ARL15 Antibody

What is ARL15 and why is it significant in current research?

ARL15 is a member of the ARF family of the Ras superfamily of small GTPases. Its significance lies in its involvement in multiple regulatory pathways, including TGFβ family signaling, adiponectin regulation, and metabolic control. The ARL15 gene locus has been implicated in numerous conditions including rheumatoid arthritis, type 2 diabetes, insulin resistance, and alterations in plasma adiponectin and HDL cholesterol levels . As a small G protein, ARL15 functions as a molecular switch, cycling between active GTP-bound and inactive GDP-bound states, allowing it to regulate various downstream pathways. Recent research has revealed its critical role in promoting the assembly of the Smad-complex, establishing ARL15 as an important modulator of TGFβ signal transduction .

What is the cellular and tissue distribution of ARL15?

ARL15 exhibits a broad tissue distribution pattern. Quantitative real-time PCR analysis of human tissues has demonstrated that ARL15 mRNA is expressed across multiple organs . At the subcellular level, GFP-tagged ARL15 localizes predominantly to the Golgi apparatus, with lower levels detected at the plasma membrane and in intracellular vesicles, suggesting its involvement in intracellular trafficking pathways . Immunocytochemical experiments have also demonstrated ARL15 localization in the kidney and endoplasmic reticulum, where it co-localizes with proteins like CNNM2 and RPN1 . This broad distribution pattern aligns with ARL15's diverse biological functions across multiple cellular systems.

What are the primary applications for ARL15 antibodies in research?

ARL15 antibodies are versatile tools that support multiple experimental approaches:

These applications enable researchers to investigate ARL15's expression, localization, interactions, and functions across various experimental systems .

How does ARL15 contribute to TGFβ family signaling?

ARL15 positively regulates TGFβ family signaling through a direct interaction with Smad4. When in its active GTP-bound state, ARL15 specifically binds to the MH2 domain of Smad4, relieving Smad4's autoinhibition, which is imposed by intramolecular interaction between its MH1 and MH2 domains . This molecular interaction enables activated Smad4 to subsequently interact with phosphorylated receptor-regulated Smads (R-Smads), forming the Smad-complex essential for TGFβ signal transduction .

Interestingly, the relationship between ARL15 and Smad4 is bidirectional. Research shows that Smad4 functions not only as an effector but also as a GTPase activating protein (GAP) for ARL15. The assembly of the Smad-complex enhances the GAP activity of Smad4 toward ARL15, accelerating GTP hydrolysis and dissociating ARL15 before the nuclear translocation of the Smad-complex . This creates a sophisticated feedback mechanism that regulates the strength and duration of TGFβ signaling.

What protein interactions have been identified for ARL15?

Several important protein interactions have been identified for ARL15:

• Smad4: ARL15 directly binds to the MH2 domain of Smad4, as identified through yeast two-hybrid screening and confirmed by immunoprecipitation assays .

• ARL6IP5 (also known as JWA and GTRAP3-18): Mass spectrometry analysis of immunoprecipitated complexes from mouse brain, adipose tissue, and 3T3-L1 cells identified ARL6IP5 as a potential interacting partner of ARL15 .

• CNNM2 and CNNM3: ARL15 has been shown to interact with CNNM proteins, with both proteins showing subcellular co-localization in the kidney. Additional co-immunoprecipitation assays demonstrated interaction between CNNM3, ARL15, and RPN1 .

• R-Smads: While ARL15 does not directly bind to R-Smads, it indirectly interacts with them through Smad4, which bridges the interaction between ARL15-GTP and phosphorylated R-Smads like Smad1, Smad2, and Smad1/5/8 .

These interactions position ARL15 as a hub connecting multiple cellular pathways, particularly in signaling and intracellular trafficking.

How does ARL15 affect gene expression in response to TGFβ signaling?

ARL15 significantly impacts the transcriptional responses to TGFβ stimulation. In MCF7 cells (representing early-stage breast cancer), overexpression of active ARL15-AL (GTP-bound form) was sufficient to upregulate transcription of N-cadherin, ID1, Snail1, p27kip1, p21cip1, Fibronectin, and α-SMA, while downregulating E-cadherin and c-Myc expression . These transcriptional changes align with TGFβ's known roles in promoting cytostasis and epithelial-mesenchymal transition (EMT).

Conversely, depletion of ARL15 in TGFβ1-treated MCF7 cells reversed these transcriptional patterns, with downregulation of genes normally upregulated by ARL15-AL overexpression and vice versa . Similar results were observed in MDA-MB-231 cells, which represent highly metastatic breast cancer cells. These findings demonstrate that ARL15 is essential for the full transcriptional response to TGFβ stimulation and highlights its potential role in cancer progression through regulation of EMT and cell cycle control genes.

What strategies are most effective for studying ARL15's GTPase activity?

Investigating ARL15's GTPase activity requires specific experimental approaches to distinguish between its active (GTP-bound) and inactive (GDP-bound) states:

• Mutational strategies: Researchers commonly use specific mutations that lock ARL15 in either GTP-bound or GDP-bound conformations. The A86L mutation (referred to as AL) creates a GTP non-hydrolyzable form, while the T46N mutation (referred to as TN) generates a GDP-bound inactive form .

