Recombinant Human G protein-regulated inducer of neurite outgrowth 1 (GPRIN1), partial

What is GPRIN1 and what are its primary functions in the nervous system?

GPRIN1 (G protein-regulated inducer of neurite outgrowth 1) functions as a downstream effector for Gα o/i/z proteins in the adult and developing brain. It plays a critical role in promoting neurite outgrowth and is highly localized to the plasma membrane and synapses where it regulates neuronal signal transduction and Ca2+ homeostasis . GPRIN1 is involved in agonist-stimulated cytoskeletal reorganization, which is crucial for both early neuronal network development and in functionally mature neurons . The protein is strongly expressed in adult mouse brain, with particular enrichment in the hippocampus, hypothalamus, habenula, and throughout the cerebral cortex .

How is GPRIN1 expression regulated in neuronal tissues?

GPRIN1 expression is partially regulated by promoter recruitment of methyl-CpG binding protein 2 (MeCP2), a transcriptional regulator that is mutated in various neurobehavioral abnormalities including Rett syndrome . This transcriptional regulation mechanism connects GPRIN1 to broader neurological development pathways. Additionally, GPRIN1 is phosphorylated by Cyclin-dependent kinase 5 (Cdk5), an essential protein known to regulate cytoskeleton remodeling, axonal guidance, and neuronal plasticity in the brain . This post-translational modification likely contributes to GPRIN1's functional capabilities in neuronal development and plasticity.

What phenotypes are observed in GPRIN1 knockout models?

GPRIN1 knockout mice are viable, allowing for detailed assessment of GPRIN1's physiological roles. Loss of GPRIN1 leads to learning deficits in vivo, suggesting its importance in cognitive function . Furthermore, GPRIN1-deficient neurons show increased sensitivity to stress conditions, which may explain the learning impairments observed in knockout models . The GPRIN1 knockout model demonstrates that this protein plays a modulatory role in brain health and disease, affecting both developmental processes and adult neuronal function.

What are recommended approaches for generating GPRIN1 knockout models?

A viable whole-body GPRIN1 knockout mouse model has been successfully generated to study its physiological role in synaptic function both ex vivo and in vivo . The methodology involves:

• Gene targeting through homologous recombination to delete the entire coding region of GPRIN1

• Confirmation of knockout through genomic PCR, RT-PCR, and Western blot analyses

• Validation of model through phenotypic characterization

For conditional knockouts, Cre-loxP technology can be employed to achieve tissue-specific or temporally-controlled GPRIN1 deletion, which is particularly useful for distinguishing developmental versus adult roles of GPRIN1.

What techniques are most effective for studying GPRIN1's role in neuronal signal transduction?

To investigate GPRIN1's role in neuronal signal transduction, researchers should consider:

• Calcium imaging: For monitoring changes in intracellular Ca2+ levels in response to various stimuli in both wild-type and GPRIN1-deficient neurons

• Electrophysiology: Patch-clamp recordings to assess the impact of GPRIN1 on spontaneous and evoked neuronal electrical activity

• Neurite outgrowth assays: Quantitative analysis of neurite extension, branching, and complexity in neuronal cultures

• Synaptic protein analysis: Immunocytochemistry and biochemical fractionation to examine synaptic protein composition and distribution

• Live-cell imaging: With fluorescently tagged proteins to track cytoskeletal dynamics in response to stimulation

These approaches have revealed that GPRIN1 affects calcium signaling after agonist-induced physiological response and contributes to spontaneous neuronal electrical activity .

How should recombinant GPRIN1 be produced and validated for functional studies?

For production of recombinant human GPRIN1:

• Expression system selection:

  • Mammalian expression systems (HEK293, CHO) are preferable for maintaining proper post-translational modifications

  • For partial GPRIN1 fragments, bacterial systems may be suitable

• Purification strategy:

  • Affinity tags (His, GST, FLAG) facilitate purification while minimizing impact on protein function

  • Size exclusion chromatography as a final polishing step ensures homogeneity

• Validation methods:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry to verify sequence integrity

  • Circular dichroism to assess proper folding

  • Functional assays to confirm biological activity:

    • G-protein binding assays

    • Neurite outgrowth induction in neuronal cultures

How does GPRIN1 contribute to learning and memory processes?

