Recombinant Rat Linker for activation of T-cells family member 2 (Lat2), partial

What is the primary function of LAT2 in T cell signaling pathways?

LAT2 (also known as NTAL or LAB in some contexts) functions as a transmembrane adaptor protein in T cells, resembling LAT (Linker for Activation of T cells) in structure but with distinct signaling properties. Unlike LAT, LAT2 does not possess a consensus binding motif for PLCγ1 and PLCγ2 . LAT2 contains a short extracellular domain, a single transmembrane region, and a cytoplasmic tail with palmitoylation cysteine residues and evolutionary conserved tyrosine-containing motifs. Five of these motifs are of the YXN type, serving as potential binding sites for the SH2 domain of the cytosolic adaptor protein Grb2 .

How does the amino acid sequence of rat LAT2 differ from human LAT2?

While the search results don't provide the complete sequence comparison between rat and human LAT2, similar transmembrane adaptor proteins show species-specific variations that affect functionality. For example, in amyloid-beta precursor protein (APP), three amino acid differences between rodent and human sequences significantly affect medically important features . In the case of LAT2, species-specific variations likely occur in key functional domains, particularly in the tyrosine-containing motifs that serve as docking sites for signaling molecules.

What expression systems are most effective for producing recombinant rat LAT2?

Based on successful recombinant protein expression strategies for rat proteins, bacterial and insect cell systems offer distinct advantages:

Bacterial expression system (E. coli):

• Suitable for producing rat proteins in soluble form, as demonstrated with rat calpain II large subunit (80 kDa)

• Yields approximately 1 mg/liter of culture

• Recommended for non-glycosylated domains of LAT2

Insect cell expression system (Baculovirus):

• Superior for mammalian proteins requiring post-translational modifications

• Can achieve high yield (up to 20% of cell protein)

• Properly processes signal peptides from rat proteins

• Recommended for full-length LAT2 or domains requiring glycosylation

What purification strategies should be employed for recombinant rat LAT2?

Purification of recombinant rat LAT2 should involve a multi-step approach:

• Initial capture: Affinity chromatography using anti-LAT2 antibodies or engineered tags (His-tag as used for rat Tie-2)

• Intermediate purification: Ion exchange chromatography to separate based on charge differences

• Polishing step: Size exclusion chromatography to achieve high purity

• Quality assessment: SDS-PAGE under reducing and non-reducing conditions to verify integrity and purity

For membrane proteins like LAT2, consider detergent solubilization before purification, using mild detergents such as CHAPS or DDM to maintain protein structure and function.

How can researchers assess the phosphorylation state of recombinant rat LAT2?

Assessment of LAT2 phosphorylation requires a multi-method approach:

Experimental design:

• In vitro phosphorylation assay: Incubate purified recombinant LAT2 with recombinant protein kinases (PKC or Src family kinases) and [γ-32P]ATP

• Mass spectrometry analysis: For identification of exact phosphorylation sites

• Phospho-specific antibodies: Western blotting using antibodies that recognize phosphorylated tyrosine residues

• Mutagenesis studies: Site-directed mutagenesis of putative phosphorylation sites (similar to the approach used for LAT2/SLC7A8)

In the case of LAT2 (SLC7A8), PKC activation upregulated transport activity without affecting phosphorylation at three potential PKC consensus sites (Thr-11, Ser-337, and Ser-487) . Similar methodologies can be applied to test LAT2 phosphorylation in the context of T cell signaling.

What experimental approaches can determine LAT2's binding partners in T cell signaling?

To identify and characterize LAT2 binding partners:

• Co-immunoprecipitation: Lysates from T cells expressing recombinant LAT2 can be immunoprecipitated, followed by mass spectrometry to identify associated proteins

• CRISPR/Cas9-based platform: This approach enables establishing the composition of LAT, CD5, and CD6 signalosomes in primary mouse T cells in approximately 4 months

• Yeast two-hybrid screening: To identify direct protein-protein interactions

• Surface plasmon resonance: For quantitative measurement of binding kinetics

• Proximity labeling approaches: BioID or APEX2 fusion proteins to identify proteins in close proximity to LAT2 in living cells

How does LAT2 signaling differ from LAT1 in T cell activation?

