Recombinant Human Probable G-protein coupled receptor 61 (GPR61)

What is GPR61 and what makes it significant for metabolic research?

GPR61 is an orphan G protein-coupled receptor belonging to the class A (rhodopsin family) of GPCRs. It is a 451 amino acid protein containing 7 transmembrane domains that transduces extracellular signals through heterotrimeric G proteins . Its significance for metabolic research stems from its predominant expression in the pituitary and appetite-regulating centers of the hypothalamus and brainstem. Mutagenesis and human genome-wide association studies have linked GPR61 to phenotypes associated with type 2 diabetes and body mass index, establishing it as a potential target for the modulation of appetite and body weight . GPR61 shares sequence similarity (28-31%) with certain histamine, adrenergic, serotonin, and dopamine receptors, placing it within the biogenic amine receptor family .

The molecular structure of GPR61, determined through cryo-EM studies, reveals key features that explain its constitutive activity and potential for therapeutic targeting. Its ability to modulate cAMP pathways without a known endogenous ligand makes it particularly intriguing for research into disorders such as cachexia and obesity .

What cellular mechanisms are involved in GPR61 signaling?

GPR61 signaling operates primarily through the cAMP pathway by activating heterotrimeric G proteins. The mechanism involves binding of the C-terminal helix of Gα to an exposed intracellular pocket of the receptor formed by activating movements of TM5 and TM6. This binding event triggers Gα to exchange GDP for GTP, leading to its activation .

In the active state, a network of polar contacts involving residues of TM3, TM6, helix 8, and ICL2 facilitates binding of the C-terminal helix of Gαs within the intracellular pocket. Specific residues like Tyr143 on TM3 play crucial roles in this interaction . The constitutive activity of GPR61 may be attributed to structural features that partially destabilize the inactive state, allowing for signal transduction in the absence of an orthosteric ligand.

Mutational studies have identified specific mutations (T92P, R236C, and R262C) that reduce this constitutive activity, as measured by cAMP production and G protein translocation assays, providing further insights into the structural determinants of GPR61 signaling .

What experimental models are most suitable for studying GPR61 function?

For studying GPR61 function, several experimental models have proven effective:

• Cell-based assays: Overexpression systems in cell lines utilizing cAMP biosensors (such as EPAC-derived FRET-based biosensors) provide a robust means to measure GPR61's constitutive activity and the effects of potential modulators or mutations . These systems allow for high-throughput screening of compounds that may affect GPR61 function.

• ebBRET-based G protein translocation assays: This orthogonal technique measures heterotrimeric Gs activation directly, complementing cAMP production experiments and providing further validation of GPR61 activity modulation .

• Surface expression assays: HiBit-tagged forms of GPR61 can be used to measure receptor surface expression and internalization dynamics in response to compounds or mutations .

The choice of model should align with specific research questions, with combinations of these approaches providing more comprehensive insights into GPR61 biology .

How can I express and purify recombinant GPR61 for structural studies?

Expression and purification of recombinant GPR61 for structural studies requires specialized approaches due to its nature as a membrane protein. Based on published structures, researchers have successfully employed the following strategy:

• Construct design: For active-state studies, GPR61 has been studied in complex with heterotrimeric G proteins (Gαs/β1/γ2). For inactive-state studies, thermostabilized E. coli apocytochrome b562 RIL (BRIL) has been rigidly fused between TM5 and TM6, replacing intracellular loop 3 (ICL3) to provide additional mass for cryo-EM alignment .

• Expression system: Insect cell expression systems (such as Sf9 or High Five cells) are typically employed for GPCR expression due to their ability to perform post-translational modifications necessary for proper folding and function.

• Solubilization and purification: After membrane extraction, the receptor is solubilized using detergents or, preferably, lipid nanodiscs to maintain a more native-like environment. Affinity chromatography (typically using polyhistidine tags) followed by size exclusion chromatography yields pure protein.

• Quality control: The functional integrity of purified GPR61 should be assessed through ligand binding assays (if available) or, in this case, constitutional activity measurements.

