SLAMF1 Human, Sf9

What is SLAMF1 and what is its basic structure?

SLAMF1 (CD150) is a type I transmembrane receptor belonging to the SLAM family, which consists of nine distinct receptors (SLAMF1-SLAMF9) expressed on hematopoietic cells. SLAMF1 has an extracellular segment consisting of two Ig-like domains (one V-like variable and one C2-like constant domain), a transmembrane segment, and a cytoplasmic tail containing multiple tyrosine-based motifs (ITSM: TxYxxI/V) . The extracellular domain of SLAMF1 enables it to form homodimers in a "head to head" fashion, meaning that binding occurs between the membrane distal Ig V-like domains as demonstrated by surface plasmon resonance (SPR) studies . Unlike SLAMF3, which has four Ig-like domains, SLAMF1 maintains the standard two-domain structure characteristic of most SLAM family members .

How does SLAMF1 function as a self-ligand in immune cells?

SLAMF1 functions primarily as a homotypic receptor, meaning it engages in self-association when identical receptors on different cells interact. This homotypic interaction initiates downstream signaling cascades that regulate immune cell functions . When SLAMF1 molecules on adjacent cells interact, their cytoplasmic domains recruit signaling adaptors, primarily from the SAP (SLAM Associated Protein) family . This interaction triggers phosphorylation of the ITSM motifs in SLAMF1's cytoplasmic domain, creating docking sites for SAP adaptors. The self-ligand property of SLAMF1 makes it particularly important during immune cell-to-cell contacts, where it contributes to the regulation of cytokine production, cellular activation states, and immune cell development .

What are the primary cell types that express SLAMF1?

SLAMF1 is predominantly expressed on hematopoietic cells, including various immune cell populations. Expression can be found on:

• T lymphocytes, particularly upon activation

• B lymphocytes

• Dendritic cells

• Macrophages

• Natural killer (NK) cells

• Platelets

The expression pattern varies depending on cell activation status and differentiation stage . In certain disease contexts, such as chronic lymphocytic leukemia (CLL), SLAMF1 expression levels have been associated with disease pathobiology and clinical outcomes . In macrophages specifically, SLAMF1 expression is critical for its function in mediating TLR4 signaling and subsequent interferon production in response to bacterial challenges .

How does the SLAMF1-SAP interaction regulate immune cell signaling?

The signaling function of SLAMF1 is largely controlled via its interaction with SAP family adaptors, which include SAP, EAT-2 (Ewing sarcoma associated transcript-2), and ERT (EAT-2-related transducer) . The SAP adaptor associates with the cytoplasmic domain of SLAMF1 through phosphorylated tyrosines within the ITSM motifs .

Upon SLAMF1 engagement and phosphorylation, SAP binds to the phosphorylated ITSMs serving two critical functions:

• It blocks the binding of inhibitory phosphatases such as SHP-1, SHP-2, and SHIP-1, preventing negative regulation

• It enhances downstream signaling by recruiting the Src family kinase Fyn

The binding of Fyn to SAP occurs via the arginine 78 residue (R78) of SAP binding to the SH3 domain of Fyn. Importantly, the R78 residue is located outside the phosphotyrosine binding pocket, allowing for simultaneous interaction between the SLAMF1 ITSM and the SH3 domain of Fyn . This unique configuration of the SLAMF1-SAP-Fyn complex leads to recruitment and phosphorylation of downstream targets such as SHIP-1 docking protein 1 (Dok1), docking protein 2 (Dok2), and the exchange factor RasGAP .

What is the role of SLAMF1 in TLR4-mediated signaling pathways?

SLAMF1 plays a critical role in TLR4-mediated signaling, particularly in the production of type I interferons during responses to Gram-negative bacteria. Research demonstrates that SLAMF1 is required for TLR4-mediated TRAM-TRIF-dependent signaling, which is essential for interferon β (IFNβ) production .