GPRIN1 plays a significant role in learning and memory as evidenced by the learning deficits observed in GPRIN1-/- mice . The mechanisms include:

• Regulation of synaptic signal transduction: GPRIN1 modulates neuronal signaling pathways critical for synaptic plasticity, the cellular basis of learning and memory

• Calcium homeostasis: GPRIN1 regulates Ca2+ dynamics, which are essential for synaptic plasticity mechanisms including long-term potentiation (LTP) and depression (LTD)

• Cytoskeletal reorganization: GPRIN1 influences agonist-stimulated cytoskeletal changes, which are necessary for structural modifications at synapses during memory formation

• Stress response modulation: GPRIN1 appears to protect neurons from stress, and its absence sensitizes neurons to stress conditions that can impair learning

These functions position GPRIN1 as an important modulator of cognitive processes with potential implications for learning disorders and cognitive decline.

How might GPRIN1 dysfunction contribute to neurodegenerative or neurodevelopmental disorders?

GPRIN1 dysfunction may contribute to various neurological disorders through several mechanisms:

• Impaired neurite outgrowth: Defects in GPRIN1 function could disrupt proper neuronal connectivity during development, potentially contributing to neurodevelopmental disorders

• Altered calcium signaling: Dysregulation of Ca2+ homeostasis is implicated in multiple neurodegenerative processes, and GPRIN1's role in calcium regulation suggests its potential involvement

• Increased neuronal vulnerability: GPRIN1 loss sensitizes neurons to stress , potentially accelerating neurodegeneration under pathological conditions

• Cytoskeletal abnormalities: As GPRIN1 regulates cytoskeletal reorganization, its dysfunction might lead to structural abnormalities associated with neurodegeneration

Given its association with MeCP2-regulated transcription , GPRIN1 dysfunction might also play a role in Rett syndrome and related neurodevelopmental disorders characterized by learning difficulties and autism-like behaviors.

What evidence supports GPRIN1's role in cancer progression and prognosis?

Recent pan-cancer analyses have identified GPRIN1 as a potential novel tumor regulator . The evidence includes:

• Expression analysis: GPRIN1 is upregulated in kidney renal papillary cell carcinoma (KIRP) and lung adenocarcinoma (LUAD)

• Prognostic correlation: Elevated GPRIN1 expression correlates with poor prognosis in these cancer types

• Regulatory network: GPRIN1 functions within a lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network that influences cancer progression

• Immune modulation: GPRIN1 expression significantly correlates with tumor immune cell infiltration, immune cell biomarkers, and immune checkpoints

This evidence suggests that GPRIN1 may influence cancer progression through complex regulatory networks and immune system interactions, making it a potential therapeutic target.

What is the proposed ceRNA regulatory network involving GPRIN1 in cancer?

The competitive endogenous RNA (ceRNA) regulatory network involving GPRIN1 in cancer, particularly in KIRP and LUAD, consists of:

This network appears to operate through the following mechanism:

• The identified lncRNAs competitively bind miR-140-3p

• This binding reduces miR-140-3p's availability to target GPRIN1 mRNA

• Consequently, GPRIN1 expression increases

• Elevated GPRIN1 then affects tumor progression and immune infiltration

This miR-140-3p-GPRIN1 axis and its upstream lncRNAs are closely related to KIRP and LUAD progression and might affect both prognosis and therapeutic outcomes in these cancer types .

How does GPRIN1 expression relate to tumor immune microenvironment?

GPRIN1 expression significantly correlates with tumor immune cell infiltration and immune checkpoint expression, suggesting its potential role in modulating the tumor immune microenvironment . Specific relationships include:

• Immune cell infiltration: GPRIN1 expression correlates with the proportion of various immune cell types in tumors as calculated using the CIBERSORT algorithm

• Immune cell biomarkers: Significant correlation between GPRIN1 expression and markers of specific immune cell populations

• Immune checkpoints: GPRIN1 expression correlates with immune checkpoint molecules including CD274 (PD-L1), CTLA4, and PDCD1 (PD-1)

These correlations suggest that GPRIN1 might influence the efficacy of immunotherapies, particularly immune checkpoint inhibitors. The relationship between GPRIN1 and the tumor immune microenvironment provides a rationale for investigating GPRIN1 as a predictive biomarker for immunotherapy response and as a potential therapeutic target to enhance immunotherapy efficacy.

