GPR161 Antibody Pair

What is GPR161 and why is it important in developmental biology research?

GPR161 (also known as RE2) is an orphan G protein-coupled receptor that plays a crucial role in developmental processes by suppressing Hedgehog signaling. In the absence of Hedgehog signals, GPR161 localizes to primary cilia and maintains downstream GLI transcription factors in their repressor forms . GPR161 functions by constitutively coupling to Gs, which elevates cAMP levels to drive PKA activity . During embryonic development, GPR161 regulates multiple processes including neural tube patterning, limb formation, and skeletal morphogenesis . Knockout studies have shown that GPR161 is essential for forelimb formation and proper limb patterning . The central importance of GPR161 in these fundamental developmental pathways makes it a significant target for developmental biology research.

What types of GPR161 antibodies are currently available for research applications?

Researchers have access to both monoclonal and polyclonal antibodies targeting GPR161:

• Monoclonal antibodies: Including mouse monoclonal IgG2a antibodies like the 1B2 clone that detects GPR161 protein of human origin .

• Polyclonal antibodies: Multiple rabbit polyclonal antibodies are available with varying reactivity profiles .

These antibodies have been validated for multiple applications including:

Most commercially available antibodies show reactivity with human and mouse GPR161, while some also recognize rat, canine, and other species variants .

How can I optimize GPR161 detection in primary cilia using immunofluorescence?

Detecting endogenous GPR161 in primary cilia can be challenging due to its naturally low expression levels. To optimize immunofluorescence detection:

• Fixation protocol: Use 4% paraformaldehyde for 10 minutes at room temperature followed by methanol fixation at -20°C for 5 minutes to preserve ciliary structures.

• Permeabilization: Use 0.1% Triton X-100 in PBS for 10 minutes to improve antibody accessibility while preserving membrane structures.

• Antibody selection: Use validated antibodies with demonstrated ciliary localization capacity (e.g., Proteintech 13398-1-AP has been widely cited for ciliary localization studies) .

• Co-staining strategy: Always co-stain with established ciliary markers (acetylated α-tubulin or ARL13B) to confirm ciliary localization.

• Signal amplification: If endogenous GPR161 signal is weak, use tyramide signal amplification or a high-sensitivity detection system.

• Modulating GRK2 activity: Treatment with GRK2 inhibitor cmpd101 can increase detectable levels of endogenous GPR161 in cilia, as GRK2 normally reduces GPR161 ciliary abundance .

• Image acquisition: Use confocal microscopy with z-stacking to properly visualize the three-dimensional structure of cilia.

Remember that endogenous GPR161 levels may be too low to detect in some cell types (e.g., NIH/3T3 cells), where the protein is present but below the detection threshold of standard immunofluorescence techniques .

What are the best experimental approaches to study GPR161 function in Hedgehog signaling?

To effectively study GPR161 function in Hedgehog signaling:

• CRISPR-mediated gene editing: Generate GPR161 knockout cell lines using multiple guide RNAs to ensure complete loss of function. This approach has been successfully used in NIH/3T3 cells to study GPR161's role in SHH sensitivity .

• Dose-response curves: When evaluating Hedgehog pathway activation, generate complete dose-response curves rather than testing single concentrations. GPR161 primarily affects the sensitivity (EC50) of cells to SHH rather than the maximum response .

• Gene expression analysis: Measure GLI1 mRNA levels by qPCR as a sensitive readout of pathway activation. GPR161 deletion shifts the SHH dose-response curve leftward, increasing ligand potency by 3-5 fold .

• Protein analysis: Monitor GLI3 processing by Western blot, examining both full-length (GLI3FL) and repressor (GLI3R) forms to understand how GPR161 affects GLI processing .

• Epistasis analysis: Use SMO antagonists like cyclopamine in GPR161-null cells to determine whether GPR161 acts upstream, downstream, or parallel to canonical signaling components .

• Reconstitution experiments: Re-express wild-type or mutant GPR161 in knockout backgrounds to determine structure-function relationships.

• cAMP assays: Use TR-FRET cAMP assays to measure changes in cAMP levels, as GPR161 functions by constitutively coupling to Gs and elevating cAMP .