Mechanistically, SLAMF1:

• Controls trafficking of TRAM (Toll receptor–associated molecule) from the endocytic recycling compartment (ERC) to phagosomes

• Physically interacts with TRAM, and this interaction is enhanced upon LPS stimulation

• Regulates TLR4-mediated signaling upstream of TBK1 and IRF3, as evidenced by decreased TBK1 and IRF3 phosphorylation in SLAMF1-silenced macrophages

• Affects MAPK pathway activation, with SLAMF1 silencing resulting in decreased phosphorylation of MAP3K/TAK1 and downstream MAPKs (p38MAPK and JNK1/2)

The importance of SLAMF1 in this pathway is further demonstrated by experiments showing that SLAMF1 overexpression enhances LPS-mediated IFNβ mRNA expression and increases phosphorylation of TBK1, IRF3, and MAPKs . This positions SLAMF1 as a key regulator of endosomal TLR4–TRAM–TRIF signaling, distinct from its role in MyD88-dependent pathways.

How does SLAMF1 function switch between activating and inhibitory roles?

This dual functionality occurs because:

• In the presence of SAP, the adaptor blocks the binding of inhibitory phosphatases (SHP-1, SHP-2, SHIP-1) to the ITSM motifs and recruits activating kinases like Fyn

• In the absence of SAP, these phosphatases can bind to the phosphorylated ITSMs, generating inhibitory signals

This phenomenon is particularly well-documented for other SLAM family members like SLAMF4 and SLAMF6, where SAP deficiency converts them from activating to inhibitory receptors . For SLAMF1 specifically, it has been observed that it can bind to SAP at low affinity even without tyrosine phosphorylation, suggesting a complex regulatory mechanism .

Additionally, the cell type and activation context influence whether SLAMF1 functions as an activating or inhibitory receptor. For example, in CD4 T cells, SLAMF1 engagement lengthens the recruitment of PKCθ to the contact site between T cells and antigen-presenting cells, enhancing NF-κB activation and IL-4 secretion .

What are the recommended methods for studying SLAMF1-TRAM interactions in macrophages?

To study SLAMF1-TRAM interactions in macrophages, researchers should consider the following methodological approaches based on published research:

• Immunoprecipitation (IP) assays:

  • Perform endogenous IPs using anti-SLAMF1 and anti-TRAM antibodies

  • Western blot analysis to detect co-precipitated proteins

  • Include appropriate controls (e.g., MyD88 as a negative control)

• RNA interference:

  • Use siRNAs targeting SLAMF1 in macrophage cell lines (e.g., THP-1) or primary human macrophages

  • Confirm knockdown efficiency by qPCR and Western blot

• Overexpression studies:

  • Transduce primary macrophages with lentiviruses encoding SLAMF1

  • Compare signaling responses between transduced and control cells

• Phosphorylation analysis:

  • Monitor phosphorylation states of downstream signaling molecules (TBK1, IRF3, MAPKs)

  • Compare phosphorylation profiles in SLAMF1-silenced, SLAMF1-overexpressing, and control cells

• Cytokine measurements:

  • Quantify IFNβ, TNF, IL-6, CXCL10, IL-1β, and IL-8 secretion using ELISA

  • Analyze mRNA expression levels by RT-qPCR

For reliable results, stimulate cells with appropriate ligands (e.g., LPS or E. coli particles) and include time-course analyses to capture both early and late signaling events.

What expression systems are most suitable for producing recombinant human SLAMF1?

While the search results don't specifically address expression systems for recombinant SLAMF1, based on the information provided and general knowledge of protein expression systems, the following approaches would be suitable for producing human SLAMF1:

• Mammalian expression systems:

  • HEK293 cells for maintaining proper glycosylation and folding

  • CHO cells for stable expression and potential scale-up

• Insect cell expression (Sf9/Sf21):

  • Baculovirus expression system in Sf9 cells offers several advantages:

    • Higher protein yields compared to mammalian cells

    • Proper folding and post-translational modifications

    • Ability to express complex proteins with disulfide bonds

• Considerations for SLAMF1 expression:

  • Include the extracellular domain (two Ig-like domains) for functional studies

  • Consider adding affinity tags (His-tag, Fc-fusion) for purification

  • For full-length protein, ensure proper transmembrane insertion

• Validation approaches:

  • Confirm proper folding using conformational antibodies

  • Verify functionality through binding assays (e.g., SPR)

  • Assess homotypic interactions that are characteristic of SLAMF1

For structural and functional studies, expression of just the extracellular domain might be preferable, while full-length protein would be necessary for cell-based assays investigating signaling pathways.

