Recombinant Human Cysteinyl leukotriene receptor 1 (CYSLTR1)

How does CYSLTR1 recognize and distinguish between different ligands?

CYSLTR1 exhibits selective binding to cysteinyl leukotrienes with a potency order of LTD4 > LTC4 > LTE4. Binding studies indicate that LTE4 likely lacks sufficient potency to significantly activate CYSLTR1 under physiological conditions .

Structural studies using crystal structures of CYSLTR1 bound to antagonists have revealed unique ligand-binding modes, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5. The receptor contains a distinct four-residue-coordinated sodium site and displays an atypical pattern of microswitches that contribute to its ligand recognition properties .

Recent crystallographic data provide molecular insights into how CYSLTR1 and CYSLTR2 achieve ligand selectivity. Computer modeling and mutagenesis studies have identified specific residues in the binding pocket that determine differential affinity for various ligands, which is crucial for developing receptor-selective antagonists .

What are the main physiological effects mediated by CYSLTR1 activation?

CYSLTR1 activation by its primary ligands (LTC4 and LTD4) triggers multiple physiological responses in both animal models and humans:

• Airway effects: Bronchoconstriction and increased hyper-responsiveness to bronchoconstrictive agents like histamine

• Vascular effects: Increased vascular permeability and edema

• Inflammatory cell recruitment: Influx of eosinophils and neutrophils to tissues

• Tissue remodeling: Smooth muscle proliferation, collagen deposition, and fibrosis

• Mucus production: Increased mucin secretion by goblet cells, goblet cell metaplasia, and epithelial cell hypertrophy

These effects collectively contribute to various inflammatory and allergic responses, particularly in asthma and other respiratory disorders, where CYSLTR1 antagonists have proven therapeutic value .

What is the tissue distribution of CYSLTR1 expression?

CYSLTR1 mRNA is widely expressed across multiple tissues and cell types, with significant expression in:

Immune cells:

• Monocytes

• Macrophages (including lung macrophages)

• Eosinophils

• Basophils

• Neutrophils

• T cells and B lymphocytes

• Mast cells

• Pluripotent hematopoietic stem cells (CD34+)

• Platelets

Structural cells:

• Airway smooth muscle cells

• Bronchial fibroblasts

• Vascular endothelial cells

• Interstitial cells of the nasal mucosa

Organs and tissues:

• Lung (particularly smooth muscle)

• Pancreas

• Small intestine

• Prostate

This broad expression pattern explains the diverse physiological and pathological roles of CYSLTR1 in inflammatory processes throughout the body.

How is CYSLTR1 internalization regulated after ligand binding?

CYSLTR1 undergoes rapid and profound internalization upon stimulation with LTD4. Unlike many other GPCRs, CYSLTR1 internalization is primarily regulated by protein kinase C (PKC) rather than arrestins. This was demonstrated in studies where:

• A C-terminal truncation mutant exhibited impaired internalization while maintaining robust signaling, identifying a critical region within amino acids 310-321 for internalization

• Pharmacological inhibition of PKC profoundly inhibited CYSLTR1 internalization while significantly increasing both phosphoinositide production and calcium mobilization

• Mutation of putative PKC phosphorylation sites within the CYSLTR1 C-tail (CysLT1RS(313-316)A) reduced receptor internalization and enhanced signaling responses

This unique regulatory mechanism distinguishes CYSLTR1 from most other GPCRs, making it the first identified receptor where PKC serves as the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization .

How does CYSLTR1 expression change in disease states?

CYSLTR1 expression is dynamically regulated in various disease conditions. In systemic lupus erythematosus (SLE), CYSLTR1 expression is significantly elevated in patients compared to healthy controls and correlates with disease activity. This elevated expression appears to be driven by DNA demethylation, suggesting epigenetic regulation of CYSLTR1 in autoimmune conditions .

In cancer, particularly breast cancer, altered CYSLTR1 expression correlates with clinical outcomes. Low CYSLTR1 expression is associated with worse survival in breast cancer patients generally, with particularly significant effects in triple-negative breast cancer (TNBC) . These findings suggest that CYSLTR1 may serve as a prognostic marker in certain cancer types.

What are the primary signaling pathways activated by CYSLTR1?

