Recombinant Human Membrane-spanning 4-domains subfamily A member 3 (MS4A3)
Description
Introduction to MS4A3
Membrane-spanning 4-domains subfamily A member 3 (MS4A3) belongs to the MS4A family of proteins, characterized by their four transmembrane domain structure. This family includes other notable members such as MS4A1 (CD20), MS4A2 (FcɛRIβ), MS4A4A, MS4A4E, and MS4A6A, all playing crucial roles in cellular processes including activation, proliferation, and development . Human MS4A3, also known as HTm4 or CD20L, shares approximately 62% amino acid identity with its mouse counterpart and shows about 20% homology with MS4A1/CD20 and MS4A2/FcɛRIβ, with the highest conservation observed in the transmembrane domains . The significance of MS4A3 lies in its restricted expression pattern within specific hematopoietic progenitor cells and its instrumental role in regulating cell cycle progression.
Protein Structure
MS4A3 features the characteristic four transmembrane domains shared by all MS4A family members. Unlike many membrane proteins that localize to the cell surface, MS4A3 is distinctively positioned as a membrane-associated protein in the perinuclear area . This unique subcellular localization suggests potential involvement in nuclear-cytoplasmic communication or regulation of nuclear processes, differentiating it from other membrane proteins and indicating specialized functions within the cell.
Expression Pattern
MS4A3 demonstrates a highly specific expression profile within the hematopoietic system, making it an excellent marker for certain lineages. Extensive analysis through techniques including qRT-PCR, database exploration, and reporter models has established that MS4A3 is predominantly expressed in:
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Bone marrow granulocyte-monocyte progenitors (GMPs)
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Common monocyte progenitors (cMoPs), albeit at lower levels
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Granulocyte progenitors in the bone marrow
Significantly, MS4A3 expression is notably absent in:
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Lymphocytes (T cells, B cells, and NK cells)
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Mature monocytes in peripheral blood
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Eosinophils
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Splenic dendritic cells (conventional DC1, DC2, and plasmacytoid DCs)
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Resident tissue macrophages (including peritoneal macrophages, alveolar macrophages, Kupffer cells, gut macrophages, microglia, and Langerhans cells)
This highly restricted expression pattern has been leveraged effectively in fate mapping studies to trace the development and differentiation of monocytes and granulocytes with unprecedented precision.
Cell Cycle Regulation
A primary function attributed to MS4A3 is its involvement in cell cycle regulation within hematopoietic cells. Research indicates that MS4A3 regulates the G1/S phase transition by controlling Cyclin-Dependent Kinase 2 (CDK2) status . This critical function positions MS4A3 as a key regulator of hematopoietic cell proliferation and differentiation, affecting the development and maintenance of myeloid lineages. The precise molecular mechanisms through which MS4A3 interacts with cell cycle machinery remain an active area of investigation with implications for both normal hematopoiesis and potential pathological conditions.
Hematopoietic Development
The restricted expression pattern of MS4A3 in granulocyte-monocyte progenitors and certain mature myeloid cells strongly indicates its involvement in myeloid development pathways. Notably, the rapid downregulation of MS4A3 expression in blood monocytes, coupled with its maintenance in granulocytes, suggests a potential role in determining cell fate decisions within the myeloid lineage . This differential regulation may contribute to the distinctive developmental trajectories and functional specializations of monocytes versus granulocytes.
Production Methods
While specific production methods for recombinant human MS4A3 are not explicitly detailed in the available literature, related MS4A family proteins are produced using various expression systems that likely apply to MS4A3 as well. Based on related MS4A family member production, recombinant MS4A3 can potentially be expressed in:
Additionally, biotinylation technologies such as the AviTag-BirA system, which has been applied to other MS4A family members, could potentially enhance recombinant MS4A3 functionality for applications requiring protein immobilization or detection .
Protein Characteristics
Recombinant human MS4A3, like other MS4A family members, would be expected to maintain its four-transmembrane domain structure when properly expressed. The protein's perinuclear localization in native settings suggests that recombinant versions for research applications may require specific subcellular targeting sequences or fusion partners to maintain proper functionality and localization in experimental systems.
MS4A3-Based Reporter and Fate Mapping Models
One of the most significant research applications of MS4A3 has been the development of sophisticated genetic reporter and fate mapping models. Researchers have created MS4A3 reporter mouse models by inserting an Ires-tdTomato sequence downstream of the Ms4a3 stop codon, enabling visualization of cells actively expressing MS4A3 .