LAT and LAT2 exhibit distinct roles in T cell signaling pathways:

LAT (LAT1):

• Operates as a positive regulator in the TCR signalosome

• Contains binding sites for PLCγ1 and PLCγ2

• Essential for phosphorylation cascades leading to T cell activation

• Mutations like LAT G135D accelerate TCR signaling and affect T cell fate determination

LAT2:

• Functions in a more regulatory capacity

• Lacks consensus binding motifs for PLCγ1 and PLCγ2

• May play a role in fine-tuning TCR signal intensity

• Contributes to the diversity and specificity of T cell responses

How does LAT2 participate in calcium signaling during T cell activation?

Based on information about related signaling proteins, LAT2 likely influences calcium signaling through the following mechanisms:

• Engagement with calcium-dependent kinases in signaling cascades

• Indirect regulation of calcium mobilization through association with adapter proteins

• Potential interaction with other signaling molecules like NFAT

As observed in the case of recombinant TCR ligands (RTLs), partial T cell activation includes calcium mobilization and NFAT activation without fully activating other pathways . LAT2 may similarly contribute to specific aspects of calcium signaling without triggering the complete activation cascade.

What are the key considerations when designing experiments using recombinant rat LAT2 in primary T cells?

When designing experiments with recombinant rat LAT2:

• Expression level control: Use titratable expression systems to prevent overexpression artifacts

• Cellular context: Consider differences between cell lines and primary T cells

• Functional readouts: Include multiple measurements (calcium flux, phosphorylation, transcriptional changes)

• Controls for specificity: Include LAT2-deficient cells and mutated forms of LAT2

• Temporal dynamics: Analyze both early (seconds to minutes) and late (hours) signaling events

For primary rat T cells, consider that peripheral CD4+CD8+ T cells exhibit poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2 , potentially affecting LAT2-mediated signaling analysis.

How can site-directed mutagenesis be employed to study LAT2 function?

Site-directed mutagenesis is a valuable approach for studying LAT2 function:

Methodological approach:

• Prediction of functional sites: Use in silico tools like ScanProsite (https://prosite.expasy.org/scanprosite/) to identify putative phosphorylation sites or protein interaction motifs

• Transmembrane region prediction: Use tools like TMHMM-2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0)[1]

• Sequence alignment: Compare LAT2 sequences across species using Multialin (http://multalin.toulouse.inra.fr/multalin/)[1]

• Mutagenesis technique: Use PrimeSTAR Max DNA polymerase or equivalent for site-directed mutagenesis

• Functional testing: Express wild-type and mutant LAT2 in appropriate cell systems to compare activities

Mutation of key tyrosine residues in YXN motifs would be particularly informative for understanding LAT2's role in assembling signaling complexes.

How conserved is LAT2 across rodent species compared to humans?

While specific comparison data for LAT2 is not provided in the search results, insights can be drawn from other transmembrane proteins:

• In the case of APP, three amino acid differences between rodent and human APP significantly affect important functional features

• For NAT (N-acetyltransferase) genes, there are strain-specific variations among inbred rat strains, with differential tissue expression patterns of rNat1, rNat2, and rNat3

LAT2 likely exhibits similar species-specific variations, particularly in regions involved in protein-protein interactions and post-translational modifications. Evolutionary conservation analysis would be essential for identifying functionally critical domains.

How does LAT2 expression vary across different rat tissues and developmental stages?

Based on patterns observed with related proteins:

Tissue expression patterns:
Different transmembrane adaptor proteins show tissue-specific expression patterns. For example, NTAL (potentially equivalent to LAT2 in some contexts) is expressed in B cells and NK cells but not in resting T cells . Similarly, semi-quantitative RT-PCR experiments demonstrate that the relative expression of rat NAT transcripts varies significantly across liver and twelve extrahepatic tissues .

Developmental regulation:
Peripheral CD4+CD8+ T cells, which may express different levels of signaling adaptors, appear among the first T cells that colonize peripheral lymphoid organs during fetal life and represent approximately 40% of peripheral T cells during the perinatal period, before decreasing to reach low values in adulthood . LAT2 expression likely follows specific developmental patterns that align with T cell maturation and functional specialization.

What are the optimal conditions for reconstituting and storing recombinant rat LAT2?

Based on protocols for other recombinant rat proteins:

Reconstitution protocol:

• Lyophilized protein should be reconstituted at a concentration of approximately 500 μg/mL in PBS

• Allow complete dissolution at room temperature with gentle agitation

• Avoid vortexing to prevent protein denaturation

• Filter using a 0.22 μm filter if necessary

Storage conditions:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, aliquot to avoid repeated freeze-thaw cycles

• For short-term storage (1-2 weeks), keep at 4°C

• For long-term storage, maintain at -80°C in the presence of a cryoprotectant (e.g., 10% glycerol)

How can researchers validate the functional integrity of recombinant rat LAT2?