For structural determination of GPR61, cryo-electron microscopy has been the method of choice, achieving resolutions of approximately 3.5 Å for the active-state complex . This allows for detailed analysis of protein-protein interactions and ligand binding modes.

What are the molecular determinants of GPR61's constitutive activity and how can they be targeted?

The constitutive activity of GPR61 appears to stem from multiple structural features that bias the receptor toward an active conformation even in the absence of ligand binding. Analysis of the active-state structure reveals several key determinants:

• N-terminal domain interactions: While the initial 44 residues of GPR61 are unresolved in the cryo-EM structure, suggesting transient interactions, these may still play a role in stabilizing the active conformation. Some portion of this peptide might bind to the extracellular surface or orthosteric site to accomplish activation .

• Transmembrane domain arrangement: The positioning of TM5 and TM6, which form the intracellular G protein binding pocket, appears to favor the active state. This arrangement involves specific networks of polar contacts and hydrophobic interactions that stabilize the activated conformation .

• Intracellular loop configuration: The conformation of ICL2 and its interaction with the G protein's C-terminal helix contributes to the stabilization of the active state. Key residues in this region form specific contacts that facilitate G protein coupling .

For targeting GPR61's constitutive activity, an inverse agonist approach has proven successful. Compound 1, a tertiary sulfonamide, has been identified as a potent and selective GPR61 inverse agonist (IC50 = 10-11 nM) that acts by binding to an allosteric pocket, blocking G protein activation by remodeling the intracellular pocket normally occupied by Gαs in the activated state . This represents a novel mechanism of GPCR inactivation that could be exploited for therapeutic development.

How do specific mutations affect GPR61 structure and function, and what can they tell us about receptor dynamics?

Specific mutations in GPR61 provide valuable insights into structure-function relationships and receptor dynamics. Studies have identified three mutations (T92P, R236C, and R262C) that significantly reduce the constitutive activity of GPR61 .

The effects of these mutations can be analyzed at multiple levels:

These mutations map to different regions of GPR61 and thus help identify multiple "hotspots" critical for maintaining constitutive activity. Understanding these structural determinants provides potential targets for rational drug design and suggests mechanisms for fine-tuning receptor activity.

What approaches are most effective for identifying and validating novel modulators of GPR61 activity?

Identifying and validating novel modulators of GPR61 activity requires a multi-faceted approach that combines high-throughput screening with detailed mechanistic validation. The most effective strategies include:

• High-throughput functional screening: Cell-based assays measuring cAMP levels in cell lines overexpressing GPR61 have successfully identified initial hits from compound libraries. This approach led to the discovery of sulfonamide-based GPR61 inverse agonists through optimization of initial hits from Pfizer's internal compound libraries .

• Orthogonal validation assays: Multiple assay formats should be employed to confirm activity:

  • cAMP production assays

  • G protein translocation assays (ebBRET-based)

  • Receptor surface expression measurements

  • Selectivity panels against related GPCRs

• Structure-based drug design: With the availability of high-resolution structures of GPR61 in both active and inactive states, rational design approaches can now be employed. The revealed allosteric binding site of the sulfonamide inverse agonist provides a template for structure-guided optimization .

• Molecular dynamics simulations: These computational approaches can predict the effects of structural modifications to lead compounds and guide medicinal chemistry efforts .

How can cryo-electron microscopy be optimized for structural studies of GPR61 in different conformational states?

Optimizing cryo-electron microscopy (cryo-EM) for structural studies of GPR61 in different conformational states presents unique challenges that require specific methodological approaches:

• Active-state stabilization: For the active-state structure, complexing GPR61 with heterotrimeric G proteins (Gαs/β1/γ2) provides sufficient mass and stability for high-resolution imaging. This approach yielded a 3.5 Å nominal resolution structure that clearly resolved all components of the complex with an architecture consistent with related active-state class A receptor structures .

