Recombinant Human Neuropeptide Y receptor type 5 (NPY5R)

What is the basic structure of human NPY5R?

NPY5R is a G-protein-coupled receptor with 7 transmembrane domains and a length of 445 amino acids. The gene encoding NPY5R is located on chromosome 4q32.2 and contains 7 exons . The protein has several aliases including NPYR5, NPY5-R, and NPYY5-R . NPY5R belongs to the neuropeptide Y receptor family, which comprises four functional subtypes (Y1R, Y2R, Y4R, Y5R) . The full protein sequence is available and contains important functional domains that mediate its interactions with ligands and downstream signaling pathways .

What are the primary ligands for NPY5R and how do they interact with the receptor?

NPY5R primarily interacts with neuropeptide Y (NPY) and peptide YY (PYY), as well as their truncated forms. Human NPY5R can be activated by NPY, PYY, NPY3-36, and PYY3-36 . The receptor-ligand interaction involves specific recognition patterns that differ between receptor subtypes. Y1R and Y4R require the full-length N terminus of NPY for full agonist activity, while Y2R can bind N-terminally truncated NPY and PYY with high affinity, and Y5R accepts peptides with deletion of the first residue .

The binding mode involves the C terminus of the peptide occupying a specific site within the transmembrane helical bundle, with multiple polar and hydrophobic interactions between specific arginine residues (R33, R35) of the peptide and receptor residues contributing to binding specificity .

What signaling pathways are activated by NPY5R?

NPY5R primarily signals through inhibition of adenylate cyclase activity . Upon activation, NPY5R couples to G proteins, particularly Gi proteins, leading to inhibition of cAMP production. In cardiac myocytes, NPY5R activation increases protein kinase C (PKC) activity and activates mitogen-activated protein kinase pathways .

Gene Set Enrichment Analysis (GSEA) of NPY5R co-expressed genes revealed involvement in several signaling pathways including JAK-STAT, Wnt, and MAPK signaling pathways . Additionally, NPY5R has been shown to influence cell cycle regulation and apoptotic pathways, with overexpression leading to G2/M phase cell cycle arrest and increased apoptosis in breast cancer cells .

What are effective methods for studying NPY5R binding kinetics?

NanoBRET-based binding assays have proven effective for studying NPY5R binding kinetics. This approach uses a nanoluciferase fused to the N terminus of the receptor as an energy donor and a fluorophore-tagged peptide ligand as an acceptor . This homogeneous, wash-free assay directly reports ligand affinities over many orders of magnitude and is sensitive to conformational changes in the binding pocket.

The methodology involves:

• Creating fusion constructs with nanoluciferase at the N-terminus of the receptor

• Validating the wild-type-like properties of tagged constructs using receptor activation assays and radioligand binding

• Measuring bioluminescence energy transfer (BRET) between the receptor and fluorophore-labeled ligands

• Distinguishing between high and low-affinity binding states

This approach has revealed biphasic binding curves with two clearly separated affinity states for peptide binding to NPY receptors, providing insights into the binding mechanisms .

How can NPY5R expression be effectively manipulated in cell culture systems?

For overexpression studies, transfection of expression plasmids containing the NPY5R gene (e.g., pcDNA-NPY5R) into cell lines has been effectively demonstrated in breast cancer cell lines like MDA-MB-231 and SK-BR-3 . The methodology involves:

• Selecting cell lines with low or absent endogenous NPY5R expression

• Constructing expression vectors containing the full NPY5R coding sequence

• Transfecting cells using standard methods (lipofection, electroporation)

• Confirming expression by RT-PCR and western blot analysis

• Conducting functional assays (proliferation, apoptosis, cell cycle) to assess the impact of NPY5R overexpression

For knockdown experiments, siRNA or shRNA approaches targeting NPY5R can be employed, followed by similar validation and functional analyses.

What functional assays are most informative for studying NPY5R activity?

Several functional assays have been effectively used to assess NPY5R activity:

• Cell Proliferation Assays:

  • CCK-8 assay has been used to measure cell proliferation in NPY5R-overexpressing cells

  • Colony formation assays provide insights into long-term effects on cell growth

• Apoptosis Assays:

  • Flow cytometry with annexin V/PI staining to quantify early and late apoptotic cells

  • Western blot analysis of apoptosis markers (cleaved caspase-9, PARP)

• Cell Cycle Analysis:

  • Flow cytometry with PI staining to determine cell cycle distribution

  • Western blot analysis of cell cycle regulators (cyclin B1, cdc25c)

• Signaling Pathway Analysis:

  • Western blot to detect phosphorylation of downstream signaling molecules

  • Reporter gene assays to measure pathway activation

• Drug Sensitivity Tests:

  • Dose-response curves with chemotherapeutic agents (e.g., doxorubicin) to assess the impact of NPY5R on drug sensitivity

What is the role of NPY5R in cancer, particularly breast cancer?