Validation of recombinant LAT2 functionality requires multiple approaches:

• Structural integrity: SDS-PAGE and Western blotting to confirm expected molecular weight and recognition by specific antibodies

• Secondary structure analysis: Circular dichroism spectroscopy to confirm proper folding

• Thermal stability: Differential scanning fluorimetry to assess protein stability

• Protein-protein interactions: Pull-down assays with known binding partners

• Cellular activity: Reconstitution experiments in LAT2-deficient cells to restore signaling functions

For membrane proteins like LAT2, reconstitution into liposomes or nanodiscs may be necessary to evaluate function in a membrane environment.

What are common challenges when expressing recombinant rat LAT2, and how can they be addressed?

Common challenges and solutions include:

How can researchers troubleshoot signaling pathway analyses involving recombinant LAT2?

When troubleshooting signaling experiments:

• Protein expression verification: Confirm LAT2 expression levels using Western blotting before functional assays

• Pathway inhibitor controls: Use specific inhibitors of various signaling components (e.g., PKC inhibitor Go6983 at 10 μM) to confirm pathway specificity

• Positive controls: Include stimuli known to activate relevant pathways (e.g., PMA at 1 μM for PKC activation)

• Time course analysis: Signal detection at multiple time points (minutes to hours) to capture transient events

• Multiple readout methods: Combine techniques (Western blotting, flow cytometry, microscopy) to verify results

• Single-cell analysis: Consider heterogeneity in cellular responses using flow cytometry or imaging approaches

Understanding that LAT2 signaling may involve partial activation of certain pathways without fully activating others can help interpret complex results.

How does LAT2 signaling contribute to immune dysregulation in rat models of autoimmunity?

While direct evidence for LAT2 in rat autoimmunity models is limited, insights can be drawn from related research:

• Mutations in transmembrane adaptor proteins can alter T cell development and activation thresholds, potentially contributing to autoimmunity

• In mice, LAT G135D mutation promotes specific phenotypic adaptations, such as upregulation of CD5 and CD6, yet by 1 year of age, female mice develop higher titers of autoantibodies along with signs of colitis

• Single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes, which are part of T cell signaling networks that may involve LAT2

In rat models, LAT2 modifications might similarly affect the balance between immune activation and tolerance, influencing susceptibility to autoimmune conditions.

What role might LAT2 play in modulating responses to recombinant therapeutic proteins in rat models?

In therapeutic contexts:

• LAT2 may influence T cell responses to recombinant proteins used therapeutically, such as recombinant interleukin-15 (IL-15)

• Alterations in LAT2 signaling could affect T cell activation thresholds, potentially modifying immune responses to protein therapeutics

• In rat models of sepsis, treatment with recombinant IL-15 significantly increased T cell and NK cell numbers and prolonged survival , processes that might involve LAT2-mediated signaling pathways

• Understanding LAT2's role could help predict or modify immune responses to recombinant protein therapies

How can CRISPR/Cas9 gene editing be used to study LAT2 function in rat primary T cells?

CRISPR/Cas9 approaches for LAT2 research:

• Knock-out studies: Design gRNAs targeting conserved exons of LAT2 to generate complete knock-outs

• Knock-in modifications: Introduce specific mutations or epitope tags into endogenous LAT2

• Domain swapping: Replace rat LAT2 domains with corresponding human sequences to study species-specific functions

• Reporter integration: Insert fluorescent proteins or luciferase to monitor LAT2 expression

For example, the CRISPR/Cas9 approach used to introduce the M139T mutation into the endogenous rat Psen1 gene can be adapted for LAT2 modifications. This would involve co-injection of Cas9 mRNA, specific sgRNAs, and oligo donors containing the desired modifications into rat zygotes.

How can single-cell analysis techniques advance our understanding of LAT2-dependent signaling heterogeneity?

Single-cell approaches provide crucial insights into signaling heterogeneity:

• Single-cell RNA-seq: Reveals transcriptional consequences of LAT2-mediated signaling across individual cells

• Mass cytometry (CyTOF): Enables simultaneous measurement of multiple phosphorylation events in single cells

• Imaging mass cytometry: Provides spatial information about LAT2 interactions in tissue contexts

• Live-cell imaging: Monitors real-time dynamics of LAT2 recruitment and signaling complex formation

• Reporter systems: Transgenic Nr4a1 and Nr4a3 reporter systems can detect differential T cell receptor signal strength , potentially revealing LAT2's impact on signaling thresholds