• Inactive-state modifications: The inactive state poses greater challenges due to the absence of the G protein complex as a fiducial marker for particle alignment. To overcome this limitation, researchers employed a strategy similar to that used for inactive Frizzled 5, where thermostabilized E. coli apocytochrome b562 RIL (BRIL) was rigidly fused between TM5 and TM6, replacing intracellular loop 3 (ICL3) . This approach requires careful optimization:

  • Ensuring continuous helicity at both fusion junctions

  • Combining knowledge from experimental structures with AlphaFold predictions to guide construct design

  • Validating constructs for proper folding and function before structural studies

• Sample preparation optimizations:

  • Lipid nanodisc reconstitution to provide a more native-like membrane environment

  • Grid optimization with different hole sizes, support films, and vitrification conditions

  • Use of Volta phase plates or energy filters to enhance contrast

• Data processing strategies:

  • Implementing motion correction and CTF estimation algorithms tailored for smaller proteins

  • Employing 3D classification approaches to separate distinct conformational states

  • Using focused refinement on specific domains to maximize local resolution

These approaches have proven successful for structural characterization of GPR61 in both its active-like complex with heterotrimeric G protein and in its inactive state bound to an inverse agonist , providing valuable insights into the molecular mechanisms of receptor activation and modulation.

What is the role of GPR61 in metabolic disorders and how might its modulation offer therapeutic benefits?

GPR61's role in metabolic disorders stems from its expression pattern and demonstrated functional effects on appetite regulation. Multiple lines of evidence support its therapeutic potential:

• Expression profile: GPR61 is predominantly expressed in the pituitary and appetite-regulating centers of the hypothalamus and brainstem, suggesting direct involvement in central regulation of feeding behavior and metabolism .

• Phenotypic associations: Mutagenesis and human genome-wide association studies have linked GPR61 to phenotypes associated with type 2 diabetes and body mass index, providing genetic evidence for its relevance to metabolic health .

• Knockout studies: GPR61 knockout mice exhibit increased fat mass and hyperphagia, indicating that GPR61 normally functions to suppress appetite. This suggests GPR61 inhibition by an inverse agonist could be used to treat wasting disorders such as cachexia .

The therapeutic potential of GPR61 modulation differs depending on the metabolic disorder:

The structural characterization of GPR61 and the discovery of a selective inverse agonist provide a foundation for developing therapeutics targeting this receptor for metabolic disorders. The unique mechanism of the sulfonamide inverse agonist, which acts through an allosteric pocket to block G protein activation, offers a novel approach to GPCR modulation that could be exploited for highly selective therapies .

What are the most effective protein expression systems for recombinant GPR61 production?

Selecting the optimal expression system for recombinant GPR61 production depends on the research objectives and downstream applications. Based on successful structural and functional studies, several effective systems have been identified:

• Mammalian cell expression: For functional studies assessing GPR61 activity, mammalian cell lines (typically HEK293) have been successfully used. These systems provide appropriate post-translational modifications and cellular machinery for proper folding and trafficking, making them ideal for:

  • cAMP signaling assays

  • G protein translocation studies

  • Surface expression analyses

  • Compound screening campaigns

• Insect cell expression: For structural biology applications requiring larger quantities of purified protein, insect cell expression systems (Sf9 or High Five cells) offer significant advantages:

  • Higher expression levels than mammalian cells

  • Appropriate post-translational modifications for proper folding

  • Scalability for purification needs

  • Compatibility with complex formation (e.g., with G proteins)

• Construct optimization strategies:

  • Inclusion of N-terminal signal sequences

  • Addition of C-terminal tags (His10 or other affinity tags)

  • Fusion partners to enhance expression (e.g., BRIL for structural studies)

  • Thermostabilizing mutations to enhance stability during purification

The choice between these systems should be guided by the specific requirements of the experiment, with consideration for protein yield, functional integrity, and downstream applications. For high-resolution structural studies by cryo-EM, insect cell expression systems combined with appropriate fusion partners have proven most effective for GPR61 .

How should molecular dynamics simulations be designed to accurately model GPR61 behavior?