NPY5R functions as a tumor suppressor in breast cancer but is frequently downregulated in tumors compared to normal tissue . Analysis of TCGA-BRCA dataset showed significantly decreased NPY5R expression in tumor tissues compared with tumor-free tissues, which was confirmed in independent GEO datasets (GSE37751, GSE5364) . Immunohistochemistry and qPCR further confirmed lower NPY5R protein and mRNA levels in breast cancer tissues compared to adjacent non-tumor tissue .

Functionally, overexpression of NPY5R in breast cancer cell lines (MDA-MB-231 and SK-BR-3) significantly suppressed cell proliferation and colony formation ability . Mechanistically, NPY5R increased apoptosis through enhanced cleavage of caspase-9 and PARP, and induced G2/M phase cell cycle arrest by decreasing key regulators cyclin B1 and cdc25c . Additionally, NPY5R overexpression enhanced the sensitivity of breast cancer cells to doxorubicin .

Gene Set Enrichment Analysis revealed that NPY5R expression is associated with negative regulation of intrinsic apoptotic signaling pathway and regulation of cell cycle G2/M phase transition .

What is the current status of NPY5R antagonists in clinical development for obesity?

Despite the initial promise of NPY5R antagonists for obesity treatment, clinical trials have shown limited efficacy. Two Y5R antagonists, MK-0557 and velneperit, have been tested in clinical trials but failed to achieve clinically meaningful reductions in obesity .

MK-0557, a potent, highly selective, orally active NPY5R antagonist, was tested in a 52-week, multicenter, randomized, double-blind, placebo-controlled trial involving 1,661 overweight and obese patients . Although weight loss was statistically significant at 52 weeks, the magnitude was not clinically meaningful . These results provide insight into the human NPY-energy homeostatic pathway and suggest that solely targeting NPY5R in future drug development programs is unlikely to produce therapeutic efficacy .

This clinical experience suggests that more complex approaches targeting multiple components of the NPY signaling system may be necessary for effective obesity treatment.

How does NPY5R expression and methylation status correlate with clinical outcomes in cancer?

NPY5R expression appears to be regulated by promoter methylation in breast cancer, with low expression correlating with poor outcomes . Random forest algorithm analysis of the GSE29431 dataset identified NPY5R as a top gene according to prognostic importance .

The correlation between NPY5R expression and clinical outcomes suggests its potential as a biomarker for breast cancer diagnosis and treatment . Low NPY5R expression has been associated with poor prognosis in breast cancer patients, indicating its potential prognostic value .

Additionally, NPY5R expression has been shown to enhance sensitivity to doxorubicin in breast cancer cells, suggesting that NPY5R status might predict response to chemotherapy . This indicates potential utility as a predictive biomarker for treatment selection.

How do the binding mechanisms differ between NPY receptor subtypes?

The binding mechanisms of NPY receptors show significant diversity despite recognizing similar ligands. Structural studies of human Y1, Y2, and Y4 receptors in complex with NPY or PP and the Gi1 protein have revealed receptor-specific recognition patterns .

Key differences include:

• N-terminus requirements: Y1R and Y4R require the full-length N-terminus of NPY for full agonist activity, while Y2R can bind N-terminally truncated NPY and PYY with high affinity, and Y5R accepts peptides with deletion of the first residue .

• C-terminal interactions: Y1R appears to form interactions with more residues at the peptide C-terminus than Y2R .

• Binding pocket configuration: Y1R and Y4R have a narrower binding cavity for certain residues (like Y36) compared to Y2R, due to differences in amino acid composition at key positions .

• Arginine interactions: R33 forms an ionic contact with D6.59 only in Y2R, while it orients more toward N6.55 and F6.54 in Y1R and Y4R . R35 makes an additional ionic interaction with E205 in ECL2 in Y2R, which is not observed in other subtypes .

These structural differences provide a molecular basis for developing selective drugs targeting specific NPY receptor subtypes.

What are the molecular mechanisms underlying NPY5R's role in chemoresistance?

NPY5R has been implicated in both promoting and inhibiting chemoresistance, depending on the cancer type. In neuroblastoma, NPY5R is involved in a pathway that protects cells from chemotherapy-induced cell death, suggesting it could be a therapeutic target .

