-translational modifications. Mammalian cell-based expression (particularly HEK293 or CHO cells) is recommended for preserving native protein conformation and function . When producing recombinant TMEM87B, consider these methodological factors:

• Expression vector selection: Vectors containing strong promoters (CMV for mammalian cells) and appropriate selection markers are essential

• Affinity tags: His-tags at either N- or C-terminus facilitate purification with minimal interference with protein function

• Signal peptide preservation: Maintaining the native signal peptide or using an optimized secretion signal

• Solubilization strategy: Proper detergent selection (e.g., DDM, CHAPS, or Triton X-100) for membrane protein extraction

• Quality control parameters: Size-exclusion chromatography to ensure monodispersity and proper folding

While bacterial expression systems offer cost advantages, they often result in misfolded TMEM87B requiring refolding procedures. Insect cell systems represent an intermediate option balancing yield and proper folding. For studies requiring native-like functionality, mammalian cell-derived TMEM87B (available commercially with >80% purity) remains the gold standard .

What experimental approaches are most effective for studying TMEM87B function in mouse models?

Several complementary experimental approaches can effectively elucidate TMEM87B function in mouse models:

• Genetic manipulation techniques:

  • CRISPR/Cas9-mediated knockout or knockin models to study loss-of-function or specific mutations

  • Conditional knockout models using Cre-loxP system for tissue-specific or temporal control

  • Transgenic overexpression models to study gain-of-function effects

• Phenotypic characterization methods:

  • Cardiac-specific assessments: Echocardiography, electrocardiography, and pressure-volume analysis for functional evaluation, especially focusing on restrictive physiology

  • Developmental analysis: Embryonic assessment at various stages to identify critical developmental windows

  • Histological examination: Tissue-specific changes in TMEM87B knockout or mutant mice

• Molecular profiling:

  • Transcriptomic analysis via RNA-Seq to identify dysregulated pathways

  • Proteomic studies to identify interaction partners and signaling networks

  • Phosphoproteomics to characterize signaling cascade changes

• Cell-based assays using primary cells from mouse models:

  • Isolation of cardiomyocytes, fibroblasts, or other relevant cell types from mutant mice

  • Assessment of cellular phenotypes: migration, proliferation, and survival

  • Signaling pathway analysis, particularly focusing on pathways implicated in cardiac development

When designing these experiments, careful consideration of genetic background effects, compensatory mechanisms, and developmental timing is essential for accurate interpretation of TMEM87B function in vivo .

How can researchers effectively detect TMEM87B-fusion proteins in experimental systems?

Detection of TMEM87B-fusion proteins requires strategic approach selection based on research objectives. For comprehensive characterization, utilize these methodological strategies:

• RNA-level detection:

  • RT-PCR with primers spanning the fusion junction (forward primer in TMEM87B and reverse primer in fusion partner)

  • RNA sequencing with computational algorithms specifically designed to detect fusion transcripts

  • NanoString technology for targeted quantification of known fusion events

• Protein-level detection:

  • Western blotting using antibodies targeting the retained domains of both fusion partners

  • Immunoprecipitation followed by mass spectrometry for unbiased identification

  • Proximity ligation assays to detect protein interactions in situ

• Structural characterization:

  • Genomic approaches including FISH or long-read sequencing to confirm chromosomal rearrangements

  • Chromosome conformation capture techniques (Hi-C, 4C) to identify genomic interactions

For the specific TMEM87B-MERTK fusion identified in cancer research, design PCR primers targeting TMEM87B exon 1 (forward) and MERTK exon 11 (reverse) to amplify across the junction point. In protein analysis, antibodies recognizing MERTK's C-terminal domain will detect both wild-type and fusion proteins, whereas antibodies against TMEM87B's signal peptide domain (amino acids 1-55) can distinguish fusion-specific events when used in combination .

What is the evidence linking TMEM87B mutations to restrictive cardiomyopathy?