Designing effective molecular dynamics (MD) simulations for GPR61 requires careful consideration of multiple parameters to ensure physiologically relevant results. Based on published methodologies, the following approach is recommended:

• Input structure preparation:

  • Start with high-resolution structural data (such as PDB: 8KGK)

  • Address any unresolved regions (e.g., the C-terminal region of GPR61 missing from the Cryo-EM structure) by either excluding them from simulations or modeling them with appropriate tools

  • Carefully check protonation states of titratable residues at physiological pH (7.4)

• Membrane environment modeling:

  • Construct a hexagonal phospholipid bilayer around the protein using a physiologically relevant lipid composition

  • Embed this bilayer in a solution of water molecules and 0.15 M NaCl to replicate typical ionic conditions

• Force field selection and parameters:

  • Employ the CHARMM36m force field for accurate modeling of molecular interactions, particularly protein-lipid and protein-solvent interactions

  • Assign protonation states at physiological pH (7.4)

• Simulation protocol:

  • Perform energy minimization using the steepest descent algorithm to eliminate steric clashes

  • Implement a multi-step equilibration process

  • Conduct production simulations under NPT conditions (constant pressure and temperature)

  • Regulate temperature at 310 K using the Nose-Hoover thermostat

  • Run multiple replicates (at least triplicates) with trajectories spanning 200+ ns each

• Analysis approaches:

  • Calculate root mean square deviations (RMSDs) to assess structural stability

  • Analyze inter-residue distances, hydrogen bonds, and salt bridges to identify key interactions

  • Monitor transmembrane domain movements and conformational changes

  • Visualize and analyze trajectories using specialized software (e.g., Chimera or VMD)

This approach has been successfully employed to characterize the effects of GPR61 mutations on receptor dynamics and function, providing valuable insights that complement experimental data .

What techniques are most reliable for measuring GPR61's constitutive activity in vitro?

Measuring GPR61's constitutive activity in vitro requires sensitive and reproducible assays that can detect signaling in the absence of a known endogenous ligand. Based on published research, the following techniques have proven most reliable:

• FRET-based cAMP biosensors: Genetically encoded, EPAC-derived FRET-based biosensors provide a direct and real-time measurement of intracellular cAMP levels, which reflect GPR61's constitutive activity through the Gs pathway. This approach has successfully detected differences between wild-type GPR61 and mutant variants with reduced activity .

• ebBRET-based G protein translocation assays: This orthogonal approach directly measures the activation and translocation of heterotrimeric Gs proteins, providing a complementary readout of receptor activity. When combined with cAMP measurements, this technique offers more comprehensive insights into signaling mechanisms .

• Surface expression monitoring: HiBit-tagged forms of GPR61 allow quantification of cell surface expression, which can provide insights into receptor trafficking and stability. Increased surface expression has been observed in response to inverse agonist treatment, attributed to compensatory overexpression following inhibition .

• Comparative assay design: To accurately measure constitutive activity, appropriate controls are essential:

  • Empty vector transfected cells as negative controls

  • Cells expressing known constitutively active GPCRs as positive controls

  • Dose-response relationships with inverse agonists to confirm specificity

What are the prospects for discovering endogenous ligands for GPR61, and what screening approaches show the most promise?

The search for endogenous ligands of GPR61 remains a significant challenge and opportunity in GPCR research. As an orphan receptor with demonstrated physiological relevance, identifying its natural ligand(s) could substantially advance our understanding of its biological function and therapeutic potential. Several promising approaches include:

• Targeted metabolomics screening: Given GPR61's expression in the hypothalamus and its role in appetite regulation, screening tissue extracts from these regions using liquid chromatography-mass spectrometry (LC-MS) could identify candidate molecules. Sequential fractionation followed by activity testing represents a classical but still valuable approach.

• Candidate-based approaches: GPR61 shares sequence similarity with biogenic amine receptors (28-31%) , suggesting that related neurotransmitters or their metabolites might serve as starting points for screening. Structural information about the orthosteric binding pocket can guide the selection of candidate molecules.

• Reverse pharmacology: The newly discovered inverse agonist (Compound 1) provides a valuable tool for competitive binding assays. Compounds that displace the inverse agonist might include the endogenous ligand or its structural mimics.

• Genetic association studies: Analyzing conditions in which GPR61 function appears altered might reveal associated metabolic pathways, narrowing the search for potential ligands.

