GIPC1 Human

What is GIPC1 and what is its molecular structure?

GIPC1 is a 333 amino acid protein (approximately 36 kDa) encoded by the GIPC1 gene in humans. The protein contains a central PDZ domain, which serves as a compact protein module mediating specific protein-protein interactions. GIPC1 was originally identified as a binding partner for the C terminus of RGS-GAIP (hence the name: GAIP Interacting Protein C-terminus), a protein involved in the regulation of G protein signaling .

The protein has been independently discovered by several research groups and thus has acquired various alternate names including synectin, C19orf3, and RGS19IP1. Within the GIPC family, GIPC1 shares approximately 60% sequence identity with its two other family members, GIPC2 and GIPC3 .

For structural analysis of GIPC1, techniques such as X-ray crystallography and NMR spectroscopy have been employed to characterize the PDZ domain and its binding interface with partner proteins. When designing experiments to study GIPC1 structure, researchers should consider using recombinant protein expression systems with affinity tags for protein purification, followed by structural biology approaches.

What are the primary interaction partners of GIPC1?

GIPC1 has been demonstrated to interact with a diverse array of receptor and cytoskeletal proteins. Key interaction partners include:

• RGS-GAIP - The original identified binding partner involved in G protein signaling

• GLUT1 receptor - Glucose transporter

• ACTN1 - Alpha-actinin-1, a cytoskeletal protein

• KIF1B - Kinesin family member 1B

• MYO6 - Myosin VI

• PLEKHG5 - Pleckstrin homology domain-containing family G member 5

• SDC4/syndecan-4 - A transmembrane proteoglycan

• SEMA4C/semaphorin-4 - Involved in axon guidance

• HTLV-I Tax - Human T-cell leukemia virus type I Tax protein

• MACC1 - Metastasis-associated in colon cancer protein 1

Methodologically, researchers investigating GIPC1 interactions should consider using techniques such as co-immunoprecipitation, yeast two-hybrid screening, mass spectrometry-based proteomics, and peptide array analysis. For instance, GIPC1's interaction with MACC1 has been confirmed through multiple complementary approaches including mass spectrometry, yeast two-hybrid assay, co-immunoprecipitation, and peptide array analysis .

How is GIPC1 expression regulated in normal tissues and disease states?

Research methodologies for studying GIPC1 expression include quantitative RT-PCR, Western blotting, and immunohistochemistry. In colorectal cancer (CRC) cell lines, a significant positive correlation (Pearson r=0.9188, P=0.0013) has been observed between GIPC1 and MACC1 expression levels . This correlation suggests potential co-regulation mechanisms that warrant further investigation.

When analyzing GIPC1 expression, it is advisable to use multiple housekeeping genes for normalization (e.g., G6PDH, GAPDH) and to validate findings across different experimental platforms. For clinical specimens, laser capture microdissection of tumor cells followed by qRT-PCR represents a methodologically robust approach, as was demonstrated in studies examining GIPC1 expression in primary CRC specimens .

What experimental approaches can reveal GIPC1's dual role as a protein binding partner and transcription factor?

GIPC1 exhibits a fascinating dual functionality: it acts both as a protein scaffold through its PDZ domain interactions and as a transcription factor binding directly to gene promoters. To investigate this dual role, researchers should implement a complementary experimental approach:

For protein-protein interactions:

• Yeast two-hybrid screening for novel interactions

• Co-immunoprecipitation followed by Western blotting for validation

• Mass spectrometry for unbiased identification of binding partners

• Pepspot analysis to map precise binding domains (as demonstrated for the GIPC1-MACC1 interaction, where amino acids 241-247 of GIPC1 were identified as binding to MACC1)

For transcription factor activity:

• Chromatin immunoprecipitation (ChIP) to identify direct DNA binding sites

• Electrophoretic mobility shift assay (EMSA) to confirm physical interactions with promoter regions

• Luciferase reporter assays with promoter constructs to quantify transcriptional effects

• Subcellular fractionation to confirm nuclear localization

Research has demonstrated GIPC1's direct binding to the MACC1 promoter through both ChIP and EMSA techniques. Specifically, GIPC1 binds to the region from the transcription start site to -60 bp in the MACC1 promoter . When performing ChIP experiments, careful selection of antibodies, appropriate controls (IgG and input DNA), and verification through multiple primer sets are essential methodological considerations.

How does GIPC1 contribute to cancer metastasis and what are the mechanisms involved?

GIPC1 promotes cancer metastasis through multiple mechanisms:

• Transcriptional regulation of metastasis-promoting genes: GIPC1 directly binds to the MACC1 promoter and drives its expression. MACC1 is a well-established prognostic indicator for metastasis formation .

• Protein-protein interactions: GIPC1 physically interacts with MACC1 protein, potentially enhancing its stability or function.

• Modulation of cell motility: Knockdown of GIPC1 reduces MACC1-induced cell migration and invasion in colorectal cancer cells .

Experimental approaches to study GIPC1's role in metastasis should include:

• RNAi-mediated knockdown using both transient siRNA and stable shRNA approaches

• In vitro cell migration and invasion assays (wound healing, transwell)

• In vivo metastasis models using xenograft techniques

• Correlation studies in patient samples comparing GIPC1 expression with metastasis outcomes

A particularly robust methodology involves intrasplenic transplantation of colorectal cancer cells in mice to assess both primary tumor growth and liver metastasis formation. Using this approach, researchers have demonstrated that GIPC1 knockdown reduces MACC1-induced tumor growth and metastasis .

What is the prognostic value of GIPC1 in cancer and how should expression data be analyzed?