This multi-faceted approach allows for comprehensive analysis of GPR161's role in Hedgehog pathway regulation.

How can I investigate the structure-function relationship of GPR161 using available antibodies?

To investigate GPR161 structure-function relationships:

• Domain-specific antibodies: Select antibodies targeting different epitopes of GPR161. The extracellular, transmembrane, and intracellular domains may have distinct functions.

• Mutant analysis: Generate GPR161 mutants (e.g., GPR161 mut1) with specific domain alterations and use antibodies to assess protein expression, localization, and function .

• Functional readouts: Combine antibody-based detection with functional assays:

  • Monitor ciliary localization using immunofluorescence

  • Assess protein-protein interactions using co-immunoprecipitation

  • Evaluate downstream signaling using phospho-specific antibodies against PKA targets

• Structure-guided approaches: The recent cryo-EM structure of GPR161-Gs complex (PDB 8SMV) revealed a unique conformation of extracellular loop 2 (ECL2) that occludes the canonical ligand binding pocket . Use this structural information to design targeted mutations and analyze them with antibodies.

• Protein segment analysis: The C-terminus of GPR161 contains an A-kinase anchoring protein (AKAP) domain that binds PKA type I regulatory subunits . Use deletion constructs and domain-specific antibodies to study the contribution of this region.

• Combining with functional readouts: Correlate antibody-based localization/detection with functional outputs like Gli transcription factor repressor formation or cAMP signaling .

This integrated approach enables detailed mechanistic studies of GPR161 structure-function relationships.

What techniques can be used to study GPR161 dynamics during Hedgehog pathway activation?

To study dynamic changes in GPR161 localization and function during Hedgehog signaling:

• Live-cell imaging: Generate fluorescently tagged GPR161 constructs and perform real-time imaging during Hedgehog stimulation. Validate key findings with antibody staining of endogenous protein.

• Pulse-chase experiments: Use antibodies to track the fate of GPR161 after Hedgehog pathway activation. Upon SHH stimulation, GPR161 exits cilia and internalizes to recycling endosomes .

• Co-localization analysis: Perform dual immunofluorescence with markers for different subcellular compartments (primary cilia, recycling endosomes, etc.) to track GPR161 movement.

• Photoactivatable or photoconvertible tags: For advanced live imaging, use photoactivatable constructs combined with antibody validation.

• FRAP (Fluorescence Recovery After Photobleaching): Measure GPR161 mobility within cilia before and after Hedgehog stimulation.

• Proximity ligation assays: Use antibodies in proximity ligation assays to detect protein-protein interactions during signaling dynamics.

• Temporal analysis: Create detailed time courses of GPR161 localization, protein-protein interactions, and downstream signaling events after Hedgehog stimulation.

These approaches enable characterization of the dynamic behavior of GPR161 during Hedgehog pathway activation and inhibition.

Why might I observe discrepancies in the molecular weight of GPR161 in Western blots?

Discrepancies in observed molecular weight of GPR161 are common and can be attributed to several factors:

• Post-translational modifications: GPR161 has a calculated molecular mass of 59 kDa, but is frequently detected at approximately 70 kDa due to post-translational modifications . These may include:

  • Glycosylation of extracellular domains

  • Phosphorylation by GRK2 and other kinases

  • Ubiquitination

• Alternative splicing: GPR161 undergoes alternative splicing, resulting in multiple isoforms that may have different molecular weights .

• Sample preparation: The method of protein extraction and denaturation can affect the apparent molecular weight:

  • Complete denaturation with SDS and reducing agents is essential

  • Membrane proteins often migrate anomalously on SDS-PAGE

  • Heat-induced aggregation can cause higher molecular weight bands

• Antibody specificity: Different antibodies may recognize distinct epitopes or isoforms, leading to apparent differences in molecular weight.

To address these issues:

• Always include positive controls with known GPR161 expression

• Use multiple antibodies targeting different epitopes

• Consider using phosphatase treatment to eliminate phosphorylation-based shifts

• Validate specificity using GPR161 knockout samples or siRNA knockdown

How can I effectively distinguish between ciliary and non-ciliary pools of GPR161?