Emerging evidence suggests TMEM87B plays a critical role in cardiac development and function, with mutations potentially contributing to restrictive cardiomyopathy (RCM). The most compelling evidence comes from a case study identifying a patient with both a maternally inherited 2q13 microdeletion encompassing TMEM87B and a paternally inherited hemizygous missense variant (c.1366A>G; p.Asn456Asp) in the remaining TMEM87B allele . This variant demonstrates multiple indicators of pathogenicity:

• Extreme rarity in population databases (ExAC: 0.00001649; EVS: 0.000077)

• High conservation scores (GERP = 5.66, phastCons = 1, PhyloP = 8.366)

• Strong predictions of pathogenicity by multiple algorithms (CADD = 29.9, PolyPhen2 = 1, MutationTaster = 1, PROVEAN = -4.92, SIFT = 0)

The variant changes a highly conserved asparagine to aspartic acid at position 456, likely affecting protein function. Importantly, the patient exhibited a clinical presentation of RCM, a rare cardiomyopathy with limited known genetic causes. Follow-up screening of 14 additional RCM patients did not identify additional TMEM87B mutations, suggesting TMEM87B might be associated with a rare form of autosomal-recessive RCM .

While this initial evidence is promising, further functional studies and additional patient cohorts are needed to definitively establish TMEM87B's causal role in RCM pathogenesis. The current evidence supports a model where TMEM87B functions in normal cardiac development, with biallelic disruption potentially leading to restrictive cardiomyopathy .

How does the TMEM87B-MERTK fusion protein contribute to oncogenic signaling?

The TMEM87B-MERTK fusion protein represents a novel oncogenic driver that leverages both structural elements and signaling capabilities to promote cancer cell survival and proliferation. This fusion joins the signal peptide of TMEM87B (amino acids 1-55) with the late extracellular domain, transmembrane, and intracellular kinase domains of MERTK (amino acids 433-1000) .

The oncogenic mechanisms of this fusion protein involve:

• Structural reorganization: The fusion retains MERTK's complete transmembrane and intracellular kinase domains while replacing most of its extracellular domain with TMEM87B's signal peptide .

• Altered expression patterns: The fusion transcript displays increased expression compared to wild-type MERTK in affected cells .

• Constitutive signaling activation: Experimental expression of TMEM87B-MERTK in cell models demonstrated:

  • Constitutively elevated phospho-Akt levels

  • Robust Erk signaling even after serum starvation

  • Maintenance of Akt signaling in growth factor-deprived conditions

• Cellular phenotype effects: In Ba/F3 cells (an IL3-dependent cell line), the fusion protein:

  • Conferred survival advantage after IL3 withdrawal

  • Enabled continued proliferation in the absence of IL3

  • Sustained long-term growth without IL3 for at least one month

• Cross-tissue applicability: Similar activation of Erk and Akt was observed when the fusion was expressed in breast-derived MCF10A cells, indicating the signaling effects are not cell-type specific .

The recurrent identification of identical TMEM87B-MERTK fusions across multiple cancer types (triple-negative breast cancer, lung squamous cell carcinoma, cervical carcinoma, and lung adenocarcinoma) suggests strong selective pressure for this specific rearrangement, highlighting its likely functional significance in oncogenesis .

What is the significance of TMEM87B in the 2q13 microdeletion syndrome?

TMEM87B occupies a critical position within the recurrent 2q13 microdeletion region, suggesting its potential contribution to the syndrome's phenotypic manifestations . The 2q13 microdeletion syndrome presents with variable features including developmental delay, craniofacial abnormalities, and cardiac defects. TMEM87B's significance in this context is multifaceted:

• Positional relevance: TMEM87B is one of the genes consistently included within the boundaries of the 2q13 microdeletion, making it a candidate contributor to the syndrome's phenotype .