CYSLTR1 activates multiple signaling pathways through its coupling to G proteins:

• G protein coupling: CYSLTR1 primarily functions as a G protein-coupled receptor that links to and activates:

  • Gq alpha subunit

  • Ga subunit (depending on the cell type)

• Downstream effectors:

  • Phosphatidylinositol-calcium second messenger system

  • PI3K/AKT/mTOR pathway activation (particularly relevant in B cells)

• Cellular responses:

  • Calcium mobilization

  • Activation of protein kinase C

  • Contraction and proliferation of smooth muscle cells

  • Eosinophil migration

  • Damage to the mucus layer in the lung

In B cells, CYSLTR1 signaling influences the BCL6-BLIMP1-XBP1 axis, which is crucial for B cell differentiation and antibody production. Inhibition of CYSLTR1 downregulates these transcription factors by suppressing the PI3K/AKT/mTOR pathway .

How does protein kinase C (PKC) specifically regulate CYSLTR1 function?

PKC plays a central and unique role in regulating CYSLTR1 function through several mechanisms:

• Receptor internalization: PKC is the primary regulator of agonist-induced internalization of CYSLTR1. Pharmacological inhibition of PKC profoundly inhibits CYSLTR1 internalization.

• Signal termination: PKC mediates rapid agonist-dependent desensitization of CYSLTR1, controlling the duration and intensity of receptor signaling.

• Phosphorylation sites: The region between amino acids 310-321 in the C-terminal tail contains putative PKC phosphorylation sites. Mutation of these sites (CysLT1RS(313-316)A) reduces receptor internalization and enhances signaling.

• Signaling intensity: PKC inhibition increases both phosphoinositide production and calcium mobilization stimulated by LTD4, suggesting PKC normally attenuates these signaling pathways.

This PKC-dependent regulation distinguishes CYSLTR1 from other GPCRs, where arrestins typically play the predominant role in receptor internalization and desensitization .

How does CYSLTR1 contribute to inflammatory pathways in immune cells?

In immune cells, CYSLTR1 signaling activates multiple inflammatory pathways:

• In B cells: CYSLTR1 activation promotes B cell differentiation into antibody-secreting cells through regulation of the BCL6-BLIMP1-XBP1 transcriptional axis. In SLE models, CYSLTR1 knockout mice exhibit reduced plasma cell frequencies and decreased lupus-like symptoms .

• In macrophages: CYSLTR1 signaling induces the release of pro-inflammatory mediators and chemokines, contributing to inflammatory cell recruitment.

• In eosinophils and neutrophils: CYSLTR1 activation promotes migration and activation of these cells, contributing to tissue inflammation.

• In T cells: CYSLTR1 modulates T cell responses, potentially contributing to inflammatory and allergic reactions.

Experiments using CYSLTR1 inhibition with antagonists like montelukast show reduction in antibody-secreting cells and plasmablasts in lupus models, demonstrating the receptor's importance in B cell-mediated immune responses .

What is the role of CYSLTR1 in systemic lupus erythematosus (SLE)?

CYSLTR1 plays a significant role in SLE pathogenesis through several mechanisms:

• Expression correlation: CYSLTR1 expression is elevated in SLE patients and correlates with disease activity, likely driven by DNA demethylation.

• B cell function: CYSLTR1 regulates B cell differentiation and antibody production through the BCL6-BLIMP1-XBP1 transcriptional axis.

• Experimental evidence: In a pristane-induced lupus model, CYSLTR1-knockout mice exhibited:

  • Reduced lupus-like symptoms

  • Decreased plasma cell frequencies

  • Attenuated immune responses

• Mechanism of action: CYSLTR1 inhibition downregulates BCL6, BLIMP1, and XBP1 in B cells by suppressing the PI3K/AKT/mTOR pathway, leading to:

  • Reduced antibody-secreting cells (ASCs)

  • Decreased immunoglobulin production

• Therapeutic potential: Montelukast (a CYSLTR1 antagonist) ameliorated SLE manifestations in two murine lupus models by reducing antibody-secreting cells and plasmablasts .

These findings suggest CYSLTR1 antagonism could represent a novel therapeutic approach for SLE treatment.

How does CYSLTR1 expression influence cancer progression?

CYSLTR1 expression appears to have important implications in cancer, particularly in breast cancer:

• Prognostic value: Low CYSLTR1 expression is associated with worse survival in breast cancer patients generally, with particularly pronounced effects in triple-negative breast cancer (TNBC) .

• Expression analysis: Studies using platforms like UALCAN, GENT2, and TCGA databases have established correlations between CYSLTR1 expression levels and clinical outcomes in breast cancer.

• Molecular networks: Co-expression analysis conducted using RNA-seq data of TNBC from the bc-GenExMiner database identified gene networks associated with CYSLTR1 function that may influence cancer progression .

• Other cancers: CYSLTR1 has been implicated in several other cancer types, including colorectal cancer where recent studies reported its involvement in both spontaneous development of colorectal cancer and in colitis-associated colon cancer models .