The plasticity of the extracellular portion of TM1 and the potential for displacement of ECL2 by a ligand, as observed in the case of rhodopsin, suggests that GPR61 could accommodate diverse ligands within its binding pocket . The diffuse density observed in this region of the cryo-EM structure indicates flexibility that might adapt to different molecular structures, complicating prediction but offering multiple possibilities for ligand discovery.

How might the structure-function relationship of GPR61 inform the development of biased signaling modulators?

The detailed structural characterization of GPR61 provides unprecedented opportunities for developing biased signaling modulators—compounds that selectively activate or inhibit specific downstream pathways. This approach could yield therapeutics with enhanced efficacy and reduced side effects.

Key structural insights that inform biased modulator development include:

• Allosteric binding sites: The discovery of Compound 1, which acts through an allosteric pocket by remodeling the intracellular G protein binding site, demonstrates the feasibility of targeting non-orthosteric sites on GPR61 . This allosteric binding mode potentially offers greater selectivity compared to orthosteric targeting.

• G protein interaction interface: The detailed mapping of interactions between GPR61 and the C-terminal helix of Gαs reveals specific contact points that could be selectively disrupted or enhanced by small molecules. These include key residues on TM3, TM6, helix 8, and ICL2 .

• Transmembrane domain dynamics: Molecular dynamics simulations of GPR61 reveal distinct conformational states that might preferentially couple to different signaling pathways. Compounds that stabilize specific conformational ensembles could bias signaling toward desired outcomes .

Development strategies for biased modulators might include:

• Structure-guided medicinal chemistry: Using the inactive-state structure with bound inverse agonist as a template for designing compounds with modified interaction profiles

• Fragment-based screening: Identifying small molecular fragments that bind to specific subpockets within the receptor and linking them to create biased modulators

• Computational docking and virtual screening: Leveraging the available structures to screen in silico for compounds predicted to make specific interactions while avoiding others

The ability to rationally design modulators with specific signaling profiles would be particularly valuable for metabolic disorders, where precisely tuned interventions might avoid unwanted effects on related physiological systems.

What insights can cross-species comparison of GPR61 provide about its evolutionary conservation and functional significance?

Cross-species comparison of GPR61 can provide valuable insights into its evolutionary history, functional conservation, and potential physiological roles. While the search results don't provide extensive information on cross-species comparison, we can outline a framework for such analysis:

• Sequence conservation analysis: Comparison of GPR61 protein sequences across species ranging from rodents to primates and other mammals can identify:

  • Highly conserved regions likely essential for core functions (transmembrane domains, G protein coupling interfaces)

  • Variable regions that might confer species-specific regulation or ligand specificity

  • Conserved post-translational modification sites

• Structural conservation: Using the human GPR61 structure as a template, homology modeling of GPR61 from different species can reveal:

  • Conservation of key structural features, particularly in the G protein binding pocket

  • Species-specific differences in potential ligand binding sites

  • Variations in extracellular loops that might affect ligand recognition or access

• Expression pattern comparison: Analysis of GPR61 expression across species can identify:

  • Conserved expression in appetite-regulating centers, supporting a fundamental role in metabolic regulation

  • Species-specific expression patterns that might reflect specialized functions

  • Developmental regulation patterns that could indicate roles beyond metabolic control

• Phenotypic correlation: Comparison of phenotypes in GPR61 knockout or mutation models across species would provide functional insights:

  • Conservation of metabolic phenotypes would strengthen the case for therapeutic targeting

  • Species-specific phenotypes might reveal additional functions or compensatory mechanisms

Such comparative analyses would be particularly valuable for translational research, helping to predict the relevance of preclinical findings to human physiology and potentially identifying model organisms that best recapitulate human GPR61 function for drug development purposes.

How can the newly discovered inverse agonist mechanism for GPR61 inform drug discovery for other constitutively active GPCRs?

