GPR119 Antibody

What is GPR119 and why is it important in metabolic research?

GPR119 is a cannabinoid receptor-like class A G protein-coupled receptor (GPCR) that plays critical roles in glucose homeostasis and feeding behavior. It is highly expressed in pancreatic β cells and intestinal enteroendocrine L cells, making it particularly relevant to metabolic research . GPR119 primarily couples to Gs proteins to activate adenylate cyclase and cyclic AMP signaling pathways. The importance of GPR119 in metabolic research stems from its ability to stimulate glucose-dependent insulin release from the pancreas and promote the secretion of incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) . Additionally, GPR119 activation has been shown to suppress food intake in rats and reduce body weight gain, suggesting its potential as a therapeutic target for metabolic disorders such as obesity and type 2 diabetes . With diabetes prevalence projected to reach 700 million cases by 2045, GPR119-targeted therapies represent a promising avenue for addressing this growing health crisis .

How does GPR119 differ structurally from other GPCRs?

GPR119 exhibits a distinctive structural feature that differentiates it from many other GPCRs—specifically, a one-amino acid shift of the conserved proline residue in transmembrane helix 5 (TM5). This shift forms an outward bulge that creates a unique hydrophobic cavity between TM4 and TM5 in the middle of the membrane . This cavity is specifically adapted for binding endogenous monounsaturated lipid metabolites, reflecting GPR119's specialized function. Phylogenetic analysis places GPR119 in the class A rhodopsin-like receptor family, with closer evolutionary relationships to adenosine and cannabinoid receptors than to other GPCR subfamilies . The full-length human GPR119 protein consists of 335 amino acids, with homologous proteins identified across various vertebrates including rats, zebrafish, and monkeys . From a signaling perspective, GPR119 features a distinctive salt bridge between intracellular loop 1 (ICL1) of the receptor and the Gβs subunit, which is critical for downstream signaling—disruption of this salt bridge eliminates cAMP production, indicating the essential role of Gβs in GPR119-mediated signaling pathways .

What detection methods are available for studying GPR119 expression?

Several methodological approaches have been developed for detecting and quantifying GPR119 expression in research settings. Flow cytometry represents a particularly effective technique for measuring cell surface expression of GPR119. In a protocol described in the literature, cells transfected with GPR119 are incubated with anti-Flag M2-fluorescein isothiocyanate antibody for 20 minutes at 4°C under light-protected conditions, followed by reaction termination with TBS buffer . The resulting cell surface expression of GPR119 can then be quantified using flow cytometry instruments such as the FACSCalibur. This approach allows for precise measurement of receptor expression levels while accounting for background fluorescence by deducting signals from negative control cells without fluorescein isothiocyanate labeling . For more comprehensive detection, researchers commonly employ a combination of techniques including Western blotting with GPR119-specific antibodies, immunohistochemistry for tissue localization studies, and quantitative PCR for mRNA expression analysis. When selecting antibodies for GPR119 detection, researchers should consider specificity validation, particularly given the structural similarities between GPR119 and other cannabinoid receptor-like GPCRs.

What expression systems are optimal for GPR119 studies?

The choice of expression system is critical for successful GPR119 research, particularly for structural and functional studies. Based on current methodologies, insect cell expression systems have proven particularly effective for high-yield production of functional GPR119 protein. Specifically, High Five cells (derived from Trichoplusia ni) using baculovirus expression vectors have been successfully employed for co-expression of human GPR119 with Gαs and Gβ1γ2 protein components . This system allows for robust expression over a 48-hour period, after which cell precipitates can be collected via centrifugation and stored frozen for subsequent purification and analysis .

For functional studies and cell-based assays, mammalian expression systems including HEK293 and CHO cells provide appropriate post-translational modifications and trafficking of GPR119. When designing expression constructs, researchers should consider incorporating epitope tags to facilitate detection and purification. The inclusion of a C-terminal strep tag on GPR119 and a his tag on Gβ1 has been reported to facilitate effective complex purification . For optimal expression and stability, modifications such as T4 lysozyme fusion may be beneficial, though researchers should verify that such modifications do not alter receptor pharmacology through appropriate control experiments .

How can researchers effectively employ antibodies for GPR119 studies?

While specific GPR119 antibody information is limited in current literature, researchers can leverage several approaches for antibody-based GPR119 studies. For flow cytometry applications, anti-tag antibodies (such as anti-Flag M2-fluorescein isothiocyanate) can be used to detect epitope-tagged GPR119 constructs on the cell surface . This approach requires careful optimization of antibody concentration, incubation time (typically 20 minutes), temperature (4°C), and protection from light to minimize photobleaching .