GIPC1 has demonstrated significant prognostic value in colorectal cancer. Methodologically, researchers analyzing GIPC1 as a prognostic biomarker should consider:

• Patient stratification: Use receiver operating characteristic (ROC)-calculated cut-offs to classify patients into high and low expressors.

• Survival analysis: Apply Kaplan-Meier survival analysis with appropriate statistical testing (log-rank test).

• Multivariate analysis: Control for confounding variables using Cox proportional hazards modeling.

• Combination with other biomarkers: Assess whether combining GIPC1 with other markers (e.g., MACC1) improves prognostic accuracy.

Research has shown that patients with high GIPC1 expression in their primary tumors have significantly shorter metastasis-free survival (MFS) times compared to those with low expression (P=0.034). Specifically, GIPC1 high expressors had a median MFS of 62.78 months (SD 51.27), whereas GIPC1 low expressors showed a median MFS of 110.27 months (SD 46.14), representing a difference of approximately 47.5 months .

The combination of MACC1 and GIPC1 expression improves patient survival prognosis, suggesting the value of multi-marker panels for clinical applications .

How does GIPC1 function in angiogenesis regulation?

GIPC1 plays a significant role in vascular development and angiogenesis through its interaction with Plexind1 signaling. Research methodologies for studying GIPC1 in angiogenesis include:

• Zebrafish models: Fish expressing Plexind1 receptors with impaired GIPC binding exhibit angiogenesis deficits and hypersensitivity to antiangiogenic drugs .

• Genetic knockout approaches: GIPC mutant fish show angiogenic impairments that can be ameliorated by reducing Plexind1 signaling .

• In vitro endothelial cell models: GIPC depletion potentiates SEMA-PLXND1 signaling in cultured endothelial cells .

The experimental evidence indicates that GIPC proteins negatively modulate Plexind1 signaling during angiogenesis. This finding is particularly relevant for understanding the molecular mechanisms that regulate blood vessel formation in development and disease contexts .

When investigating GIPC1's role in angiogenesis, researchers should consider using both genetic approaches (knockdown/knockout) and pharmacological interventions (antiangiogenic drugs), ideally in combination with in vivo imaging techniques to visualize vascular development in real-time.

What are the optimal experimental systems for studying GIPC1 function?

Based on the research literature, the following experimental systems have proven valuable for investigating GIPC1:

Cell line models:

• Colorectal cancer cell lines (SW620, SW480, HT29, WIDR, HCT116, HCT15, HCA7, DLD1, Caco-2, SW48) have been used successfully to study GIPC1's role in cancer biology .

• Endothelial cells are appropriate for investigating GIPC1's function in angiogenesis .

Animal models:

• Zebrafish models provide an excellent system for studying GIPC1's role in vascular development .

• Mouse xenograft models, particularly intrasplenic injection followed by liver metastasis assessment, are valuable for cancer-related studies .

Genetic manipulation approaches:

• RNA interference (siRNA for transient knockdown, shRNA for stable knockdown)

• CRISPR-Cas9 gene editing for knockout or knock-in studies

• Overexpression systems using appropriate promoters (e.g., CMV for high expression)

When selecting an experimental system, researchers should consider the specific aspect of GIPC1 biology they aim to investigate and choose models that best recapitulate the relevant physiological or pathological context.

How can researchers effectively target GIPC1 for therapeutic development?

Several methodological approaches show promise for GIPC1-targeted therapeutic development:

• RNAi-based approaches: Gene-specific silencing using siRNA or shRNA has demonstrated efficacy in reducing GIPC1 expression and its downstream effects on tumor growth and metastasis .

• Short peptide inhibitors: Peptides designed to interfere with specific GIPC1 protein-protein interactions represent a potential therapeutic strategy .

• Small molecule discovery: Screening platforms can be employed to identify compounds that disrupt GIPC1's interaction with binding partners or inhibit its transcription factor activity.

• Combination approaches: Targeting both GIPC1 and its effector proteins (e.g., MACC1) might provide synergistic anti-tumoral and anti-metastatic effects .

For validation of GIPC1-targeting therapies, comprehensive evaluation should include:

• In vitro assessment of target engagement and functional outcomes

• In vivo efficacy studies in appropriate animal models

• Biomarker analysis to identify patient populations most likely to benefit

• Mechanistic studies to understand the molecular basis of therapeutic effects

What are the unresolved questions regarding GIPC1 biology and function?

Despite significant advances in understanding GIPC1, several important questions remain:

• How is GIPC1's dual role as protein interactor and transcription factor regulated? What determines whether GIPC1 functions in the cytoplasm or nucleus?

• What is the complete set of genes transcriptionally regulated by GIPC1 beyond MACC1?

• How do post-translational modifications affect GIPC1 function and localization?

• What is the evolutionary conservation of GIPC1 function across species?

• How does GIPC1 interact with other signaling pathways beyond those already identified?

Addressing these questions will require integrated approaches combining genomics, proteomics, structural biology, and functional studies in relevant model systems.

How can technological advances enhance GIPC1 research?

Emerging technologies that may advance GIPC1 research include:

• Single-cell sequencing to understand cell-type specific functions of GIPC1

• Spatial transcriptomics to map GIPC1 expression in tissue microenvironments

• CRISPR-based screens to identify synthetic lethal interactions with GIPC1

• Cryo-electron microscopy to resolve GIPC1 protein complexes at high resolution

• Genome-wide CRISPR activation/inhibition to map GIPC1 regulatory networks

• Proteomics approaches to comprehensively identify GIPC1 interactors under various conditions

These technologies, when applied to GIPC1 research, have the potential to reveal new insights into its function and identify novel therapeutic opportunities.