• Nucleotide loading experiments: Using non-hydrolyzable GTP analogs like guanosine 5′-[β,γ-imido]triphosphate (GMPPNP) helps maintain ARL15 in its active conformation during biochemical assays .

• Interaction-based detection: Since only GTP-bound ARL15 interacts with certain partners (like Smad4), co-immunoprecipitation with these partners can serve as an indirect measure of ARL15's activation state .

• Downstream signaling readouts: Monitoring transcriptional targets of TGFβ/BMP pathways provides functional readouts of ARL15 activity, as active ARL15 enhances Smad-dependent transcription .

These approaches allow researchers to correlate ARL15's nucleotide-binding state with its biological functions, providing mechanistic insights into its role in various cellular processes.

What are reliable methods to validate ARL15 antibody specificity?

Ensuring antibody specificity is crucial for obtaining reliable results in ARL15 research. Several validation approaches are recommended:

• Genetic controls: The gold standard validation employs tissues from Arl15 knockout mice as negative controls for western blotting and immunohistochemistry. This approach confirms signal specificity, as demonstrated in research utilizing homozygous Arl15 knockout mouse brain as a negative control .

• RNA interference validation: Knockdown of ARL15 using siRNA or shRNA provides a complementary approach to validate antibody specificity. For example, research has shown that siRNA-mediated knockdown of Smad2 but not Smad3 significantly reduced the band detected by anti-Smad2/3 antibody in HEK293T cells .

• Recombinant protein controls: Using purified recombinant ARL15 protein as a positive control helps establish the correct molecular weight and antibody reactivity pattern.

• Multiple detection methods: Confirming results across different techniques (western blotting, immunohistochemistry, immunoprecipitation) strengthens confidence in antibody specificity.

• Proper antigen retrieval for IHC: For immunohistochemistry applications, appropriate antigen retrieval methods are crucial. TE buffer at pH 9.0 is suggested, with citrate buffer at pH 6.0 as an alternative .

These validation approaches collectively provide robust confirmation of antibody specificity, essential for accurate interpretation of experimental results.

How can researchers effectively study ARL15's role in adipocyte biology?

Investigating ARL15's functions in adipocyte biology requires specialized experimental approaches:

• Conditional knockdown models: Utilizing conditional knockdown of Arl15 in murine 3T3-L1 (pre)adipocytes has been instrumental in demonstrating ARL15's role in adiponectin secretion and adipogenesis .

• Adipocyte differentiation assays: Monitoring the effect of ARL15 manipulation on adipogenesis through markers of differentiation and lipid accumulation assays helps elucidate its role in adipocyte development .

• Secretion assays: Measuring secreted adipokines (particularly adiponectin) in control versus ARL15-depleted adipocytes reveals ARL15's specific role in adipokine trafficking and secretion .

• Subcellular localization studies: Using GFP-tagged ARL15 to track its distribution in differentiating and mature adipocytes helps identify its association with specific organelles involved in protein secretion .

• Protein-protein interaction analyses: Immunoprecipitation followed by mass spectrometry has successfully identified ARL15-interacting proteins in adipose tissue, providing insights into its molecular mechanisms .

These methodologies have revealed that ARL15 plays distinct roles at different stages of adipocyte biology, affecting both differentiation of preadipocytes and specialized secretory functions of mature adipocytes .

How do genetic variants in ARL15 contribute to metabolic disorders?

Genome-wide association studies (GWAS) have identified the ARL15 gene locus as significantly associated with multiple metabolic traits:

• Adiponectin regulation: Variants in ARL15 influence circulating adiponectin levels, linking ARL15 to insulin sensitivity and metabolic regulation .

• Insulin resistance: Common genetic variants at the ARL15 locus are associated with plasma insulin concentrations .

• Lipid metabolism: ARL15 variants correlate with HDL cholesterol levels and obesity risk .

• Rare pathogenic variants: Sequencing of ARL15 in severely insulin-resistant patients identified rare heterozygous variants with potential clinical significance . These included:

  • An early nonsense mutation in a patient with femorogluteal lipodystrophy and non-classical congenital adrenal hyperplasia

  • An essential splice site mutation in a patient with partial lipodystrophy and history of childhood yolk sac tumor

The association between ARL15 variants and lipodystrophy is particularly significant, given experimental evidence showing ARL15's role in adipocyte differentiation and adiponectin secretion . These findings suggest that human ARL15 haploinsufficiency may predispose to lipodystrophy through impaired adipogenesis and altered adipokine secretion.

What is the relationship between ARL15 and BMP signaling pathways?

While most research has focused on ARL15's role in TGFβ signaling, evidence indicates it also participates in BMP (Bone Morphogenetic Protein) signaling:

• Promotion of BMP Smad-complex assembly: ARL15 facilitates the assembly of BMP Smad-complexes, similar to its role in TGFβ signaling .

• Essential for BMP-dependent transcription: Experimental data show ARL15 is required for the full activity of BMP R-Smad-dependent transcription .