Conversely, in breast cancer, NPY5R overexpression enhanced the sensitivity of cells to doxorubicin . The mechanistic basis for this sensitization involves:

• Enhanced apoptotic signaling: NPY5R increases cleaved caspase-9 and PARP levels, promoting apoptotic cell death .

• Cell cycle regulation: NPY5R induces G2/M phase arrest, which can enhance sensitivity to certain chemotherapeutic agents .

• Signaling pathway modulation: NPY5R influences several signaling pathways including JAK-STAT, which may affect cellular responses to chemotherapy .

These opposing roles in different cancers highlight the context-dependent function of NPY5R and suggest that personalized approaches may be necessary when targeting NPY5R in cancer therapy.

How does the complex interplay between different NPY receptors affect physiological outcomes?

The four NPY receptor subtypes (Y1R, Y2R, Y4R, Y5R) often work in concert to mediate various physiological processes, with complex interactions affecting outcomes. While each receptor has distinct binding properties and tissue distribution, their overlapping expression and activation by similar ligands create intricate regulatory networks .

In cancer biology, different NPY receptors may have opposing effects. For example, NPY1R has been shown to inhibit prostate cancer progression , while Y5R antagonism inhibited proliferation in some breast cancer cell lines but had no effect on others .

The differential expression patterns of NPY receptors across tissues and developmental stages, combined with their ability to form homo- and heterodimers, add further complexity to this system. Understanding this interplay is crucial for developing effective therapeutic strategies targeting the NPY system.

What are the key considerations for developing selective NPY5R modulators?

Developing selective NPY5R modulators presents several challenges due to the high homology between NPY receptor subtypes and their overlapping ligand preferences. Key considerations include:

• Structural insights: Leveraging structural differences in binding pockets between receptor subtypes is crucial. For example, Y1R and Y4R have narrower binding cavities for certain residues compared to Y2R . These differences can be exploited to design selective modulators.

• Critical binding residues: Mutational studies have identified residues critical for NPY5R binding and function. For instance, R35 of NPY makes specific interactions with receptor residues that differ between subtypes . Targeting these unique interaction points can enhance selectivity.

• Allosteric sites: Exploring allosteric binding sites distinct from the orthosteric (peptide-binding) pocket may offer greater selectivity between receptor subtypes.

• Function vs. binding selectivity: A compound may bind to multiple NPY receptor subtypes but elicit functional responses only through specific subtypes. Functional assays are therefore essential to complement binding studies when characterizing selectivity.

• In vivo validation: Due to compensatory mechanisms between NPY receptors, in vitro selectivity may not translate to in vivo efficacy, as demonstrated by the limited clinical efficacy of selective Y5R antagonists in obesity treatment .

How can contradictory findings about NPY5R function in different cancer models be reconciled?

Contradictory findings regarding NPY5R function in cancer can be reconciled through several methodological approaches:

• Context-dependent analysis: NPY5R's effects appear to be highly context-dependent. In breast cancer, it functions as a tumor suppressor , while in other contexts, it may promote cell survival . Systematic analysis across multiple cancer types and models is needed.

• Expression level consideration: The relative expression levels of NPY5R and other NPY receptors may determine the net effect of receptor modulation. Quantitative analysis of receptor expression across models is essential.

• Signaling pathway integration: NPY5R activates multiple downstream pathways, including JAK-STAT, Wnt, and MAPK . The predominant pathway activated may depend on the cellular context and determine the functional outcome.

• Co-receptor and heterodimer analysis: NPY receptors can form homo- and heterodimers, potentially altering signaling properties. Analysis of receptor complexes rather than individual receptors may explain contradictory findings.

• Epigenetic regulation: NPY5R is subject to epigenetic regulation, particularly promoter methylation . Different methylation patterns across cancer types may contribute to functional differences.

Addressing these factors through comprehensive, multi-omics approaches can help resolve conflicting observations and lead to a more nuanced understanding of NPY5R's role in cancer.

What are the most robust approaches for validating NPY5R as a therapeutic target?