How can researchers effectively measure SLAMF1-dependent IFNβ production in response to bacterial stimulation?

Based on the research methodologies described in the search results, researchers can effectively measure SLAMF1-dependent IFNβ production using these approaches:

• Gene expression analysis:

  • Quantify IFNβ mRNA levels using RT-qPCR at various time points following bacterial stimulation

  • Compare expression in SLAMF1-silenced, SLAMF1-overexpressing, and control cells

• Protein secretion measurements:

  • Measure IFNβ secretion in cell culture supernatants using ELISA

  • Include time-course analysis to capture both early and sustained responses

• Downstream signaling readouts:

  • Monitor STAT1 phosphorylation (Y701) as a readout of IFNβ binding to IFNAR

  • Assess expression of IFN-inducible genes like CXCL10

• Bacterial stimulation protocols:

  • Use purified LPS for specific TLR4 activation

  • Alternatively, employ E. coli particles for a more physiological stimulus

  • Consider heat-killed bacteria for safety and experimental consistency

• Signaling pathway analysis:

  • Examine phosphorylation of IRF3, which is critical for early IFNβ transcription

  • Assess TBK1 phosphorylation, which acts upstream of IRF3

  • Analyze MAPK pathway activation (p38MAPK, JNK1/2) that contributes to AP-1 activity and IFNβ expression

This multi-faceted approach allows researchers to comprehensively assess the contribution of SLAMF1 to IFNβ production at both transcriptional and post-transcriptional levels.

What is the role of SLAMF1 in bacterial infection and host defense?

SLAMF1 plays a crucial role in host defense against Gram-negative bacterial infections through several mechanisms:

• Enhancement of type I interferon production:

  • SLAMF1 is required for TLR4-mediated induction of IFNβ in response to Gram-negative bacteria

  • This makes it a potential target for controlling inflammatory responses against these pathogens

• Regulation of bacterial killing:

  • SLAMF1-deficient bone marrow-derived macrophages (BMDMs) show deficiencies in bacterial killing

  • This defect is associated with reduced production of reactive oxygen species (ROS) in response to E. coli

• NOX2 regulation:

  • Mouse SLAMF1 positively regulates NOX2 activity by forming a complex with beclin-1–Vps34–Vps15–UVRAG

  • This contributes to ROS production and subsequent bacterial clearance

• TLR4 signaling from phagosomes:

  • SLAMF1 acts as a critical regulator of TLR4-mediated signaling from the phagosome

  • It interacts with TRAM adapters and class I Rab11 family interacting proteins (FIPs)

  • This interaction facilitates recruitment of adapters to the TLR4 signaling complex

These functions position SLAMF1 as an important component of innate immune responses to Gram-negative bacterial infections, with potential implications for therapeutic interventions targeting bacterial sepsis and inflammation.

How is SLAMF1 involved in cancer progression and immunotherapy?

The search results provide limited direct information about SLAMF1's role in cancer specifically, but do mention SLAM family receptors as potential targets for cancer immunotherapy:

• Expression in hematological malignancies:

  • SLAMF1 has been studied in the pathophysiology of chronic lymphocytic leukemia (CLL) and lymphoma

  • It acts as a negative regulator of IL-10 expression and secretion on B-cell CLL surfaces

  • This negative regulation is associated with favorable clinical outcomes

• Signaling pathway modulation:

  • The interaction of SLAMF1 and CD180 receptor pathways contributes to the pathobiology of B cell CLL through selective suppression of Akt and MAPK signaling

  • This suggests SLAMF1 may influence cancer cell survival and proliferation pathways

• Potential as immune checkpoint:

  • There is growing evidence that SLAM-family receptors have been involved in cancer progression

  • They have been heralded as novel immune checkpoints on T cells, suggesting potential for immunotherapeutic targeting

• Connection to viral oncogenesis:

  • SLAMF1 has been implicated in Epstein-Barr virus (EBV)-positive B cell contexts, suggesting a potential role in virus-associated lymphomagenesis

While specific therapeutic approaches targeting SLAMF1 in cancer were not detailed in the search results, its role in modulating immune responses and potential function as an immune checkpoint makes it a promising target for cancer immunotherapy research, particularly in hematological malignancies.