More advanced MS4A3-based Cre-Rosa TdT fate mapper models have been developed to trace the lineage of cells that have expressed MS4A3 at any point during their development. These innovative models offer several distinct advantages:
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They efficiently trace blood monocytes (97%) and granulocytes (100%)
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They do not trace lymphocytes or tissue dendritic cells
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They allow precise quantification of monocyte contribution to resident tissue macrophage populations during both homeostasis and inflammatory conditions
These tools have revolutionized the study of monocyte fate and function in various physiological and pathological contexts.
Key Research Findings Using MS4A3 Tools
Studies employing MS4A3-based fate mapping approaches have yielded critical insights into monocyte biology and macrophage development:
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The contribution of monocytes to resident tissue macrophage populations varies significantly by tissue type, sex, and age
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MS4A3 expression is rapidly downregulated in circulating blood monocytes while maintained in certain other myeloid lineages
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MS4A3-based models provide unambiguous discrimination between monocytes and dendritic cells in lineage tracing studies
These findings have substantially clarified the complex dynamics of monocyte differentiation and macrophage maintenance across various physiological and pathological states, addressing longstanding questions in the field of myeloid cell biology.
Therapeutic Potential
Given its role in cell cycle regulation and specific expression in certain hematopoietic lineages, MS4A3 represents a promising target for therapeutic interventions addressing disorders of hematopoietic cell proliferation or differentiation. Future research directions could explore:
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MS4A3-targeted strategies for treating hematological malignancies
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Development of MS4A3-based biomarkers for diagnosing and monitoring hematopoietic disorders
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Application of MS4A3 in regenerative medicine approaches involving hematopoietic stem cells
Advanced Fate Mapping Applications
The established utility of MS4A3 in fate mapping studies could be further expanded to address more complex questions in immunology and cell biology:
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Investigation of monocyte contributions to tissue macrophage populations in various disease models
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Analysis of monocyte differentiation dynamics in response to different inflammatory stimuli
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Development of more sophisticated lineage tracing systems based on MS4A3 expression patterns
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please clearly indicate your preference in the order notes, and we will prepare the product accordingly.
Note: All our protein products are shipped with standard blue ice packs. If you require dry ice shipping, please notify us in advance. Additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms offer a longer shelf life of 12 months at -20°C/-80°C.
Target Background
- Research findings indicate that cell surface expression of MS4A3 is a valuable marker for distinguishing granulocyte/macrophage lineage-committed progenitors from other lineage-related cells in early human hematopoiesis. This suggests MS4A3 is a useful tool for monitoring the early stages of myeloid differentiation in human hematopoiesis. PMID: 29274779
- A 7-gene signature, including MPO, CEACAM8, CRISP3, MS4A3, CEACAM6, HEMGN, and MMP8, was identified to accurately predict the primary prefibrotic myelofibrosis group with a sensitivity of 100% and a specificity of 89%. PMID: 27579896
- Our data highlight MS4A3 as a novel direct target of EVI1 in human myeloid cells, suggesting its repression plays a role in EVI1-mediated tumor aggressiveness. PMID: 25886616
- The C-terminal domain of HTm4 is essential for its role in cell cycle regulation. PMID: 15830103
- Elevated levels of HTm4 are associated with neoplasms. PMID: 19818099
Q&A
What is MS4A3 and what cellular processes does it regulate in the hematopoietic system?
MS4A3 is a transmembrane protein belonging to the membrane-spanning 4-domains subfamily A. Recent research has revealed that MS4A3 functions as an important regulator of myeloid differentiation rather than a G1/S phase inhibitor as previously thought. MS4A3 primarily promotes endocytosis of common β-chain (βc) cytokine receptors after GM-CSF/IL-3 stimulation, which enhances downstream signaling pathways that drive cellular differentiation . MS4A3 represents an early marker of myeloid lineage commitment, with expression levels increasing throughout myeloid differentiation . MS4A family proteins broadly function as cell surface signaling and/or intracellular adaptor proteins in immune and epithelial cells, with many members showing upregulation during cellular differentiation processes .
How does MS4A3 expression differ across hematopoietic lineages and differentiation stages?
