Recombinant Mouse 2-oxoglutarate receptor 1 (Oxgr1)

What is the molecular structure and classification of mouse OXGR1?

Mouse OXGR1 (also known as GPR99) belongs to the Class A (Rhodopsin) family of G-protein-coupled receptors, specifically within the alicarboxylic acid receptors subfamily . The receptor contains seven transmembrane domains typical of GPCRs, with Leu124 residing in the third transmembrane helix, where its side chain protrudes into the receptor's central core . The protein structure reveals that this leucine residue is surrounded within 4Å by predominantly hydrophobic amino acid side chains (five of seven), creating a critical structural feature of the receptor .

What is the tissue distribution of OXGR1 in mouse models?

OXGR1 exhibits specific tissue distribution patterns with significant expression in cardiovascular and renal systems. In the kidney, OXGR1 is localized to Type B and non-A non-B intercalated cells of the cortical collecting duct (CCD), with enrichment in the luminal membrane . Western blot analysis confirms OXGR1 expression in wild-type mouse hearts, while it is almost completely absent in OXGR1 knockout (OXGR1−/−) mice . This specific expression pattern suggests specialized physiological roles in these tissues.

What are the primary ligands and activation mechanisms for mouse OXGR1?

The primary endogenous ligand for OXGR1 is α-ketoglutarate (αKG), a citric acid cycle intermediate . In experimental settings, OXGR1 activation can be achieved with 1 mM αKG, which has been demonstrated to increase 45Ca2+ influx and stimulate ERK phosphorylation in expression systems . More recent research has also identified cysteinyl leukotriene, particularly leukotriene E4, as another potential ligand for OXGR1, suggesting multiple activation pathways for this receptor .

How can I express and detect recombinant mouse OXGR1 in heterologous systems?

For heterologous expression of mouse OXGR1, the Xenopus oocyte system has proven effective. This experimental approach involves:

• Preparation of OXGR1 cRNA for microinjection

• Injection of 50 ng OXGR1 cRNA into Xenopus oocytes

• Incubation for 72 hours at 17.5°C in MBS (Modified Barth's Solution)

• Protein extraction using RIPA buffer containing protease inhibitors (6 μl per oocyte)

• Processing through cycles of vortexing, freezing at -80°C, thawing, and centrifugation

• Clarified protein extraction and quantification using the BCA method

For detection, MYC-tagged OXGR1 can be visualized using:

• Confocal immunofluorescence microscopy to observe surface expression

• Immunoblot analysis using anti-MYC antibodies (20 μg of extract protein mixed with SDS load buffer containing β-mercaptoethanol)

What functional assays are available to measure OXGR1 activation?

Several validated functional assays can assess OXGR1 activation:

What are the established protocols for generating OXGR1 knockout models?

OXGR1 knockout mice have been successfully generated and characterized for functional studies. These models display normal cardiac size and function under basal conditions but exhibit enhanced responses to pathological stimuli . The generation of such models involves:

• Genetic ablation of the Oxgr1 gene

• Confirmation of knockout via Western blot analysis of cardiac tissue

• Phenotypic characterization under both basal and stressed conditions

• Comparison with wild-type littermates as controls

For in vitro studies, adenoviral vectors expressing OXGR1 (Ad-OXGR1) can be used to achieve OXGR1 overexpression in neonatal rat cardiomyocytes (NRCM), with Ad-LacZ serving as a control vector .

What is the role of OXGR1 in cardiac physiology and pathophysiology?

OXGR1 functions as a negative regulator of pathological cardiac hypertrophy. Research evidence supporting this function includes:

• OXGR1−/− mice exhibit an enhanced hypertrophic response following transverse aortic constriction (TAC), with significantly increased:

  • Heart weight/body weight ratio (HW/BW)

  • Cardiomyocyte cross-sectional area (WT TAC: 460.2 ± 6.8 μm2 vs. KO TAC: 549.6 ± 6.7 μm2)

  • Expression of hypertrophic marker Atrial Natriuretic Peptide (ANP)

• OXGR1−/− mice show impaired cardiac function after TAC:

  • Increased cardiac posterior wall thickness

  • Significantly reduced cardiac fractional shortening and ejection fraction

  • Evidence of maladaptive cardiac remodeling

• Conversely, adenoviral-mediated OXGR1 overexpression in cardiomyocytes reduces phenylephrine-induced hypertrophy, confirming its protective role .

These findings suggest that OXGR1 plays a crucial role in suppressing maladaptive cardiac hypertrophy and preserving contractile function during pressure overload stress.

How does OXGR1 regulate renal function in mouse models?

In the kidney, OXGR1 regulates Pendrin-mediated anion exchange activity in intercalated cells:

• OXGR1 is expressed in Type B and non-A non-B intercalated cells of the cortical collecting duct (CCD)

• When activated by αKG, OXGR1 increases Pendrin-mediated Cl−/HCO3− exchange through:

  • A Ca2+-dependent, PKCα- or PKCδ-dependent pathway

  • Alternatively, via a PKC-independent, SPAK-mediated pathway

• This regulation contributes to:

  • Maintaining systemic volume homeostasis

  • Mediating base secretion in response to systemic base load

  • Facilitating Cl− reabsorption during systemic volume depletion

Recent genetic evidence suggests OXGR1 may also be involved in calcium oxalate nephrolithiasis, indicating a potential role in mineral metabolism and kidney stone formation .