• Functional evidence: The identification of a hemizygous pathogenic variant in TMEM87B (c.1366A>G; p.Asn456Asp) in a patient with 2q13 microdeletion who developed restrictive cardiomyopathy suggests TMEM87B's haploinsufficiency may contribute specifically to the cardiac manifestations of the syndrome .

• Dosage sensitivity: While a heterozygous deletion (haploinsufficiency) may contribute to subtle developmental abnormalities, the case study suggests that a "second hit" affecting the remaining TMEM87B allele might be necessary for severe phenotypes like restrictive cardiomyopathy .

• Genotype-phenotype correlation: The variable expressivity observed in 2q13 microdeletion syndrome might be partially explained by varying effects on TMEM87B function, either through different deletion boundaries or through additional variants in the remaining allele .

• Developmental importance: The association with cardiac defects highlights TMEM87B's potential role in cardiac development, making it particularly relevant to the congenital heart defects observed in some patients with 2q13 microdeletion syndrome .

Understanding TMEM87B's specific contribution to 2q13 microdeletion syndrome will require additional functional studies and larger patient cohorts with detailed genotype-phenotype correlations .

How can TMEM87B be targeted for therapeutic development in associated pathologies?

Therapeutic targeting of TMEM87B and its pathological variants requires strategic approaches based on its structure and disease mechanisms. Several potential therapeutic strategies warrant investigation:

• For TMEM87B loss-of-function in cardiomyopathy:

  • Gene therapy approaches to restore functional TMEM87B expression in cardiac tissue

  • Small molecule chaperones to rescue misfolded TMEM87B variants (like p.Asn456Asp)

  • Targeting downstream pathways affected by TMEM87B deficiency once identified through omics studies

• For oncogenic TMEM87B-MERTK fusions:

  • Selective MERTK kinase inhibitors to block constitutive signaling from the fusion protein

  • Proteolysis-targeting chimeras (PROTACs) specifically designed to degrade the fusion protein

  • Antisense oligonucleotides or siRNAs targeting the unique fusion junction

• Discovery pipeline considerations:

  • High-throughput screening using cell models expressing TMEM87B variants

  • Structure-based drug design once the three-dimensional structure is resolved

  • Phenotypic screening approaches in disease-relevant assays

For TMEM87B-MERTK fusions specifically, preliminary data suggests targeting the MERTK kinase domain could be effective. In experimental models, the fusion protein activates Akt and Erk signaling pathways, suggesting inhibitors of these pathways might reverse oncogenic effects . The unique junction between TMEM87B and MERTK also presents an opportunity for highly specific therapeutic targeting that would spare wild-type proteins, potentially reducing off-target effects .

What are the key considerations for designing CRISPR-based knockout studies of TMEM87B?

Designing effective CRISPR-based knockout studies for TMEM87B requires careful consideration of multiple factors to ensure valid and interpretable results:

• Guide RNA (gRNA) design strategies:

  • Target early exons (particularly exons 1-2) to ensure complete loss of function

  • Design multiple gRNAs targeting different exons to confirm phenotypic consistency

  • Perform in silico analysis to minimize off-target effects

  • Consider the 19 coding exons of TMEM87B when designing comprehensive knockout strategies

• Control considerations:

  • Generate paired isogenic control lines from the same parental cells

  • Include rescue experiments with wild-type TMEM87B to confirm specificity

  • Create compound heterozygous knockouts to model patient mutations (e.g., 2q13 deletion + point mutation)

• Phenotypic analysis framework:

  • For cardiac-focused studies, assess contractility, calcium handling, and response to stress

  • Examine developmental timing effects if studying embryonic development

  • Perform comprehensive transcriptomic analysis to identify affected pathways

  • Evaluate protein interaction networks disrupted by TMEM87B loss

• System-specific considerations:

  • For cellular models: Choose relevant cell types (cardiomyocytes, breast epithelial cells)

  • For animal models: Consider developmental lethality possibility and implement conditional knockout strategies

  • For human iPSC models: Differentiate into disease-relevant cell types before phenotypic analysis

• Validation approaches:

  • Confirm knockout at DNA (sequencing), RNA (RT-PCR), and protein (Western blot) levels

  • Address potential compensatory mechanisms by examining related family members

  • Perform complementation studies with specific TMEM87B domains to map functional regions

These considerations ensure robust and reproducible CRISPR-based studies that accurately reflect TMEM87B's biological functions and disease relevance .