What are the implications of SLAMF1 dysfunction in immune deficiencies?

While the search results don't detail specific SLAMF1 dysfunction in immune deficiencies, they provide insights into how SLAM family receptors and their associated adaptors contribute to immune regulation and deficiencies:

• SAP-related immunodeficiency:

  • Defects in SLAM family members and SAP adaptors have been implicated in causing immune deficiencies

  • This is exemplified in patients with X-linked lymphoproliferative (XLP) disease, where SAP undergoes a loss of function mutation

  • Since SLAMF1 function is regulated by SAP, SAP deficiency would impact SLAMF1-mediated signaling

• Impact on cytokine production:

  • SLAMF1 contributes to cytokine production, particularly IFNβ, IL-6, and CXCL10

  • Dysfunction could lead to impaired cytokine responses to bacterial infections

• Effects on T cell and B cell function:

  • SAP deficiency has been shown to inhibit T follicular helper (TFH) cell polarization, B cell functions, and germinal center formation

  • This can lead to defects in humoral immunity

  • Since SLAMF1 interacts with SAP, SLAMF1 dysfunction might contribute to these phenotypes

• Bacterial clearance deficiencies:

  • SLAMF1 plays a role in bacterial killing through regulation of ROS production

  • Dysfunction could impair the ability to clear Gram-negative bacterial infections

The complex interplay between SLAMF1 and its signaling partners suggests that dysfunction could manifest in various immune deficiencies, particularly those affecting cytokine production, B cell responses, and bacterial clearance mechanisms.

How does SLAMF1 interaction with other SLAM family members affect immune responses?

The search results don't specifically address SLAMF1 interactions with other SLAM family members, but we can infer potential interactions and effects based on the information provided:

Further research is needed to directly investigate how SLAMF1 functions in the context of other SLAM family members and how these interactions shape immune responses in different cellular contexts.

What are the latest findings on SLAMF1 post-translational modifications and their functional significance?

The search results don't specifically detail the post-translational modifications of SLAMF1, but they do provide information about phosphorylation-dependent signaling that suggests important regulatory mechanisms:

• Tyrosine phosphorylation:

  • The cytoplasmic domain of SLAMF1 contains ITSM motifs (TxYxxI/V) that undergo phosphorylation

  • This phosphorylation creates docking sites for SAP family adaptors

  • SLAMF1 can reportedly bind to SAP at low affinity even without tyrosine phosphorylation, suggesting complex regulation

• Phosphorylation-dependent protein interactions:

  • Phosphorylated ITSMs create binding sites for multiple proteins including:

    • SAP family adaptors (SAP, EAT-2, ERT)

    • SH2 domain-containing phosphatases (SHP-1, SHP-2, SHIP-1)

    • The inhibitory kinase Csk

• Signaling pathway regulation:

  • The formation of SLAMF1-SAP-Fyn complex leads to recruitment and phosphorylation of downstream targets

  • This includes SHIP-1 docking protein 1 (Dok1), docking protein 2 (Dok2), and RasGAP

While specific details about other post-translational modifications (glycosylation, ubiquitination, etc.) are not provided in the search results, the phosphorylation-dependent regulation of SLAMF1 appears to be central to its function in immune signaling.

What computational approaches can predict SLAMF1 binding partners and signaling networks?

The search results don't specifically address computational approaches for studying SLAMF1, but based on the information provided about SLAMF1 structure and signaling, several computational methods would be valuable:

These computational approaches would complement experimental studies and help generate hypotheses about SLAMF1 function in various immune contexts.