The novel mechanism of GPR61 inhibition by the sulfonamide inverse agonist represents a significant advance in our understanding of GPCR modulation and offers valuable insights for drug discovery across the GPCR superfamily. This mechanism—binding to an allosteric pocket that remodels the intracellular G protein binding site—presents several broadly applicable principles:

• Targeting intracellular binding pockets: The mechanism reveals that functionally selective modulation can be achieved by targeting the intracellular face of GPCRs, rather than the more traditional extracellular orthosteric site. This approach could be particularly valuable for:

  • Other orphan receptors lacking known ligands

  • GPCRs with highly conserved orthosteric sites where selectivity is challenging

  • Constitutively active receptors involved in disease states

• Structure-based design principles: The GPR61-inverse agonist complex structure provides a template for rational design of modulators targeting similar mechanisms in other GPCRs:

  • Identification of conserved intracellular pocket architectures

  • Design of scaffolds that can access and stabilize inactive conformations

  • Exploitation of receptor-specific features for selectivity

• Pharmacological implications: The potency and selectivity achieved with Compound 1 (IC50 = 10-11 nM) demonstrates that this mechanism can yield highly effective modulators . For drug discovery, this suggests:

  • Screening libraries against intracellular pockets of constitutively active GPCRs

  • Repurposing existing chemical scaffolds to target similar mechanisms

  • Developing assays specifically designed to detect G protein-competitive binding

• Therapeutic applications: This mechanism provides a new paradigm for addressing diseases involving constitutively active GPCRs, which are implicated in various conditions including:

  • Metabolic disorders

  • Certain cancers with GPCR mutations

  • Endocrine disorders with receptor overactivation

The heretofore undescribed mechanism of GPCR inactivation demonstrated by GPR61 and its inverse agonist expands the repertoire of approaches for therapeutic modulation of this important receptor family .

What are the current technical challenges in GPR61 research and how might they be addressed?

GPR61 research faces several significant technical challenges that limit our comprehensive understanding of this receptor. These challenges, along with potential solutions, include:

• Lack of known endogenous ligands:

  • Challenge: The absence of identified natural ligands significantly hampers functional characterization and pharmacological studies .

  • Solutions:

    • Implement unbiased screening approaches using tissue extracts from regions of high GPR61 expression

    • Develop computational methods to predict potential ligands based on structural information

    • Utilize the discovered inverse agonist as a tool for competitive binding studies

• Limited availability of selective tool compounds:

  • Challenge: Until recently, no selective modulators of GPR61 were available for mechanistic studies.

  • Solutions:

    • The newly discovered sulfonamide inverse agonist (Compound 1) provides an important starting point

    • Develop derivatives with altered pharmacokinetic properties for in vivo studies

    • Design labeled versions of Compound 1 for binding and localization studies

• Difficulties in structural characterization:

  • Challenge: GPR61's nature as a membrane protein complicates structural studies.

  • Solutions:

    • The successful cryo-EM approach using G protein complexes for active states and BRIL fusion for inactive states provides effective templates

    • Further optimize constructs for crystallization attempts to achieve higher resolution

    • Explore alternative stabilization strategies for capturing different conformational states

• Translating in vitro findings to in vivo physiology:

  • Challenge: Connecting molecular mechanisms to physiological functions remains difficult.

  • Solutions:

    • Develop conditional and tissue-specific knockout models

    • Create knock-in models expressing mutations that alter constitutive activity

    • Develop PET ligands based on the inverse agonist scaffold for in vivo imaging

• Reconciling contradictory functional data:

  • Challenge: Different assay systems sometimes yield conflicting results, as seen with the R236C and R262C mutations that showed effects in cAMP assays but not consistently in G protein translocation assays .

  • Solutions:

    • Implement multiple orthogonal assays in parallel

    • Standardize expression levels and experimental conditions across laboratories

    • Develop more sensitive assays for detecting subtle changes in receptor conformation and activity

Addressing these challenges will require multidisciplinary approaches combining structural biology, pharmacology, molecular biology, and computational methods.

How reliable are current experimental models for studying GPR61 function, and what improvements are needed?