MS4A3 exhibits a highly lineage-specific expression pattern across hematopoietic cells. Single-cell sequencing analyses have demonstrated that MS4A3 is highly expressed in common monocyte progenitors (cMoPs) but not in common dendritic cell progenitors (CDPs), making it a potential molecular marker to distinguish monocytic from dendritic cell lineages . Unlike other lineage markers such as Cx3cr1 and Lyz2 that have been previously used in fate-mapping approaches, MS4A3 shows more restricted expression to the granulocyte-monocyte progenitor (GMP) lineage . Expression analyses confirm that MS4A3 levels progressively increase during normal myeloid differentiation, suggesting its crucial role in this process .
How does MS4A3 expression influence leukemic stem/progenitor cell behavior in chronic myeloid leukemia?
Meta-analysis of CML transcriptomes has identified low MS4A3 expression as a universal characteristic across three critical aspects of CML pathophysiology: LSPC quiescence, BCR-ABL1-independent tyrosine kinase inhibitor (TKI) resistance, and transformation to blast phase (BP) . This expression pattern suggests MS4A3 plays a central role in regulating LSPC differentiation status. When MS4A3 levels are suppressed, LSPCs can evade βc cytokine-induced differentiation signals and maintain a more primitive, TKI-insensitive state . Experimental validation has confirmed that knockdown of MS4A3 in CML CD34+ cells increases clonogenicity and protects colony formation upon BCR-ABL1 inhibition, while MS4A3 overexpression produces opposite effects, decreasing colony formation and enhancing sensitivity to TKI treatment .
What molecular mechanisms are involved in MS4A3 downregulation in CML stem cells?
Research has identified several mechanisms responsible for MS4A3 suppression in CML stem cells. These include aberrant DNA methylation patterns and dysregulation through a MECOM-C/EBPε transcriptional axis . The abnormal suppression of MS4A3 appears to be a critical alteration that helps LSPCs maintain stemness and resist differentiation signals. This suppression represents a unifying mechanism underlying both CML quiescence in chronic phase and the differentiation block characteristic of blast phase CML . The correlation between MS4A3 expression and CML survival has been analyzed in patient cohorts, suggesting its potential value as a prognostic biomarker .
How does MS4A3 influence cytokine receptor signaling and what downstream pathways are affected?
MS4A3 regulates myeloid cell differentiation through a specific mechanism involving cytokine receptor endocytosis and signaling. Upon GM-CSF or IL-3 stimulation, MS4A3 promotes the endocytosis of common β-chain (βc) cytokine receptors, which enhances downstream signal transduction . Specifically, MS4A3 knockdown abrogates GM-CSF/IL-3-induced full activation of pERK and pSTAT5 Y694 signaling pathways, while MS4A3 overexpression amplifies these signals . Structure-function analyses have revealed that proper bundling of MS4A3's four transmembrane domains is critical for controlling receptor endocytosis, possibly by facilitating surface receptor clustering . Additionally, the N-terminal cytoplasmic domain appears to play a role in the interaction between MS4A3 and GM-CSF receptor .
What genetic approaches are most effective for studying MS4A3 function in hematopoietic cells?
Several complementary genetic approaches have proven effective for investigating MS4A3 function. For in vitro studies, doxycycline-inducible MS4A3 shRNAs provide temporal control over MS4A3 knockdown, allowing researchers to observe resulting phenotypes in colony formation assays and signaling studies . Lentiviral transduction of primary CD34+ cells with these constructs enables manipulation of MS4A3 expression in patient-derived cells . For more permanent genetic modification, CRISPR/Cas9-mediated deletion of MS4A3/Ms4a3 has been utilized .
For in vivo lineage tracing, the Ms4a3Cre fate-mapper mouse model has been developed by inserting an Ires-Cre cassette downstream of the Ms4a3 stop codon and crossing with Rosa26tdTomato reporter strain . This model specifically and efficiently fate-maps granulocytes and monocytes, allowing detailed study of monocyte-derived cell populations . These genetic tools collectively provide robust platforms for dissecting MS4A3 biology across multiple experimental systems.
What assays are most informative for measuring MS4A3-dependent cellular responses?
Several functional assays have proven particularly informative for analyzing MS4A3-dependent cellular responses. Colony-forming assays represent a key method for assessing the impact of MS4A3 manipulation on clonogenicity and TKI sensitivity of hematopoietic progenitors . Long-term culture initiating cell (LTC-IC) assays provide insights into the effects of MS4A3 on stem cell self-renewal capacity .