For structural studies, nanobodies (such as Nb35) that stabilize the receptor-G protein complex have proven valuable. These can be produced in E. coli expression systems, purified through affinity and gel filtration chromatography, and incorporated during complex formation to enhance stability for techniques such as cryo-EM . When designing immunoprecipitation protocols for GPR119, researchers should consider the hydrophobic nature of this transmembrane protein, which may require optimization of detergent conditions to maintain protein stability while preserving antibody-epitope interactions. Cross-validation with multiple detection methods is recommended to confirm specificity of antibody-based GPR119 detection.

What are the key considerations when designing GPR119 mutagenesis experiments?

Mutagenesis experiments have been instrumental in elucidating the structure-function relationships of GPR119. When designing such experiments, researchers should first identify key residues involved in ligand binding or receptor activation based on structural data. Several residues have been identified as critical for GPR119 function, including F157^ECL2^ and W265^7.39^, where mutation to alanine completely abolishes activation response . Similarly, mutations of W238^6.48^ to alanine result in complete loss of activation effect, highlighting the importance of π-π interactions in receptor function .

Other important considerations include residues V85^3.32^ and F241^6.51^, where alanine substitutions significantly decrease cAMP accumulation but do not completely eliminate function . The hydrophobic cavity formed by residues T86^3.33^, A89^3.36^, A90^3.37^, V93^3.40^, L94^3.41^, I136^4.56^, and L169^5.43^ is critical for agonist interaction . Substitution of polar amino acid T86^3.33^ with non-polar glycine or alanine abolishes agonist potency, while substituting hydrophobic L94^3.41^ with negatively charged aspartic acid leads to complete loss of agonist potency .

When reporting mutagenesis results, researchers should use standardized nomenclature (e.g., Ballesteros-Weinstein numbering for GPCRs) and include appropriate positive and negative controls to account for potential expression level differences between mutants.

What techniques have been most effective for determining GPR119 structure?

Cryo-electron microscopy (cryo-EM) has emerged as the most effective technique for determining high-resolution structures of GPR119 in complex with various ligands and G proteins. This technique has successfully resolved structures of GPR119-Gs complexes bound to agonists including AR231453, MBX-2982, and APD597 at resolutions approaching 2.8 Å . The cryo-EM workflow typically involves:

• Expression and purification of the GPR119-Gs complex, often stabilized with nanobody Nb35

• Vitrification of purified samples on cryo-EM grids

• Data collection using high-end electron microscopes

• Image processing including motion correction, CTF estimation, and particle picking

• Ab initio reconstruction and heterogeneous refinement

• Non-uniform refinement for final resolution improvement

The resulting density maps allow for detailed modeling of the 7TM elements of GPR119, the Gs heterotrimer, and the bound agonist in the orthosteric pocket . Notably, this approach has revealed that each GPR119 agonist adopts a specific binding mode, with distinguishable interaction patterns that correlate with their pharmacological properties. For model building and refinement, researchers typically use existing structures as initial models (e.g., using PDB: 7WCM as a starting point), followed by iterative manual building in COOT and real-space refinement in PHENIX .

How do different agonists interact with the GPR119 binding pocket?

APD597 interacts with the binding pocket through several key mechanisms:

• Its dimethylpyridine moiety forms π-π interactions with F157^ECL2^ and W265^7.39^

• The pentamethylpyrimidine tail, piperidine, and isopropyl carboxylate moieties interact with a hydrophobic cluster including T86^3.33^, A89^3.36^, A90^3.37^, V93^3.40^, L94^3.41^, I136^4.56^, and L169^5.43^

• Pentamethylcytosine forms a hydrogen bond with W265^7.39^ and engages in π-π interaction with W238^6.48^

MBX-2982 shows similar interactions but with notable differences, particularly in the positions of Q65^2.64^ and E261^7.35^, which come into closer proximity to its tetrazole moiety . AR231453 features distinct interactions, with its 2-fluoro group engaging in a halogen-π interaction with F241^6.51^ and its 4-methylsulfonyl group forming a hydrogen bond with F157^ECL2^ .

The potency of agonists is significantly influenced by specific structural features. For APD597, the methyl group plays a crucial role, as its removal leads to a 20-100 fold reduction in activation potency . Similarly, the 5-position of the central pyrimidinyl moiety is particularly sensitive to substituent size and polarity .

What structural features determine GPR119 coupling specificity to G proteins?