What intracellular signaling pathways are activated by OXGR1?

OXGR1 engages multiple signaling cascades upon activation:

Of particular importance is the interaction between OXGR1 and CSN5 (also known as JAB1), which appears to regulate STAT3 activation. CSN5 also interacts with TYK2, a major upstream regulator of STAT3, suggesting that OXGR1 may regulate the pro-hypertrophic STAT3 pathway via interaction with the CSN5-TYK2 complex .

What is the evidence linking OXGR1 to cardiovascular diseases?

Evidence linking OXGR1 to cardiovascular diseases comes primarily from pressure overload studies in mouse models:

• OXGR1 functions as a suppressor of pathological cardiac hypertrophy, with knockout mice showing:

  • Enhanced hypertrophic response to TAC

  • Reduced contractile function (decreased fractional shortening and ejection fraction)

  • Increased maladaptive cardiac remodeling

• The metabolic context is significant as α-ketoglutarate, the OXGR1 ligand, is elevated in the serum of heart failure patients, suggesting potential physiological relevance

• Mechanistically, OXGR1 inhibits the pro-hypertrophic STAT3 pathway, potentially via its interaction with CSN5 and TYK2

This evidence indicates that OXGR1 could represent a novel therapeutic target for pathological cardiac hypertrophy, with receptor agonists potentially offering cardioprotective effects.

How is OXGR1 implicated in kidney stone formation?

Recent genetic studies have identified OXGR1 as a candidate disease gene for human calcium oxalate nephrolithiasis:

• Exome sequencing and directed sequencing of the OXGR1 locus in a worldwide nephrolithiasis/nephrocalcinosis cohort revealed rare, potentially deleterious OXGR1 variants

• Structural modeling showed that one variant (L124R substitution) affects the third transmembrane helix of OXGR1, potentially reducing protein stability (predicted pseudoΔΔG = −2.7)

• The co-segregation of these variants with disease phenotypes supports a causal relationship between OXGR1 dysfunction and kidney stone formation

These findings suggest that OXGR1 may play a previously unrecognized role in renal calcium handling and mineral metabolism, with implications for understanding and treating nephrolithiasis.

How do experimental conditions affect OXGR1 function in recombinant systems?

When studying recombinant mouse OXGR1, several experimental factors critically influence function and interpretation:

• Expression system selection:

  • Xenopus oocytes have proven suitable for OXGR1 functional expression

  • Expression levels must be carefully monitored as they affect baseline activity

  • Co-expression with potential effector proteins (e.g., Pendrin) requires optimization of relative expression levels

• Activation conditions:

  • αKG concentration (typically 1 mM) must be standardized across experiments

  • Response timing varies between immediate events (calcium influx) and delayed responses (gene expression)

  • Temperature sensitivity of GPCR signaling requires strict control (17.5°C for oocytes)

• Data interpretation challenges:

  • The effects of OXGR1 stimulators can be obscured at near-maximal activity levels

  • Background signaling in expression systems must be accounted for

  • Complex interplay between multiple signaling pathways requires careful experimental design

What protein-protein interactions modulate OXGR1 function?

Key protein interactions regulate OXGR1 signaling and function:

• CSN5/JAB1 interaction:

  • Identified as an OXGR1 interacting partner via yeast two-hybrid screening

  • Confirmed by immunoprecipitation analysis

  • Mediates regulation of downstream signaling pathways

  • Known to control protein phosphorylation, ubiquitination, and nuclear-cytoplasmic translocation

• TYK2 connection:

  • CSN5 interacts with TYK2, a major upstream regulator of STAT3

  • Suggests OXGR1 may regulate STAT3 pathway via the CSN5-TYK2 complex

  • Provides mechanistic explanation for enhanced STAT3 activation in OXGR1−/− hearts

• Signaling complex formation:

  • OXGR1 likely forms part of larger signaling complexes

  • These complexes may include G-proteins, kinases, and scaffold proteins

  • Complex assembly and stability affects signaling efficiency and specificity

Understanding these interactions is crucial for developing therapeutic approaches targeting OXGR1 function.

What are the current contradictions and knowledge gaps in OXGR1 research?

Despite significant advances, several important questions remain unresolved:

• Ligand specificity concerns:

  • OXGR1 was initially classified as a P2Y receptor responsive to adenosine/AMP

  • Later identified as an α-ketoglutarate receptor

  • More recently described as a receptor for cysteinyl leukotriene (leukotriene E4)

  • The physiological relevance and context-dependency of these multiple ligands require clarification

• Tissue-specific functions:

  • While cardiac and renal functions are partially characterized, OXGR1's role in other tissues remains unclear

  • Expression data from single-cell RNA sequencing suggests broader tissue distribution

  • The physiological significance of OXGR1 in these additional tissues is unknown

• Signaling pathway integration:

  • How OXGR1 signaling integrates with other metabolic and physiological pathways

  • Whether OXGR1 function changes under different metabolic states

  • The complete downstream effectors and their tissue-specific activities

• Therapeutic potential:

  • Whether OXGR1 agonists could provide cardioprotection remains speculative

  • Potential off-target effects of OXGR1 modulation need evaluation

  • Development of tissue-specific targeting strategies is still in its infancy