How can researchers differentiate between primary and secondary effects of TMEM87B disruption in experimental models?

Differentiating between primary and secondary effects of TMEM87B disruption presents a significant challenge in functional genomics. Researchers should implement these methodological approaches to distinguish direct consequences from downstream effects:

• Temporal analysis strategies:

  • Implement inducible knockout or knockdown systems (e.g., Tet-On/Off, auxin-inducible degron)

  • Perform time-course experiments with dense early timepoints (minutes to hours post-disruption)

  • Utilize pulse-chase labeling to track protein synthesis and degradation kinetics

  • Compare acute versus chronic TMEM87B disruption phenotypes

• Multi-omics integration approaches:

  • Combine transcriptomics, proteomics, and metabolomics at matched timepoints

  • Apply causal network analysis algorithms to infer directionality of effects

  • Identify immediate protein interaction partners through proximity labeling (BioID, APEX)

  • Monitor post-translational modification changes as early indicators of signaling perturbation

• Rescue experiment design:

  • Structure-function analysis with domain-specific mutants to map functional regions

  • Complementation with orthologous TMEM87B from other species to identify conserved functions

  • Targeted rescue of specific downstream pathways to assess their contribution to the phenotype

• Contextual controls:

  • Compare TMEM87B disruption effects across multiple cell types and tissues

  • Assess phenotypes under various stress conditions to unmask context-dependent functions

  • Implement parallel disruption of related genes to identify pathway-specific versus TMEM87B-specific effects

In the case of TMEM87B-MERTK fusion, researchers distinguished primary oncogenic effects by isolating the specific contribution of constitutive MERTK signaling through targeted inhibition of downstream Akt and Erk pathways . Similarly, for TMEM87B's potential role in RCM, developmental timing analyses would help distinguish between direct effects on cardiac development versus secondary adaptive responses .

What are the critical knowledge gaps in understanding TMEM87B structure-function relationships?

Despite emerging evidence of TMEM87B's biological significance, several critical knowledge gaps limit our understanding of its structure-function relationships:

• Three-dimensional structure determination:

  • No high-resolution crystal or cryo-EM structure of TMEM87B currently exists

  • The arrangement and functional significance of the six transmembrane domains remain theoretical

  • Potential conformational changes during protein function are unknown

  • Structural impact of disease-associated variants (e.g., p.Asn456Asp) remains speculative

• Protein interaction network:

  • Direct binding partners in physiological conditions are largely uncharacterized

  • Whether TMEM87B forms homo- or heteromultimers is unknown

  • Potential scaffold functions for signaling complexes have not been investigated

  • Tissue-specific interaction differences have not been mapped

• Post-translational regulation:

  • Phosphorylation sites and their functional significance remain uncharacterized

  • Potential glycosylation patterns that may affect protein localization or function

  • Half-life and degradation pathways regulating TMEM87B levels

  • Subcellular trafficking mechanisms and localization signals

• Tissue-specific functions:

  • Different roles of TMEM87B across tissues (cardiac, neural, others)

  • Developmental stage-specific functions during embryogenesis

  • Cell type-specific expression patterns within complex tissues

  • Functional redundancy with related transmembrane proteins

• Mechanistic basis for disease associations:

  • How TMEM87B contributes to cardiac development and RCM pathogenesis

  • Mechanism by which the TMEM87B portion of the TMEM87B-MERTK fusion affects oncogenic potential

  • Whether TMEM87B function extends to other cellular processes affected in 2q13 microdeletion syndrome

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, proteomics, developmental biology, and disease modeling to fully understand TMEM87B's physiological roles and pathological mechanisms.