The reliability of current experimental models for studying GPR61 function varies considerably, with each approach presenting specific strengths and limitations that influence data interpretation. Critical evaluation of these models reveals several areas for improvement:

• Overexpression systems:

  • Current approach: Most functional studies utilize cell lines overexpressing GPR61, which provide clear signals for assay development .

  • Limitations: Artificial overexpression may alter receptor trafficking, interactions with signaling components, and constitutive activity levels.

  • Improvements needed:

    • Development of stable cell lines with physiologically relevant expression levels

    • CRISPR-edited cell lines expressing tagged endogenous GPR61

    • Primary cell cultures from tissues with native GPR61 expression

• Functional readouts:

  • Current approaches: FRET-based cAMP biosensors and ebBRET G protein translocation assays provide information on canonical signaling pathways .

  • Limitations: These may miss non-canonical signaling or pathway-specific effects.

  • Improvements needed:

    • Broader profiling of signaling pathways (e.g., β-arrestin recruitment, ERK activation)

    • Phosphoproteomic analysis to identify all affected signaling nodes

    • Transcriptomic studies to identify downstream gene expression changes

• Animal models:

  • Current understanding: GPR61 knockout mice exhibit increased fat mass and hyperphagia .

  • Limitations: Global knockouts may trigger compensatory mechanisms; species differences may limit translational relevance.

  • Improvements needed:

    • Conditional and tissue-specific knockout models

    • Humanized mouse models expressing human GPR61

    • Models with altered GPR61 activity (e.g., through mutation or pharmacological modulation)

• Structural models:

  • Current approach: Cryo-EM structures provide valuable insights but represent static snapshots .

  • Limitations: Dynamic processes and transitional states are missed; some regions remain unresolved.

  • Improvements needed:

    • Time-resolved structural studies

    • Combined NMR-cryo-EM approaches to capture dynamic information

    • Improved computational models integrating experimental constraints

• Comparing data across models:

  • Current challenge: Different experimental systems sometimes yield contradictory results .

  • Improvements needed:

    • Standardized protocols and reporting

    • Multi-center validation studies

    • Development of reference compounds and assays

The field would benefit significantly from establishing consensus guidelines for GPR61 research, including standardized assays, reference compounds, and reporting formats to enhance data comparison across different laboratories and experimental models.

How might advanced integrated approaches enhance our understanding of GPR61's role in metabolic regulation?

Advanced integrated approaches combining multiple technologies and disciplines offer the potential to substantially deepen our understanding of GPR61's role in metabolic regulation. Future research directions should focus on:

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data from GPR61-expressing tissues under different metabolic conditions

  • Correlating GPR61 expression and activity with global metabolic signatures

  • Identifying metabolites that co-vary with GPR61 activity as potential endogenous ligands

• Systems biology modeling:

  • Developing computational models of appetite regulation circuits incorporating GPR61 signaling

  • Predicting the effects of GPR61 modulation on whole-body energy homeostasis

  • Simulating the impact of different feeding states on GPR61 activity and downstream effects

• Advanced imaging technologies:

  • Implementing PET imaging with labeled GPR61 ligands to visualize receptor distribution and occupancy in vivo

  • Using CRISPR-based tagging of endogenous GPR61 for high-resolution cellular imaging

  • Employing optogenetic or chemogenetic approaches to selectively activate GPR61-expressing neurons

• Genetic approaches in model organisms:

  • Creating conditional and inducible GPR61 knockout/knockin models

  • Employing CRISPR-based screens to identify genes that modify GPR61 phenotypes

  • Developing humanized mouse models expressing human GPR61 for translational studies

• Clinical correlation studies:

  • Analyzing GPR61 variants in population cohorts with metabolic phenotyping

  • Examining GPR61 expression in post-mortem samples from patients with metabolic disorders

  • Correlating circulating metabolites with GPR61 variants to identify potential biomarkers

Integration of these approaches would provide a comprehensive view of GPR61's role in metabolic regulation, potentially revealing:

• Temporal aspects of GPR61 signaling during feeding and fasting cycles

• Cell-type specific functions in different brain regions

• Interaction with other appetite-regulating pathways

• Novel therapeutic opportunities for precision medicine approaches to metabolic disorders

What potential exists for developing allosteric modulators of GPR61 with improved pharmacological properties?