The structural basis for GPR119 coupling to G proteins involves specific interaction interfaces that determine signaling specificity. A critical feature is the salt bridge formed between intracellular loop 1 (ICL1) of GPR119 and the Gβs subunit . Disruption of this salt bridge eliminates cAMP production, highlighting the essential role of Gβs in GPR119-mediated signaling . This distinctive feature suggests that both the α and β subunits of the G protein complex contribute to effective coupling.

The GPR119-Gαs interaction interface is formed primarily through the Ras domain of Gαs, while the α-helix domain appears to have flexibility as indicated by missing density in structural studies . Upon agonist binding, GPR119 undergoes conformational changes that create a suitable interface for G protein coupling. These changes include shifts in transmembrane helices, particularly TM6, which are characteristic of GPCR activation .

Experimental approaches to study coupling specificity include:

• Site-directed mutagenesis of key interface residues

• Assessment of coupling efficacy through cAMP accumulation assays

• Structural determination of complex formation using cryo-EM

• Measurement of downstream signaling events

These approaches have collectively demonstrated that GPR119 primarily couples to Gs proteins, leading to activation of adenylate cyclase and cyclic AMP signaling . This coupling specificity underlies the receptor's ability to stimulate insulin secretion and incretin release, which are central to its potential as a therapeutic target for metabolic disorders.

How does GPR119 activation influence incretin and insulin secretion?

GPR119 activation triggers a cascade of physiological responses that ultimately enhance both incretin and insulin secretion. In pancreatic β cells, activation of GPR119 directly stimulates glucose-dependent insulin release through a Gs-coupled pathway that increases intracellular cAMP levels . This cAMP accumulation enhances glucose-stimulated insulin secretion by potentiating calcium-dependent exocytosis of insulin granules.

In intestinal enteroendocrine cells, particularly L-cells, GPR119 activation promotes the secretion of incretin hormones including GLP-1 and GIP . These incretins then act on pancreatic β cells to further enhance glucose-dependent insulin secretion, creating a coordinated response to manage glucose homeostasis. This dual mechanism—direct action on β cells and indirect action via incretin hormones—makes GPR119 a particularly attractive therapeutic target for type 2 diabetes, as it addresses multiple aspects of the disease pathophysiology .

Research approaches to study these effects include:

• Measurement of cAMP accumulation in cell-based assays

• Quantification of insulin secretion from pancreatic β cell lines or isolated islets

• Assessment of GLP-1 and GIP release from intestinal cell lines or primary cultures

• In vivo studies measuring plasma incretin and insulin levels following GPR119 agonist administration

These investigations have demonstrated that targeting the GPR119/incretin axis represents a promising approach for developing novel therapeutics for metabolic disorders that could potentially address the needs of patients who struggle to achieve optimal blood sugar control with existing treatments .

What are the methodological challenges in translating GPR119 research from in vitro to in vivo systems?

Translating GPR119 research from in vitro cellular systems to in vivo models presents several methodological challenges. A primary consideration is the pharmacokinetic properties of GPR119 agonists. Many endogenous GPR119 agonists exhibit relatively low efficacy in cell experiments, along with poor stability and solubility, limiting their utility in clinical research . While synthetic agonists like APD597 offer improved solubility and pharmacokinetic profiles, optimizing drug delivery remains challenging.

• Species differences in GPR119 expression and function between humans and animal models

• Potential off-target effects of GPR119 agonists in complex physiological systems

• Development of biomarkers that accurately reflect GPR119 engagement and activation

• Assessment of long-term efficacy and safety profiles in chronic administration scenarios

Addressing these challenges requires complementary approaches, including the use of humanized animal models, careful validation of biomarkers, and development of increasingly selective agonists based on detailed structural understanding of the receptor-ligand interactions.

How can antibody-based detection methods enhance GPR119 tissue distribution studies?

Antibody-based detection methods provide valuable tools for investigating GPR119 tissue distribution, though specific information about GPR119 antibodies is limited in current literature. Flow cytometry using fluorescently labeled antibodies represents an established approach for quantifying GPR119 expression on cell surfaces . This technique can be adapted for tissue distribution studies by analyzing primary cell populations isolated from different tissues.