• Potential mediator of TGFβ/BMP pathway crosstalk: Given that TGFβ and BMP pathways often have antagonistic roles in development and disease, ARL15 may mediate competition between these pathways for the common Smad4 partner .

This dual role in both TGFβ and BMP signaling positions ARL15 as a potential regulatory node in determining the balance between these sometimes opposing pathways. Further research is needed to fully understand how ARL15 may contribute to pathway-specific responses and cross-regulation.

How does ARL15 contribute to cancer biology through TGFβ signaling modulation?

ARL15's role in modulating TGFβ signaling has significant implications for cancer biology:

• Epithelial-mesenchymal transition (EMT) regulation: Overexpression of active ARL15 in MCF7 cells upregulates EMT markers (N-cadherin, Snail1, Fibronectin, α-SMA) while downregulating E-cadherin, promoting a more invasive phenotype .

• Cell cycle control: ARL15 affects expression of cell cycle regulators including p27kip1, p21cip1, and c-Myc, potentially influencing the cytostatic effects of TGFβ signaling .

• Cell-type specific responses: ARL15's effects were observed in both early-stage (MCF7) and highly metastatic (MDA-MB-231) breast cancer cell lines, suggesting its involvement across different cancer stages .

• Therapeutic implications: Understanding ARL15's contribution to TGFβ signaling in cancer cells may identify new therapeutic opportunities, particularly in cancers where the TGFβ pathway has switched from tumor-suppressive to tumor-promoting roles.

The observation that ARL15 depletion can reverse TGFβ-induced transcriptional changes suggests it might represent a potential therapeutic target in cancers where aberrant TGFβ signaling drives progression.

What are common challenges in detecting endogenous ARL15 expression?

Researchers often encounter specific challenges when working with endogenous ARL15:

• Antibody specificity: Ensuring antibody specificity is critical, as demonstrated by the use of knockout tissue controls in validation studies .

• Multiple isoforms: At least three isoforms of ARL15 are known to exist, which can complicate band interpretation in western blots .

• Optimal dilution range: For western blotting, ARL15 antibodies typically require optimization within a 1:200-1:1000 dilution range .

• Tissue-specific expression levels: Expression levels vary across tissues, requiring adjustment of detection protocols for different sample types.

• Post-translational modifications: As a GTPase, ARL15's detection may be affected by its nucleotide-binding status and potential modifications.

• Antigen retrieval for IHC: For immunohistochemistry applications, specific antigen retrieval conditions (TE buffer pH 9.0 or alternatively citrate buffer pH 6.0) are recommended for optimal results .

Addressing these challenges requires careful optimization and the use of appropriate positive and negative controls in each experimental system.

What experimental strategies differentiate between functions of ARL15-GTP and ARL15-GDP forms?

Distinguishing the functions of active versus inactive ARL15 requires specific experimental approaches:

• Expression of nucleotide-binding mutants: Using the constitutively active A86L mutant (AL, GTP-bound form) versus the inactive T46N mutant (TN, GDP-bound form) allows comparison of functional outcomes .

• Interaction partner analysis: Only ARL15-GTP (not ARL15-GDP) interacts with Smad4, providing a readout for the active state. For example, research showed that exogenously expressed GFP-tagged Arl15-AL, but not Arl15-TN, pulled down endogenous Smad4 .

• Transcriptional readouts: Comparing the effects of ARL15-GTP versus ARL15-GDP on TGFβ-responsive gene expression provides functional differentiation between these states .

• Subcellular localization differences: Potential differences in localization between active and inactive forms can be visualized using tagged constructs and cellular fractionation .

• In vitro nucleotide loading: For biochemical assays, loading recombinant ARL15 with non-hydrolyzable GTP analogs versus GDP allows direct comparison of biochemical properties .

These approaches have collectively demonstrated that the GTP-bound form of ARL15 is responsible for its biological activity in promoting TGFβ and BMP signaling.

How can researchers optimize co-immunoprecipitation protocols for studying ARL15 interactions?

Optimizing co-immunoprecipitation (co-IP) for ARL15 interaction studies requires several specific considerations:

• Nucleotide supplementation: For studying GTP-dependent interactions, supplementing lysates with non-hydrolyzable GTP analogs like GMPPNP stabilizes ARL15 in its active conformation. Research has shown that endogenous Smad4 pulled down substantially more endogenous Arl15 in the presence of GMPPNP than GDP .

• Stimulation conditions: TGFβ1 treatment significantly increases the interaction between Smad4 and ARL15, as demonstrated by enhanced co-immunoprecipitation following TGFβ stimulation .

• Validation controls: Using tissue from homozygous Arl15 knockout mice provides an excellent negative control for specificity of co-IP experiments .

• Detergent conditions: Mild detergents are preferable to preserve protein-protein interactions, especially for membrane-associated proteins like ARL15.

• Cross-validation: Confirming interactions through reverse co-IP (using antibodies against the partner protein) strengthens confidence in results, as shown in studies where both ARL15-GFP was used to pull down Smad4 and endogenous Smad4 was used to pull down ARL15 .

These optimized approaches have successfully identified and characterized several key interaction partners of ARL15, including Smad4, ARL6IP5, and components of the CNNM protein family .