Robust validation of NPY5R as a therapeutic target requires a multi-faceted approach:

• Genetic validation:

  • CRISPR/Cas9-mediated knockout studies in relevant cell lines and animal models

  • Conditional knockout models to assess tissue-specific effects

  • Rescue experiments to confirm specificity of observed phenotypes

• Pharmacological validation:

  • Development of highly selective NPY5R modulators with demonstrated target engagement

  • Dose-dependent correlation between target engagement and phenotypic effects

  • Comparison of multiple structurally distinct modulators to rule out off-target effects

• Clinical correlation:

  • Analysis of NPY5R expression and activation in patient samples

  • Correlation of NPY5R status with disease progression and treatment response

  • Biomarker development to identify patient populations likely to respond to NPY5R modulation

• Combination approaches:

  • Assessment of synergy between NPY5R modulation and standard therapies

  • Evaluation of combinations targeting multiple NPY receptor subtypes

  • Investigation of rational combinations addressing potential resistance mechanisms

• Pathway analysis:

  • Confirmation of pathway modulation in response to NPY5R targeting

  • Identification of potential compensatory mechanisms

  • Development of companion biomarkers for pathway activation/inhibition

The failure of Y5R antagonists in obesity treatment despite strong preclinical rationale highlights the importance of thorough target validation across multiple platforms and models before advancing to clinical studies.

How might emerging single-cell technologies advance our understanding of NPY5R biology?

Single-cell technologies offer unprecedented opportunities to understand NPY5R biology at a more granular level:

• Single-cell RNA sequencing can reveal cellular heterogeneity in NPY5R expression across tissues and within tumors, potentially identifying specific cell populations where NPY5R plays critical roles.

• Single-cell proteomics can map NPY5R protein levels and post-translational modifications, providing insights into receptor regulation beyond transcriptional control.

• Spatial transcriptomics can preserve information about the spatial distribution of NPY5R-expressing cells within tissues, revealing important microenvironmental influences on receptor function.

• CyTOF (mass cytometry) coupled with NPY5R-specific antibodies can simultaneously analyze multiple parameters in NPY5R-expressing cells, revealing correlations between receptor expression and cellular states.

• Single-cell ATAC-seq can identify open chromatin regions regulating NPY5R expression, providing insights into its epigenetic control in different cell types.

These technologies could help resolve contradictions in current NPY5R research by revealing cell type-specific functions and regulatory mechanisms that are obscured in bulk analyses.

What are the implications of NPY5R genetic variants for personalized medicine approaches?

While the search results don't provide specific information about NPY5R genetic variants, we can discuss the potential implications based on our understanding of receptor biology:

• Pharmacogenomic considerations: Genetic variants in NPY5R may affect ligand binding, G-protein coupling, or receptor expression, potentially influencing individual responses to drugs targeting this receptor. Identifying such variants could help stratify patients for clinical trials and personalized treatment approaches.

• Disease susceptibility: Variants affecting NPY5R function might contribute to individual differences in susceptibility to conditions where the receptor plays a role, such as obesity, eating disorders, or certain cancers. Genetic screening could help identify at-risk individuals.

• Biomarker development: Specific NPY5R variants could serve as biomarkers for disease prognosis or treatment response, particularly in cancers where NPY5R has been implicated as a tumor suppressor .

• Drug development: Understanding the functional consequences of common NPY5R variants could inform drug development strategies, potentially leading to variant-specific therapeutic approaches.

• Systems biology approaches: Integrating NPY5R variant data with other genetic and environmental factors could provide a more comprehensive picture of disease mechanisms and treatment responses.

As genomic sequencing becomes more routine in clinical settings, the relevance of NPY5R variants in personalized medicine approaches will likely become clearer.

What are the most reliable protein expression systems for recombinant NPY5R production?

While the search results don't directly address recombinant NPY5R production systems, we can infer appropriate approaches based on general practices for G-protein coupled receptors:

• Mammalian expression systems:

  • HEK293 cells are widely used for GPCR expression due to proper folding and post-translational modifications

  • CHO cells offer stable expression and are suitable for large-scale production

  • These systems are particularly appropriate for functional studies requiring native-like receptor conformation

• Insect cell expression:

  • Baculovirus-infected Sf9 or Hi5 insect cells can produce higher yields of membrane proteins

  • This system has been successfully used for structural studies of other NPY receptors

  • Suitable for purification and structural biology applications

• Yeast expression systems:

  • Pichia pastoris and Saccharomyces cerevisiae can express functional GPCRs

  • Advantages include cost-effectiveness and ease of scale-up

  • May require optimization for proper folding and trafficking

• Cell-free expression systems:

  • Emerging option for difficult-to-express membrane proteins

  • Allows rapid screening of constructs and conditions

  • Direct incorporation into nanodiscs or liposomes possible

Each system has advantages and limitations, and the choice depends on the specific research goals, whether for functional assays, binding studies, or structural analyses of NPY5R.