• Constitutive activity: Interestingly, high constitutive G protein signaling activity of CYSLTR2 mutants (a related receptor) has been associated with uveal melanoma and other cancer types, though the role of CYSLTR receptors in cancer remains complex and sometimes contradictory .

What is known about CYSLTR1's role in bone metabolism?

Studies investigating CYSLTR1's role in bone metabolism have yielded important insights:

What therapeutic approaches target CYSLTR1 in inflammatory conditions?

Several therapeutic approaches targeting CYSLTR1 have been developed:

• FDA-approved antagonists:

  • Montelukast

  • Zafirlukast

  • Pranlukast

  These selective CYSLTR1 antagonists are widely prescribed as antiasthmatic drugs and for allergic rhinitis .

• Mechanism of action: Crystal structures of CYSLTR1 bound to zafirlukast and pranlukast have revealed their binding modes, showing:

  • Lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5

  • Specific interactions with key residues in the binding pocket

• Emerging applications:

  • SLE treatment: Montelukast ameliorated SLE manifestations in mouse models

  • Cancer therapy: Targeting CYSLTR1 in certain cancers where it influences progression

  • Neurodegenerative disorders: CysLTR2-selective or CysLTR1/CysLTR2 dual antagonists show promise for brain injury and neurodegenerative conditions

• Limitations: Current antagonists demonstrate low effectiveness in some patients and exhibit various side effects, highlighting the need for more targeted approaches .

The reported crystal structures of CYSLTR1 provide important templates for rational discovery of safer and more effective drugs with desired selectivity profiles .

What approaches are used to generate recombinant CYSLTR1 protein for research?

Researchers use several methods to produce recombinant CYSLTR1 protein:

• Expression systems:

  • E. coli: The full-length human CYSLTR1 (1-337aa) can be expressed in E. coli with an N-terminal His tag .

  • Mammalian cells: For functional studies requiring proper folding and post-translational modifications.

  • Insect cells: Used for structural studies requiring higher protein yields with eukaryotic processing.

• Purification strategies:

  • Affinity chromatography using His-tag

  • Size exclusion chromatography

  • Crystallization procedures for structural studies

• Storage considerations:

  • Recommended storage at -20°C/-80°C

  • Lyophilized forms with 6% Trehalose in Tris/PBS-based buffer, pH 8.0

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol for long-term storage

  • Avoiding repeated freeze-thaw cycles

• Quality control:

  • SDS-PAGE to confirm purity (>90%)

  • Functional assays to verify ligand binding and signaling activity

What are the most effective methods for generating CYSLTR1 knockout models?

CRISPR-Cas9 has emerged as the preferred method for generating CYSLTR1 knockout models, as demonstrated in recent studies:

• Target design:

  • Selection of highly specific target sequences (sgRNAs) using software like CRISPRdirect

  • Targeting coding regions, particularly exon 4 of Cysltr1

  • Careful screening for potential off-target effects in genes like Plcl1, Pde10a, Mcc, and Ppargc1b

• CRISPR-Cas9 delivery:

  • Electroporation of Cas9/sgRNA complexes into one-cell fertilized eggs

  • Parameters: 30V (3 msec ON + 97 msec OFF) × 6 (±) using an electroporator

• Verification strategies:

  • PCR genotyping (for example, a 112-bp deletion mutation was detected)

  • DNA sequencing to confirm frameshift or in-frame mutations

  • Sequencing of potential off-target sites to ensure specificity

  • Functional validation through calcium flux assays in response to LTD4

• Types of mutations generated:

  • Frameshift mutations resulting in premature stop codons (Cysltr1 KO)

  • In-frame deletions affecting critical domains like the first extracellular loop (Cysltr1 Δ105)

The generation of multiple types of mutations (null and domain-specific) allows for more comprehensive functional studies of CYSLTR1.

What experimental approaches are used to study CYSLTR1 signaling?

Researchers employ various techniques to investigate CYSLTR1 signaling:

• Calcium mobilization assays:

  • Intracellular calcium flux measurements in response to ligands like LTD4

  • Used to assess receptor functionality and ligand potency

  • Particularly valuable for validating knockout or mutant receptors

• Phosphoinositide (PI) production assays:

  • Measuring phosphoinositide generation following receptor activation

  • Evaluating effects of inhibitors or mutations on signaling intensity

• Receptor internalization studies:

  • Tracking receptor trafficking after ligand binding

  • Comparing wild-type and mutant receptors to identify domains critical for internalization

  • Investigating the effects of kinase inhibitors on receptor trafficking

• Transcriptomic analysis:

  • RNA-seq to identify genes regulated by CYSLTR1 signaling

  • Co-expression analysis to construct gene networks associated with CYSLTR1 function

  • Enrichment analysis to identify pathways affected by CYSLTR1 activation or inhibition

• Inhibitor studies:

  • Using selective antagonists (montelukast, zafirlukast, pranlukast)

  • Applying pathway-specific inhibitors (PI3K and mTOR inhibitors)

  • Comparing effects in wild-type versus receptor-deficient systems to distinguish on-target from off-target effects

These methods collectively provide a comprehensive picture of CYSLTR1 signaling mechanisms and their physiological consequences.