For analyzing MS4A3's effects on differentiation, flow cytometry assessment of surface markers (CD34, CD38) following cytokine stimulation can reveal how MS4A3 levels influence differentiation trajectories . To examine receptor dynamics, endocytosis assays measuring surface retention of cytokine receptors (GM-CSFR, IL-3R) after ligand stimulation help quantify MS4A3's impact on receptor trafficking . Intracellular phospho-kinase staining for pERK and pSTAT5 allows measurement of how MS4A3 influences signal strength downstream of cytokine stimulation . These complementary assays provide a comprehensive assessment of MS4A3's functional impact on hematopoietic cells.
How can Ms4a3Cre fate-mapping models advance our understanding of monocyte lineage development?
The Ms4a3Cre fate-mapper mouse model represents a significant advance for tracing monocyte-derived cells with high specificity. By inserting an Ires-Cre cassette downstream of the Ms4a3 stop codon and crossing with Rosa26tdTomato reporter mice, researchers have created a system where cells that have expressed Ms4a3 are permanently labeled with tdTomato . This model specifically marks granulocytes and monocytes while excluding dendritic cells, providing superior lineage discrimination compared to previous models using Cx3cr1 or Lyz2 .
The enhanced specificity of the Ms4a3Cre system allows researchers to precisely identify monocyte-derived cells in various tissues and inflammatory conditions, resolving previous limitations in distinguishing monocyte-derived cells from other myeloid populations . This model enables more accurate mapping of cellular ontogeny and fate decisions in the myeloid compartment, offering new insights into developmental hematopoiesis and inflammatory responses that could not be achieved with less specific markers.
What approaches show promise for therapeutic targeting of MS4A3 in leukemia?
Experimental evidence suggests that restoration of MS4A3 expression or function represents a promising therapeutic strategy for targeting TKI-resistant leukemic stem cells. Researchers have manufactured prototype MS4A3-loaded liposomal nanoparticles with a median size of approximately 150 nm, using CD62L coating for targeted delivery to CD34+ cells . These nanoparticles successfully delivered MS4A3-EGFP protein to target cells, with approximately 11% of cells retaining the protein for 24 hours and 6% for 48 hours after a single dose .
In functional studies with CD34+ cells from TKI-resistant accelerated phase CML patients, MS4A3 nanoparticles reduced the CD34+CD38− stem cell population while increasing more differentiated CD34+CD38+ and CD34−CD38+ populations . This was accompanied by reduced colony formation compared to control nanoparticles without MS4A3 payload . These proof-of-concept experiments demonstrate that therapeutic delivery of MS4A3 to leukemic stem/progenitor cells is feasible and may overcome differentiation blockade in TKI-resistant disease.
How might structural modifications of recombinant MS4A3 optimize its therapeutic efficacy?
Structure-function analyses of MS4A3 have revealed critical domains that could be targeted for optimization in therapeutic applications. Research indicates that proper bundling of MS4A3's four transmembrane domains is essential for its control of receptor endocytosis . Deletion of any transmembrane domain compromises MS4A3's function, suggesting that maintaining intact transmembrane architecture is crucial for any recombinant protein design .
The N-terminal cytoplasmic domain also appears to contribute to MS4A3's interaction with GM-CSF receptor, though its deletion only mildly reduces function . Future therapeutic development might focus on creating minimally modified recombinant MS4A3 proteins that maintain these critical structural features while potentially enhancing stability, cell penetration, or resistance to degradation. Optimization of delivery systems, such as refining the nanoparticle formulations already tested in prototype form, will also be essential for translating MS4A3-based therapies toward clinical applications in leukemia treatment.
What is the relationship between MS4A3 expression and response to combination therapies in refractory leukemia?
The discovery that MS4A3 influences sensitivity to differentiating cytokines suggests important implications for combination therapy approaches in refractory leukemia. Since MS4A3 knockdown reduces sensitivity to GM-CSF/IL-3-induced differentiation while preserving CD34+ leukemic stem/progenitor cells, patients with low MS4A3 expression might benefit from targeted therapies that bypass this resistance mechanism .
Combination approaches might include MS4A3 restoration therapy (via nanoparticle delivery) alongside conventional TKIs, potentially sensitizing otherwise resistant leukemic stem cells to differentiation-inducing signals . Alternatively, targeting downstream pathways normally activated by MS4A3-dependent receptor endocytosis might provide a complementary approach. Future clinical research should investigate whether MS4A3 expression levels could serve as a biomarker for predicting response to differentiation-inducing therapies, potentially guiding personalized treatment decisions for patients with TKI-resistant disease .
