ptprsa Antibody

What are PTPRD and PTPRSA and how do they function in cellular signaling?

Protein tyrosine phosphatases receptor type δ (PTPRD) and receptor type S (PTPRSA) belong to the receptor protein tyrosine phosphatase (RPTP) family, which are critical regulators of signal transduction . These transmembrane proteins function by counterbalancing tyrosine kinases - removing phosphate groups from specific protein substrates. In certain cellular contexts, PTPRD has been shown to elicit tumor-promoting functions, including elevating SRC activity and promoting metastasis . RPTPs contain extracellular domains that interact with ligands, transmembrane regions, and intracellular phosphatase domains that execute the dephosphorylation activity.

What are the structural characteristics of these receptor phosphatases?

These RPTPs are characterized by:

• An ectodomain (extracellular region) containing fibronectin (FN) domains and other recognition motifs

• A transmembrane domain anchoring the protein in the cell membrane

• Cytoplasmic phosphatase domains responsible for catalytic activity

PTPRD contains specific domains that can be targeted by antibodies, including the FN domain segment that is recognized by antibody RD-43 .

How do antibodies targeting RPTPs differ from traditional protein tyrosine phosphatase inhibitors?

While traditional PTP inhibitors often target the enzymatic active site, this approach has proven challenging for therapeutic development. As Nicholas Tonks explains: "People have targeted kinases for 25, 30 years... But many challenges remain. In cancer, patients will respond to these sorts of kinase inhibitors and then, after a period of time, resistance develops" .

Antibody-based approaches offer an alternative strategy by:

• Targeting the ectodomain rather than the intracellular catalytic site

• Inducing dimerization that inhibits phosphatase activity

• Potentially promoting receptor degradation

• Providing higher specificity than small molecule inhibitors

What validation methods should be employed when using PTPR antibodies?

Based on research with PTPRD antibodies, comprehensive validation should include:

• Flow cytometry to confirm specific labeling of cells expressing the target protein

• Immunofluorescence to validate binding at expected membrane locations

• Western blot detection with appropriate controls

• Testing in knockout models to confirm specificity

• Cross-reactivity testing with related RPTP family members

For example, researchers validated PTPRD antibodies by demonstrating their ability to specifically label 293T cells expressing PTPRD using flow cytometry, and confirmed membrane localization through immunofluorescence .

What cell models are appropriate for studying PTPR antibody effects?

When selecting cell models, researchers should consider:

• Endogenous expression levels of the target PTPR

• Expression of relevant downstream effectors (e.g., SRC for PTPRD studies)

• Availability of knockout variants for control experiments

For PTPRD studies, CAL51 breast cancer cells were selected based on analysis of DepMap RNA-seq data showing low MTSS1 and high PTPRD levels, consistent with a tumor type that would likely respond to PTPRD antibodies . Researchers should similarly identify appropriate models for PTPRSA based on expression profiles.

How can researchers measure functional outcomes of PTPR antibody treatment?

Key methodological approaches include:

• Monitoring phosphorylation status of downstream substrates (e.g., SRC phosphorylation at Tyr527 for PTPRD)

• Tracking receptor degradation via immunoblotting over time courses

• Conducting functional assays such as cell invasion assays

• Comparing effects of bivalent antibodies vs. monovalent Fab fragments

• Analyzing receptor dimerization through biochemical techniques

How do antibodies induce PTPR dimerization and what are the functional consequences?

The bivalent nature of antibodies enables them to induce protein dimerization. For PTPRD:

• Antibody RD-43 binds to the FN domain segment of PTPRD's ectodomain

• This binding causes two PTPRD molecules to dimerize

• Dimerization inhibits phosphatase activity

• The inhibition of phosphatase activity leads to increased phosphorylation of substrates (e.g., SRC)

• Subsequently, the antibody-PTPRD dimer complex is degraded through lysosomal and proteasomal pathways

The requirement for bivalent binding was demonstrated using monovalent Fab fragments (RD-43 MS-Fab), which bound PTPRD but did not cause degradation or SRC inhibition unless cross-linked with secondary antibodies .

What signaling pathways are affected by PTPR antibody-induced inhibition?

For PTPRD antibodies:

• SRC signaling is a primary affected pathway

• Inhibition of PTPRD via antibody RD-43 suppresses SRC-dependent cell invasion in breast cancer cells

• Treatment with RD-43 inhibited SRC activity as measured by phosphorylation status

• These effects mimicked those observed following chemically induced dimerization of PTPRD

Researchers studying PTPRSA should similarly investigate relevant downstream pathways based on known PTPRSA substrates.

What is the time course of PTPR degradation following antibody binding?

For PTPRD:

• Noticeable reduction in protein levels occurs within 1 hour of RD-43 treatment

• Further degradation continues with extended antibody exposure

• The degradation pathway is independent of secretase cleavage

• Both lysosomal and proteasomal pathways contribute to degradation

This rapid timeline suggests active degradation mechanisms rather than simply preventing new protein synthesis.

What are the optimal conditions for Western blot applications with PTPR antibodies?

Based on available data for PTPRSA antibody (PACO61350):

Researchers should note that observed band sizes may differ from predicted molecular weights due to post-translational modifications, proteolytic processing, or dimerization .

How should researchers troubleshoot non-specific binding or weak signal issues?

When encountering technical difficulties:

• For non-specific binding: Optimize antibody dilution, increase blocking stringency, validate in knockout models

• For weak signals: Increase protein loading, optimize transfer conditions, verify target expression in samples

• For unexpected band patterns: Consider degradation products, isoforms, or post-translational modifications

• For inconsistent results: Standardize sample preparation protocols and antibody handling procedures

What controls are essential for experiments using PTPR antibodies?

Critical controls include:

• Knockout/knockdown controls: As demonstrated with PTPRD antibodies in CAL51 PTPRD knockout cells

• Isotype controls: To rule out non-specific effects of antibody backbone

• Monovalent fragment controls: To confirm dimerization-dependent mechanisms

• Secondary-only controls: To identify background binding in immunostaining

• Cross-linking controls: Using secondary antibodies to restore functionality to Fab fragments

How do PTPR antibodies show potential as therapeutic agents?

Research with PTPRD antibodies demonstrates several mechanisms relevant to therapeutic development:

• Inhibition of phosphatase activity through ectodomain targeting rather than active site inhibition

• Induction of receptor degradation, providing sustained inhibition

• Suppression of cancer-promoting pathways such as SRC signaling

• Reduction of cell invasion in metastatic breast cancer models

As noted in the research: "Together, these findings demonstrate that manipulating RPTP function via antibodies to the extracellular segments has therapeutic potential" .

What disease contexts might benefit from PTPR-targeting antibodies?

Based on the research with PTPRD:

• Metastatic breast cancers with high PTPRD expression

• Cancer types with elevated SRC activity dependent on PTPR regulation

• Potentially other conditions where aberrant protein tyrosine phosphorylation contributes to pathology

Researchers studying PTPRSA would need to identify specific disease contexts where this receptor plays a significant role.