The discovery of an allosteric binding site for the sulfonamide inverse agonist of GPR61 opens significant opportunities for developing modulators with enhanced pharmacological properties. Future development could focus on:

• Structure-guided optimization:

  • Using the elucidated binding mode of Compound 1 to design derivatives with:

    • Improved brain penetration for targeting central GPR61

    • Enhanced selectivity against related GPCRs

    • Modified pharmacokinetic properties for different administration routes

  • Exploring different chemical scaffolds that maintain key interactions with the allosteric binding site

• Functional diversity:

  • Developing a spectrum of modulators beyond inverse agonists:

    • Neutral antagonists that block potential endogenous ligands without affecting constitutive activity

    • Partial inverse agonists for fine-tuned modulation

    • Positive allosteric modulators that might enhance activity of yet-to-be-discovered endogenous ligands

  • Creating biased modulators that selectively affect specific downstream pathways

• Novel binding approaches:

  • Exploring additional allosteric sites identified through computational mapping

  • Developing bitopic ligands that simultaneously engage multiple binding sites

  • Creating covalent modulators for prolonged engagement with GPR61

• Advanced delivery strategies:

  • Tissue-targeted delivery systems to concentrate modulators in appetite-regulating centers

  • Stimuli-responsive formulations that release modulators under specific physiological conditions

  • Combinations with other appetite-regulating agents for synergistic effects

• Translational considerations:

  • Species differences in the allosteric binding site must be addressed

  • Patient stratification based on GPR61 variants might be necessary

  • Different modulators may be needed for obesity versus cachexia

The unique mechanism of the discovered sulfonamide inverse agonist, which acts by remodeling the intracellular G protein binding site, provides a novel paradigm for GPCR modulation that could yield highly selective therapeutics with reduced off-target effects . This represents a significant advance in the field of GPCR pharmacology with broad implications beyond GPR61.

What methodological advances are needed to fully characterize the conformational dynamics of GPR61?

Fully characterizing the conformational dynamics of GPR61 requires methodological advances that can capture the receptor's behavior across multiple timescales and conditions. Current approaches provide valuable but incomplete information, and several technological developments would enhance our understanding:

• Time-resolved structural methods:

  • Development of time-resolved cryo-EM approaches to capture short-lived conformational intermediates

  • Implementation of temperature-jump or ligand-jump techniques compatible with structural determination

  • Application of serial femtosecond crystallography at X-ray free electron lasers (XFELs) if suitable crystals can be obtained

• Enhanced spectroscopic approaches:

  • Site-specific incorporation of fluorescent unnatural amino acids for direct monitoring of conformational changes

  • Application of double electron-electron resonance (DEER) spectroscopy to measure distances between labeled sites during activation

  • Development of 19F NMR approaches for GPR61 to monitor specific conformational changes in solution

• Advanced computational methods:

  • Implementation of enhanced sampling techniques for molecular dynamics simulations

  • Development of machine learning approaches to predict conformational transitions from limited experimental data

  • Integration of experimental constraints from multiple techniques into computational models

• Single-molecule techniques:

  • Application of single-molecule FRET to monitor conformational changes in individual GPR61 molecules

  • Development of force spectroscopy approaches to characterize the energetics of conformational transitions

  • Implementation of high-speed atomic force microscopy to visualize structural dynamics

• Native environment characterization:

  • Methods to study GPR61 dynamics in native membrane environments rather than detergent or simplified lipid systems

  • Approaches to monitor conformational changes in living cells with high temporal and spatial resolution

  • Techniques to assess the impact of membrane composition on GPR61 dynamics

These methodological advances would address critical questions about GPR61 dynamics:

• The sequence of conformational changes during activation and inhibition

• The energy landscape governing transitions between different states

• The influence of membrane environment on receptor behavior

• The molecular determinants of constitutive activity

• The conformational effects of the inverse agonist and potential undiscovered ligands

Integrating information from these complementary approaches would provide a comprehensive view of GPR61 dynamics essential for understanding its function and developing more effective modulators.