What high-throughput approaches are recommended for comprehensive GPRIN1 functional analysis?

For comprehensive functional analysis of GPRIN1, consider the following high-throughput approaches:

• Transcriptomics:

  • RNA-seq to identify genes differentially expressed in GPRIN1-deficient vs. wild-type neurons

  • Single-cell RNA-seq to resolve cell type-specific responses to GPRIN1 manipulation

• Proteomics:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS) to identify GPRIN1 interacting partners

  • Phosphoproteomics to characterize GPRIN1-dependent signaling pathways and phosphorylation targets of Cdk5

• Functional genomics:

  • CRISPR/Cas9 screens to identify genes that modify GPRIN1-dependent phenotypes

  • CRISPRi/CRISPRa approaches for tunable modulation of GPRIN1 expression

• Imaging:

  • High-content screening for neurite outgrowth and morphological parameters

  • Automated calcium imaging to assess neuronal activity in GPRIN1-manipulated networks

• Bioinformatics:

  • Network analysis to integrate multi-omics data

  • Machine learning approaches to identify patterns in GPRIN1-dependent gene expression or cellular phenotypes

How can researchers effectively analyze the relationship between GPRIN1 and cytoskeletal reorganization?

To effectively analyze GPRIN1's role in cytoskeletal reorganization:

• Live imaging approaches:

  • Fluorescently tagged cytoskeletal proteins (actin, tubulin) in neurons with manipulated GPRIN1 expression

  • Super-resolution microscopy to visualize fine cytoskeletal structures at growth cones and synapses

  • FRAP (Fluorescence Recovery After Photobleaching) to measure cytoskeletal dynamics

• Biochemical analyses:

  • F-actin/G-actin ratio determination in GPRIN1-manipulated neurons

  • RhoGTPase activity assays, as these are key regulators of cytoskeletal dynamics

  • Assessment of cytoskeletal post-translational modifications (tubulin acetylation, actin phosphorylation)

• Functional assays:

  • Quantitative analysis of growth cone morphology and dynamics

  • Measurement of neurite retraction/extension rates in response to stimuli

  • Analysis of synapse formation and stability in long-term cultures

• Interactome mapping:

  • BioID or APEX proximity labeling to identify cytoskeletal proteins in GPRIN1's proximity

  • Yeast two-hybrid or mammalian two-hybrid screens focused on cytoskeletal components

Given that GPRIN1 is involved in agonist-stimulated cytoskeletal reorganization crucial for neuronal network development , these approaches would provide valuable insights into its molecular mechanisms.

What computational methods should be used to analyze GPRIN1-related gene networks in neurological and cancer contexts?

To analyze GPRIN1-related gene networks across different contexts, researchers should employ:

• Differential co-expression network analysis:

  • Identification of gene modules co-regulated with GPRIN1 in neurological versus cancer contexts

  • Detection of context-specific network hubs and regulators

• Pathway enrichment and ontology analysis:

  • Gene Set Enrichment Analysis (GSEA) to identify biological processes associated with GPRIN1 expression

  • Comparison of enriched pathways between neurological and oncological datasets

• Integration of multi-omics data:

  • Weighted gene co-expression network analysis (WGCNA) combining transcriptomic, proteomic, and epigenomic data

  • Multi-layer network visualization to represent different types of molecular interactions

• Cancer-specific computational approaches:

  • Survival analysis using Cox proportional hazards models to evaluate GPRIN1's prognostic value

  • CIBERSORT algorithm application to assess the relationship between GPRIN1 expression and immune cell infiltration

  • ceRNA network prediction to identify miRNAs and lncRNAs interacting with GPRIN1 mRNA

• Deep learning approaches:

  • Neural network models to predict GPRIN1 function from sequence or expression data

  • Feature importance analysis to identify key determinants of GPRIN1-dependent phenotypes

These computational methods would facilitate the identification of context-specific functions and regulatory mechanisms of GPRIN1, providing insights into its dual roles in neurological development and cancer progression.