For more comprehensive tissue mapping, immunohistochemistry (IHC) and immunofluorescence (IF) techniques can be employed. These approaches require:

• Careful fixation and sectioning of tissue samples

• Antigen retrieval to expose epitopes potentially masked during fixation

• Blocking of non-specific binding sites

• Incubation with validated primary antibodies against GPR119

• Detection using labeled secondary antibodies or amplification systems

• Counterstaining to visualize tissue architecture

When designing such studies, researchers should include appropriate positive controls (tissues known to express GPR119, such as pancreatic islets and intestinal L-cells) and negative controls (tissues not expected to express GPR119 or sections stained with isotype control antibodies). Validation of antibody specificity is particularly important and can be accomplished through parallel analysis of GPR119 knockout tissues or cells, or through correlation with mRNA expression data from PCR or in situ hybridization studies.

How do mutations in GPR119 affect agonist binding and receptor activation?

Mutagenesis studies have provided crucial insights into the relationship between GPR119 structure and function. Key findings from these studies reveal distinct roles for different amino acid residues in agonist binding and signal transduction. Critical residues for GPR119 function include:

These findings demonstrate that mutations can affect receptor function through multiple mechanisms. Some mutations disrupt direct ligand binding interactions, while others may alter the conformational changes required for receptor activation and G protein coupling. For instance, T86^3.33^ appears to play a critical role in forming interactions with the pentamethylcytosine moiety of APD597, as mutations to non-polar residues abolish agonist potency . Similarly, the π-π interaction between W238^6.48^ and pentamethylcytosine appears essential for receptor activation, as evidenced by the complete loss of activity when this residue is mutated to alanine .

The differential effects of mutations on receptor response to different agonists (e.g., APD597 versus MBX-2982) highlight the complexity of GPR119 activation mechanisms and suggest opportunities for developing agonists with tailored pharmacological profiles.

What are the latest advances in developing selective GPR119 agonists for research applications?

Recent structural insights into GPR119-agonist interactions have accelerated the development of increasingly selective agonists for research applications. Current approaches focus on optimizing three key aspects of agonist design:

• Structure-activity relationship refinement: Detailed structural studies have revealed that minor modifications to agonist structures can significantly impact potency and selectivity. For instance, the methyl group in APD597 plays a crucial role in activation, as its removal leads to a 20-100 fold reduction in potency . Similarly, the 5-position of the central pyrimidinyl moiety demonstrates sensitivity to substituent size and polarity .

• Targeting specific binding pocket regions: The GPR119 binding pocket can be divided into three distinct regions, each contributing differently to agonist interaction . Selective targeting of these regions through rational design can enhance specificity and potentially modulate signaling outcomes.

• Optimizing pharmacokinetic properties: Beyond potency and selectivity, developing agonists with favorable solubility, stability, and metabolic profiles is essential for effective research applications. APD597 demonstrates advantages in this regard, exhibiting good solubility and producing high-concentration hydroxyl metabolites with extended half-lives .

How can researchers investigate biased signaling in GPR119-mediated pathways?

Investigating biased signaling in GPR119-mediated pathways requires sophisticated experimental approaches that can distinguish between different downstream signaling outcomes. While GPR119 primarily couples to Gs proteins to activate adenylate cyclase and cyclic AMP signaling , there may be additional signaling pathways or biased signaling mechanisms that remain to be fully characterized.

A comprehensive approach to investigating biased signaling includes:

• Multiple readout assays: Researchers should employ assays measuring different signaling outcomes, including:

  • cAMP accumulation for Gs-mediated signaling

  • Calcium mobilization for potential Gq-mediated responses

  • β-arrestin recruitment using bioluminescence resonance energy transfer (BRET)

  • ERK1/2 phosphorylation to capture convergent signaling outputs

• Comparative analysis of different agonists: Different ligands may preferentially activate specific signaling pathways. Comparative studies of endogenous ligands and synthetic agonists such as APD597, MBX-2982, and AR231453 can reveal biased signaling profiles.

• Structural studies of different active states: The structures reported for GPR119 in complex with various agonists reveal subtle differences in receptor conformation . These structural variations may correlate with different signaling outcomes and provide insights into the molecular basis of biased signaling.

• Mutagenesis of key signaling interface residues: The salt bridge between ICL1 of GPR119 and Gβs plays a critical role in signaling . Targeted mutations at this interface and other potential signaling interfaces could reveal residues specifically involved in distinct signaling pathways.

• Tissue-specific signaling analysis: Different cellular contexts may support different signaling outcomes due to varying expression of G proteins and other signaling components. Comparing GPR119 signaling in pancreatic β cells versus intestinal L cells could reveal context-dependent signaling biases.

These approaches collectively provide a framework for characterizing the complex signaling landscape of GPR119 and may identify opportunities for developing biased agonists that selectively activate beneficial pathways while minimizing unwanted effects.