How does the structure of CYSLTR1 determine ligand selectivity and antagonist design?

Advanced structural studies have provided crucial insights into CYSLTR1 ligand selectivity:

• Crystal structures: Structures of CYSLTR1 bound to antagonists (zafirlukast and pranlukast) have revealed:

  • Unique ligand-binding modes with lateral access to the orthosteric pocket between TM4 and TM5

  • An atypical pattern of microswitches involved in receptor activation

  • A distinct four-residue-coordinated sodium site that influences receptor conformation

• Structure-activity relationships:

  • Comprehensive mutagenesis studies have identified key residues determining ligand selectivity

  • Computer modeling accurately recapitulates binding of dozens of known ligands

  • Structure-based explanations for structure-activity relationships of 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid scaffold derivatives

• Receptor selectivity determinants:

  • Molecular comparison between CYSLTR1 and CYSLTR2 has revealed critical differences in binding pockets

  • These differences explain the selectivity profiles of various antagonists

  • Provide templates for designing receptor-selective or dual antagonists with desired properties

• Disease-related variants:

  • Structural studies have shed light on the effects of disease-related single nucleotide variants on receptor function

  • Help explain how mutations alter receptor signaling in conditions like asthma

These structural insights serve as templates for rational design of new generation antagonists with improved selectivity and efficacy profiles.

What is the relationship between CYSLTR1 and autophagy regulation?

Recent research has uncovered intriguing connections between CYSLTR1 and autophagy:

• CYSLTR1 inhibition and autophagy induction:

  • CYSLTR1 antagonist application increases autophagic flux in retinal pigment epithelial cells (ARPE-19)

  • This effect has been observed with antagonists like zafirlukast (ZK)

• Cellular consequences:

  • CYSLTR1 inhibition for 24 hours using zafirlukast decreased quantities of:

    • Autophagosomes

    • Late endosomes/lysosomes

    • Aggregated proteins

    • Reactive oxygen species (ROS)

• Cell surface expression correlations:

  • Cells expressing CYSLTR1 on the surface (SE+) showed increased levels of:

    • Autophagosomes

    • Late endosomes/lysosomes

    • Aggregated proteins

    • Autophagy activity

  • These cells simultaneously displayed decreased ROS formation

• Implications for cellular aging:

  • High levels of plasma membrane-localized CYSLTR1 indicate increased amounts of aggregated protein

  • This appears to raise the rate of autophagic flux as a compensatory mechanism

  • CYSLTR1 antagonism potentially mimics physiological conditions observed in CYSLTR1 SE+ cells

  • This mechanism could potentially dampen cellular aging processes

These findings suggest CYSLTR1 inhibition may have therapeutic potential beyond inflammatory conditions, particularly in disorders associated with protein aggregation.

What are the emerging roles of CYSLTR1 in non-inflammatory processes?

Beyond its well-established role in inflammation, CYSLTR1 is emerging as a regulator of several non-inflammatory processes:

• Cellular homeostasis and protein quality control:

  • CYSLTR1 inhibition reduces levels of aggregated proteins in cells

  • The receptor influences the endosomal-lysosomal system (ELS) function

  • May affect cellular waste degradation via autophagy regulation

• Cancer biology beyond inflammation:

  • CYSLTR1 expression levels correlate with survival outcomes in breast cancer

  • The receptor may influence cancer cell proliferation, migration, or other tumorigenic processes

  • Expression patterns may serve as prognostic markers in certain cancer types

• B cell differentiation and antibody production:

  • CYSLTR1 modulates B cell differentiation through the BCL6-BLIMP1-XBP1 transcriptional axis

  • This affects antibody-secreting cell development independent of acute inflammatory responses

  • Suggests a role in normal humoral immunity regulation

• Potential neuroprotective effects:

  • CYSLTR2-selective or CYSLTR1/CYSLTR2 dual antagonists show promise for brain injury and neurodegenerative disorders

  • May involve mechanisms beyond classical inflammatory pathways

• Metabolic regulation:

  • Emerging evidence suggests CYSLTR1 may influence metabolic processes

  • Further investigation needed to fully characterize these functions