How might single-cell approaches advance our understanding of TMEM87B function?

Single-cell technologies offer unprecedented opportunities to resolve TMEM87B function across heterogeneous cell populations and developmental trajectories:

• Single-cell transcriptomics applications:

  • Mapping TMEM87B expression across cell types in developing heart and other tissues

  • Identifying co-expressed gene networks to infer functional pathways

  • Tracking compensatory transcriptional responses to TMEM87B disruption

  • Reconstructing developmental trajectories to pinpoint when TMEM87B function is critical

• Single-cell proteomics approaches:

  • Quantifying TMEM87B protein levels in rare cell populations

  • Measuring post-translational modifications in a cell type-specific manner

  • Correlating TMEM87B levels with activation of downstream signaling pathways

  • Detecting cell-specific protein interaction networks

• Spatial transcriptomics integration:

  • Mapping TMEM87B expression in tissue context to identify spatial patterns

  • Correlating expression with morphological features during heart development

  • Identifying neighboring cell interactions potentially mediated by TMEM87B

  • Localizing TMEM87B-expressing cells within pathological tissues

• Single-cell multi-omics strategies:

  • Combined DNA sequencing with transcriptomics to correlate genetic variation with expression

  • Simultaneous profiling of chromatin accessibility and gene expression to identify regulatory mechanisms

  • Integrating protein and transcript measurements to detect post-transcriptional regulation

• Lineage tracing applications:

  • Tracking fate of TMEM87B-expressing cells during development

  • Monitoring clonal expansion of cells carrying TMEM87B-MERTK fusions in cancer models

  • Studying compensatory proliferation following TMEM87B disruption

For RCM research specifically, single-cell approaches could identify the cardiac cell populations most affected by TMEM87B disruption and reveal cell-autonomous versus non-cell-autonomous effects . In cancer contexts, these approaches could track the emergence and expansion of cells harboring TMEM87B fusion events and characterize their unique molecular signatures .

What experimental models would best advance our understanding of TMEM87B-associated cardiomyopathy?

Developing optimal experimental models for TMEM87B-associated cardiomyopathy requires strategic selection and design approaches:

• Genetically engineered mouse models:

  • Knock-in model of the p.Asn456Asp variant to directly model the patient mutation

  • Conditional knockout using cardiac-specific Cre drivers (αMHC-Cre, Nkx2.5-Cre)

  • Inducible systems to distinguish developmental versus adult functions

  • Combined models with 2q13 microdeletion to recapitulate compound genetic effects

• Human iPSC-derived cardiomyocyte models:

  • Patient-derived iPSCs from individuals with TMEM87B mutations

  • CRISPR-engineered isogenic lines with specific TMEM87B variants

  • 3D cardiac organoids to model tissue-level phenotypes

  • Long-term culture systems to capture late-onset restrictive physiology

• Zebrafish models:

  • CRISPR knockout or knockdown of TMEM87B ortholog

  • High-throughput screening platform for testing genetic interactions

  • Real-time visualization of cardiac development and function

  • Rescue experiments with wild-type versus mutant human TMEM87B

• Ex vivo systems:

  • Langendorff heart preparations from mutant mice

  • Engineered heart tissues incorporating TMEM87B-mutant cells

  • Biomechanical testing systems to measure restrictive physiology parameters

• Molecular and cellular assays:

  • Calcium handling measurements in isolated cardiomyocytes

  • Sarcomere organization and contractility assessments

  • Fibrosis development in cardiac fibroblast co-culture systems

  • Electrophysiological characterization using patch-clamp techniques

The ideal approach would integrate multiple models, particularly comparing findings between mouse models and human iPSC-derived cardiomyocytes to identify conserved mechanisms. Given the association with restrictive cardiomyopathy, special attention should be paid to diastolic function parameters, calcium handling, and fibrosis development across these models .