Distinguishing between ciliary and non-ciliary GPR161 pools is crucial for understanding its function and regulation:

• High-resolution imaging techniques:

  • Super-resolution microscopy (STED, STORM, PALM) to precisely localize GPR161

  • Confocal microscopy with careful z-stack acquisition and 3D reconstruction

• Biochemical fractionation:

  • Isolate ciliary fractions using established protocols

  • Compare with total membrane and cytosolic fractions by Western blot

• Dual immunofluorescence approach:

  • Co-stain with ciliary markers (ARL13B, acetylated tubulin)

  • Use membrane markers (Na+/K+ ATPase) and endosomal markers (RAB11) to identify non-ciliary pools

• Quantitative analysis:

  • Calculate ciliary enrichment factors (ratio of ciliary to non-ciliary signal)

  • Use intensity profiles along the ciliary axoneme

• Genetic approaches:

  • Create GPR161 mutants defective in ciliary localization (like GPR161 mut1)

  • Compare phenotypes between ciliary localization-defective and wild-type GPR161

• Selective permeabilization:

  • Use digitonin for selective plasma membrane permeabilization while leaving ciliary membranes intact

  • Compare with full permeabilization using Triton X-100

• Proximity labeling:

  • Use APEX2 or BioID fused to ciliary proteins to selectively label ciliary proteins

These approaches allow for clear distinction between ciliary and extraciliary pools of GPR161, which is essential for understanding its role in the Hedgehog pathway.

How can GPR161 antibodies be used to investigate its role in developmental disorders and disease models?

GPR161 antibodies can be powerful tools for investigating developmental disorders:

• Neural tube defects: GPR161 knockout embryos exhibit neural tube ventralization . Use antibodies to:

  • Characterize GPR161 expression patterns in normal and pathological neural tube development

  • Correlate GPR161 localization with Hedgehog pathway activity markers

  • Identify potential misregulation in human neural tube defect samples

• Limb formation disorders: GPR161 is essential for forelimb formation and proper digit patterning . Use antibodies to:

  • Map expression patterns during critical developmental windows

  • Correlate with markers of limb field specification (Tbx5)

  • Analyze GPR161 in polydactyly and other limb formation disorder models

• Skeletal morphogenesis: GPR161 promotes osteoblastogenesis and regulates endochondral bone formation . Applications include:

  • Studying GPR161 expression in growth plate development

  • Analyzing interactions with Ihh signaling in chondrocyte differentiation

  • Investigating bone collar and trabecular bone formation disorders

• Ciliopathies: As a ciliary protein, GPR161 may be implicated in ciliopathies. Research approaches include:

  • Screening ciliopathy patient samples for altered GPR161 localization

  • Investigating GPR161 interactions with known ciliopathy proteins

  • Analyzing GPR161 in models of Joubert syndrome, Bardet-Biedl syndrome, etc.

• Cancer models: Hedgehog pathway dysregulation is implicated in multiple cancers. GPR161 antibodies can be used to:

  • Assess GPR161 expression in tumor samples

  • Correlate with Hedgehog pathway activation markers

  • Investigate potential prognostic value

These applications enable detailed investigation of GPR161's role in both developmental disorders and disease processes.

What are the current technical limitations in studying GPR161 and how might they be overcome?

Several technical challenges exist in GPR161 research:

• Low endogenous expression levels:

  • Challenge: Endogenous GPR161 is often below detection threshold in standard assays

  • Solutions: Use signal amplification methods like tyramide signal amplification, RNAscope for mRNA detection, or generate knock-in tagged versions at endogenous loci

• Dynamic regulation and transient interactions:

  • Challenge: GPR161 undergoes rapid trafficking between compartments

  • Solutions: Develop better live imaging tools, use optogenetic control of Hedgehog pathway components, apply single-molecule tracking techniques

• Orphan receptor status:

  • Challenge: The endogenous ligand for GPR161 is unknown

  • Solutions: Perform unbiased ligand screens, use the recent cryo-EM structure to guide virtual screening approaches, develop sensors for GPR161 activation

• Multiple functional domains:

  • Challenge: GPR161 has both GPCR and AKAP functions that are difficult to separate

  • Solutions: Generate domain-specific mutations, use domain-specific antibodies, develop selective inhibitors

• Species-specific differences:

  • Challenge: GPR161 function may vary between model organisms

  • Solutions: Generate species-specific antibodies, perform careful cross-species validation, develop humanized models

• Antibody cross-reactivity:

  • Challenge: Ensuring antibody specificity across applications

  • Solutions: Validate with knockout controls, use multiple antibodies targeting different epitopes, perform careful titration experiments

Future technological developments that might address these limitations include:

• CRISPR-based endogenous tagging strategies

• Improved proximity labeling techniques

• Advanced microscopy methods with higher spatial and temporal resolution

• More sensitive mass spectrometry approaches for interaction studies

• Computational modeling based on the recent structural data

Addressing these technical challenges will enable more comprehensive understanding of GPR161 biology.

How can I investigate the interplay between GPR161 and other components of the Hedgehog signaling pathway?

To investigate interactions between GPR161 and other Hedgehog pathway components:

• Epistasis analysis: Systematically knock down or inhibit pathway components in GPR161-null backgrounds:

  • Use SMO antagonists (cyclopamine) to determine dependence on canonical signaling

  • Analyze GLI processing in different genetic backgrounds

  • Test PKA inhibitors to determine the relationship with downstream effectors

• Protein-protein interactions:

  • Use co-immunoprecipitation with GPR161 antibodies to identify interacting partners

  • Perform proximity labeling experiments (BioID, APEX) with GPR161 as the bait

  • Validate interactions using complementary approaches (FRET, BiFC)

• Localization studies:

  • Analyze co-localization of GPR161 with PTCH1, SMO, and other pathway components

  • Track dynamic changes in localization upon pathway activation

  • Determine how GPR161 affects the localization of other components

• Functional output measurements:

  • Quantify GLI activator/repressor ratios in different contexts

  • Measure target gene expression changes

  • Analyze developmental outcomes in model systems

• Structural studies:

  • Use the recent cryo-EM structure of GPR161-Gs complex to guide interaction studies

  • Investigate potential for direct interactions with other pathway components

• Temporal dynamics:

  • Create detailed time courses of protein interactions and modifications

  • Analyze the sequence of events during pathway activation and inhibition

This multi-dimensional approach provides comprehensive insight into how GPR161 integrates with the broader Hedgehog signaling network.

What methods are most effective for studying GPR161's dual role in cAMP signaling and PKA anchoring?

GPR161 has two distinct but related functions: Gs-coupled cAMP generation and PKA anchoring through its AKAP domain . To study these functions:

• Selective domain disruption:

  • Generate mutants that selectively disrupt G protein coupling while preserving PKA binding

  • Create AKAP domain mutants that maintain G protein coupling

  • Use antibodies to confirm expression and proper localization of mutant proteins

• Functional separation of signaling outputs:

  • Measure cAMP levels using FRET-based sensors or TR-FRET cAMP assays

  • Analyze PKA substrate phosphorylation using phospho-specific antibodies

  • Compare outcomes in wild-type versus domain-specific mutants

• Localization studies:

  • Use antibodies to determine co-localization of GPR161 with PKA regulatory subunits

  • Analyze how mutations affect localization of both GPR161 and PKA

  • Study dynamics of complex formation upon pathway activation

• Protein-protein interaction analyses:

  • Use co-immunoprecipitation to determine binding partners

  • Analyze how different mutations affect interaction profiles

  • Perform in vitro binding assays with purified components

• Comparative signaling analyses:

  • Study how GPR161 mutations differentially affect GLI processing versus target gene expression

  • Compare with other GPCRs that lack AKAP domains

  • Analyze phenotypic outcomes in developmental contexts

• Biosensor development:

  • Create FRET-based sensors to monitor GPR161-PKA interactions

  • Develop proximity sensors for localized cAMP production

This integrated approach enables dissection of GPR161's dual role in cAMP generation and PKA anchoring, providing insight into how these functions cooperate in Hedgehog pathway regulation.