DRD1 Antibody

What is DRD1 and what biological systems express it?

Dopamine receptor D1 (DRD1) is the most abundant form of dopamine receptor in the central nervous system. It belongs to the D1-like class of dopamine receptors, which also includes the D5 receptor, while the D2-like class comprises D2, D3, and D4 subtypes . DRD1 plays crucial roles in various physiological processes by stimulating adenylyl cyclase, activating cyclic AMP-dependent protein kinases, modulating D2 receptor activity, and regulating neuronal growth and differentiation .

While predominantly expressed in the caudate putamen in humans, recent research has demonstrated that DRD1 and other dopamine pathway components are also expressed in normal human lung tissue . This wider distribution suggests broader physiological functions beyond the nervous system.

What are the molecular characteristics of DRD1?

DRD1 is a G protein-coupled receptor with the following key characteristics:

The variation between calculated and observed molecular weight is likely due to post-translational modifications. There are two variants of this gene resulting from alternate transcription initiation sites .

What are the recommended applications for DRD1 antibodies?

DRD1 antibodies have been validated for multiple experimental applications, with specific dilution recommendations:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results as performance may vary by sample type and experimental conditions .

How should DRD1 antibodies be properly stored and handled?

Proper storage is crucial for maintaining antibody functionality:

Most DRD1 antibodies should be stored at -20°C and remain stable for one year after shipment . The standard formulation includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For unconjugated antibodies, aliquoting is generally unnecessary for -20°C storage, though smaller size preparations (e.g., 20μl) may contain 0.1% BSA .

For conjugated antibodies, such as PE-conjugated anti-DRD1, the solution should be stored undiluted between 2°C and 8°C . Always check the lot-specific concentration and expiration information from the manufacturer's Certificate of Analysis.

What are the best practices for validating DRD1 antibody specificity?

Validation of DRD1 antibody specificity is essential for reliable research outcomes:

• Knockout/Knockdown Controls: Use DRD1 knockout or knockdown samples as negative controls. Several publications have used this approach to validate antibody specificity .

• Peptide Competition Assay: Pre-incubate the antibody with the immunogen peptide (e.g., SSHHEPRGSISKDC peptide corresponding to amino acid residues 372-385 of rat DRD1) to verify signal elimination .

• Multi-application Validation: Confirm consistent results across different applications (WB, IHC, IF) using the same antibody.

• Cross-species Testing: Verify expected reactivity with samples from humans, mice, and rats as indicated in reactivity profiles .

• Molecular Weight Verification: Confirm that the observed band appears at the expected molecular weight range (50-75 kDa for DRD1) .

What protocols are recommended for using DRD1 antibodies in flow cytometry?

For flow cytometric analysis of DRD1:

The recommended protocol involves using 5 μl of PE-conjugated anti-DRD1 antibody per million cells in 100 μl staining volume, or 5 μl per 100 μl of whole blood . The PE-conjugated anti-DRD1 antibody is excited by blue (488 nm) or green/yellow-green lasers (532 nm/561 nm) .

When analyzing DRD1 expression in heterogeneous cell populations, include appropriate isotype controls (e.g., Mouse IgG2b, κ for clone L205G1) to distinguish specific from non-specific binding . Additionally, consider using DRD1-transfected cells as positive controls to establish proper gating strategies.

What is the current understanding of DRD1-DRD2 heteromers and implications for experimental design?

The existence and functional significance of DRD1-DRD2 heteromers remains controversial:

Current evidence suggests that despite the ability of D1 and D2 receptors to interact under certain conditions, they do not readily form functional heteromers under physiological conditions . Research using BRET biosensors has shown that while D1 and D2 receptors can activate their cognate G proteins (Gαs and Gαi1 respectively), they do not form heteromers that activate Gαq .

Proximity-ligation assays (PLA) revealed virtually absent signals for D1-D2 heteromers in the nucleus accumbens of wild-type, D1 receptor KO, and D2 receptor KO mice . Only when both receptors are dramatically overexpressed—either in HEK cells or in vivo—do they appear to physically interact .

For experimental design, these findings suggest:

• Caution in interpreting results assuming DRD1-DRD2 heteromer formation

• Need for appropriate controls when studying receptor interactions

• Critical evaluation of previous literature claiming heteromer-specific functions

• Consideration of expression levels when studying potential interactions

How is DRD1 associated with cancer progression and what techniques are used to study this relationship?

Recent research has revealed unexpected roles for DRD1 in cancer biology:

DRD1 has been identified as a negative regulator of disease progression in Non-Small Cell Lung Cancer (NSCLC) . Loss of DRD1 expression through promoter hypermethylation appears to be a common feature in NSCLC patients and is associated with worse survival outcomes .

At the cellular level, DRD1 affects proliferation by inhibiting the activation of Epidermal Growth Factor Receptor (EGFR) and Mitogen-Activated Protein Kinase 1/2 (ERK1/2) . Additionally, DRD1 has been found to regulate the expression of Programmed Death-Ligand 1 (PD-L1) in lung cancer cells, suggesting potential implications for immunotherapy responses .

Techniques used to study DRD1 in cancer include:

• RNA sequencing (RNAseq) and RT-PCR to measure expression levels

• Methylation analysis to assess epidermal silencing

• In vitro functional experiments for cell proliferation

• In vivo tumor growth studies

• Transcriptome and kinome analysis in DRD1-modulated cells

• Western blotting and immunoprecipitation to study effects on EGFR and ERK pathways

What are the key considerations when troubleshooting inconsistent DRD1 antibody results in Western blotting?

When facing inconsistent results with DRD1 antibodies in Western blotting:

• Sample Preparation: Brain tissue samples may require specific handling; incubating mouse brain tissue at 37°C has been shown to improve detection .

• Molecular Weight Variability: DRD1 has a calculated molecular weight of 49 kDa but is typically observed at 50-75 kDa due to post-translational modifications . Variations outside this range may indicate specificity issues.

• Antibody Dilution: Titrate between 1:500-1:2000 for Western blotting to determine optimal concentration for your specific sample .

• Blocking Conditions: Optimize blocking buffers as DRD1 detection may be sensitive to excessive blocking.

• Species Differences: Verify antibody compatibility with your species of interest. Most validated DRD1 antibodies show reactivity with human, mouse, and rat samples .

• Epitope Accessibility: Different antibodies target different epitopes (e.g., amino acid residues 372-385 of rat DRD1) , which may affect detection efficiency depending on protein conformation.

• Positive Controls: Include 37°C incubated mouse brain tissue as a positive control, which has been verified for DRD1 detection .

What methodologies are effective for studying DRD1 signaling pathways?

To effectively study DRD1 signaling pathways:

• BRET Biosensor Technique: This approach allows measurement of G-protein activation based on conformational changes between α and γ subunits . This technique has been validated to accurately reflect activation of G proteins in response to receptor stimulation.

• Complementation-Based Resonance Energy Transfer (CODA-RET): This method can measure G-protein coupling specifically from defined receptor dimers, useful when studying potential heteromeric interactions .

• Proximity-Ligation Assay (PLA): Effective for detecting receptor complexes ex vivo, this technique can be used to investigate physical interactions between DRD1 and other proteins .

• Adenylyl Cyclase Activation Assays: Since DRD1 stimulates adenylyl cyclase and activates cAMP-dependent protein kinases , measuring cAMP levels provides functional readout of receptor activity.

• Phosphorylation Analysis: Examining downstream phosphorylation events, particularly of ERK1/2, can provide insights into DRD1 signaling cascades, especially in cancer models .

How does DRD1 expression and function differ between neuronal and non-neuronal tissues?

While DRD1 has been extensively studied in neuronal contexts, research now reveals important roles in non-neuronal tissues:

The functions appear to be tissue-specific:

• In neurons: Primarily involved in neurotransmission, synaptic plasticity, and behavioral regulation

• In lung tissue: Functions as a negative regulator of cell proliferation by inhibiting EGFR activation and ERK1/2 signaling

• In cancer cells: Regulates PD-L1 expression, potentially influencing immune responses

This expanding understanding suggests that experimental approaches should be tailored to the specific tissue context, with different positive controls and expected outcomes based on the system under study.

What are the implications of DRD1 promoter methylation as a biomarker in cancer?

The discovery that DRD1 promoter hypermethylation is associated with worse survival in NSCLC patients has important implications:

Research has shown that loss of DRD1 expression through promoter hypermethylation is a common feature in NSCLC . This epigenetic modification appears to silence DRD1, removing its inhibitory effect on cell proliferation and EGFR activation.

Methodological considerations for studying DRD1 methylation include:

• Methylation-specific PCR techniques to quantify promoter methylation

• Correlation analysis between methylation status and expression levels

• Survival analysis to assess prognostic value

• Comparison with established biomarkers

• Evaluation of methylation patterns across different cancer subtypes

The potential use of DRD1 methylation as a biomarker may provide valuable prognostic information for NSCLC patients and could inform treatment strategies, particularly for those receiving EGFR-targeted therapies or immunotherapies targeting the PD-1/PD-L1 axis .

What are the key factors to consider when using DRD1 antibodies in multi-color flow cytometry?

For multi-color flow cytometry applications involving DRD1:

• Fluorophore Selection: PE-conjugated anti-DRD1 antibodies (such as clone L205G1) are excited by blue (488 nm) or green/yellow-green lasers (532 nm/561 nm) . Design your panel to minimize spectral overlap with these channels.

• Panel Design: Consider the expression level of DRD1 in your target cells. Since DRD1 may have variable expression, pair it with a brighter fluorophore if expression is expected to be low.

• Compensation Controls: Include single-stained controls for each fluorophore in your panel to enable proper compensation, especially important when PE has spectral overlap with other fluorochromes.

• Titration: The standard recommendation of 5 μl per million cells should be validated for your specific experimental system .

• Controls: Include FMO (Fluorescence Minus One) controls to set proper gates, particularly important when examining shifts in DRD1 expression rather than discrete positive/negative populations.

• Sample Preparation: For neuronal tissues or samples where DRD1 is predominantly intracellular, ensure your fixation and permeabilization protocol maintains epitope accessibility.

How can researchers effectively compare results from different DRD1 antibody clones?

When comparing results obtained using different DRD1 antibody clones:

• Epitope Mapping: Different antibodies target different epitopes. For instance, some target the intracellular region (e.g., residues 372-385 of rat DRD1) , while others may target extracellular domains. Document the specific epitope targets to understand potential differences in detection.

• Cross-Validation: When transitioning between antibody clones, run parallel experiments with both antibodies to establish correlation factors.

• Standard Samples: Include standard positive controls (e.g., mouse brain tissue for WB , DRD1-transfected cells for flow cytometry ) in all experiments to normalize between different antibodies.

• Isotype Matching: When comparing different clones, ensure matched isotype controls (e.g., Mouse IgG2b, κ for clone L205G1 , Rabbit IgG for polyclonal antibodies ).

• Application-Specific Validation: An antibody performing well in one application (e.g., Western blot) may not perform equally in another (e.g., immunohistochemistry). Validate each clone for your specific application.

• Batch Normalization: Record lot numbers and include internal controls to account for batch-to-batch variations within the same clone.