GPC4 Human

What is the basic molecular structure of human GPC4?

Human Glypican-4 is a GPI-anchored heparan sulfate proteoglycan with an approximately 220 kDa total molecular weight, containing a 60 kDa protein core. The mature protein is encoded by the GPC4 gene (also known as K-glypican) and shares high sequence homology with mouse (97%) and rat (96%) orthologs . The full protein consists of the sequence from Ala23 to Ser529 (Accession # O75487), with alternative splice isoforms identified that may lack the N-terminal 70 amino acids including the signal peptide .

What are the primary expression patterns of GPC4 in human tissues?

GPC4 expression has been documented across multiple tissue types with notable presence in:

• Brain tissue, particularly in lateral ventricles surrounding the telencephalon, dentate gyrus, proliferating neuroepithelial cells, and neural precursors during development

• Kidney tissue, where expression levels correlate with renal function

• Adipose tissue, with differential expression between fat depots

• Adrenal gland tissue
  Expression patterns vary significantly during development and under various pathological conditions, making it a dynamically regulated protein across different physiological contexts.

What are the established functional roles of GPC4 in normal human physiology?

GPC4 serves multiple physiological functions through tissue-specific mechanisms:

• Neural development: Inhibits dopaminergic differentiation of neurons and contributes to excitatory synapse development through a 30 kDa cleaved form that binds in cis to PTP sigma

• Metabolic regulation: Functions as an insulin-sensitizing adipokine, with soluble forms released by adipocytes that enhance insulin receptor signaling and support adipocyte differentiation

• Growth factor interaction: Binds to basic fibroblast growth factor (bFGF), potentially modulating its signaling pathways

• Kidney function: Serum levels correlate with glomerular filtration rate and kidney health indicators

How does post-translational processing affect GPC4 functionality?

GPC4 undergoes several critical post-translational modifications that significantly impact its biological activities:

• Proteolytic processing: A 30 kDa cleaved form of GPC4 has distinct functions in neural systems, specifically binding to PTP sigma and contributing to excitatory synapse development and function . This suggests that proteolytic processing creates functionally distinct forms of the protein.

• Heparan sulfate attachment: As a proteoglycan, the pattern and degree of heparan sulfate modification likely influences GPC4's interaction with growth factors and receptors, though the exact regulatory mechanisms remain under investigation.

• GPI-anchor processing: The membrane-bound form can be cleaved to release soluble GPC4, which has distinct biological activities, particularly in metabolic contexts where it circulates and influences insulin signaling .
  Researchers investigating GPC4 function should consider which form of the protein (full-length, cleaved, soluble, or membrane-bound) is relevant to their specific biological question.

What explains the apparently contradictory effects of GPC4 in different cancer types?

Recent survival analysis using TCGA data reveals that GPC4 expression has divergent effects across various cancer types . This dichotomy may be explained by:

• Tissue context dependency: GPC4 interacts with different signaling pathways depending on the cellular environment and available binding partners.

• Isoform expression patterns: Different cancer types may express distinct GPC4 isoforms or post-translationally modified variants.

• Dual functionality in proliferation: Functional genomics assays in glioblastoma versus non-small cell lung cancer models show opposite effects on proliferation, suggesting cancer type-dependent activities .

• Interaction with cancer-specific pathways: GPC4 may enhance or suppress different oncogenic pathways depending on which are dominant in a specific cancer type.
  This divergent behavior highlights the importance of cancer-specific analysis when targeting GPC4 in therapeutic approaches.

How do soluble versus membrane-bound forms of GPC4 differ in their biological activities?

The distinct biological activities of different GPC4 forms include:

What are the optimal methods for detecting different forms of GPC4 in biological samples?

Detection of GPC4 requires careful consideration of protein forms and expression levels:

• Western blotting:

  • For full-length GPC4: Use antibodies targeting conserved domains; run samples on 6-10% gels to accommodate the ~220 kDa glycosylated form

  • For cleaved forms: Use antibodies specific to N or C-terminal regions; run samples on higher percentage gels (12-15%)

  • Include deglycosylation controls to distinguish core protein from glycosylated forms

• ELISA-based detection:

  • Commercial recombinant GPC4 standards are available for calibration curves

  • Consider carrier-free preparations when BSA might interfere with assay performance

  • For serum measurements, validated clinical assays have established reference ranges for conditions like kidney disease

• Imaging techniques:

  • Infrared spectral imaging has been successfully applied to study GPC4 in tissue samples

  • Immunofluorescence should include permeabilization optimization to detect both membrane and internal pools

What expression systems are most appropriate for recombinant GPC4 production?

When producing recombinant GPC4 for research, several systems offer distinct advantages:

• Mammalian expression systems:

  • Preferred for maintaining native glycosylation patterns

  • Human embryonic kidney (HEK) cells using vectors like pCMV6-AC-GFP have been validated

  • Selection typically uses neomycin/G418 resistance

  • Consider carrier-free formulations for applications where BSA might interfere

• Fusion protein approaches:

  • Fc chimera proteins (human IgG1 Pro100-Lys330) maintain solubility and facilitate purification

  • C-terminal tags like GFP allow tracking of expression and localization

• Reconstitution considerations:

  • Lyophilized preparations typically reconstitute in PBS at 200 μg/mL

  • Avoid repeated freeze-thaw cycles to maintain activity

  • For long-term storage, aliquot and maintain at -20°C to -80°C

What are the key considerations for designing GPC4 knockdown or overexpression experiments?

When manipulating GPC4 expression levels experimentally:

• Knockdown strategies:

  • siRNA approaches should target conserved exons to affect all splice variants

  • CRISPR-Cas9 targeting requires careful guide RNA design to avoid off-target effects

  • Validation should include both mRNA and protein level assessments due to GPC4's post-translational regulation

• Overexpression approaches:

  • Commercial expression plasmids with verified sequences are available (e.g., RG202022)

  • Consider using inducible systems to control expression timing and magnitude

  • Verify proper processing and localization of the overexpressed protein, as artificial systems may alter GPC4 processing

• Physiologically relevant models:

  • Cell type specificity is critical given GPC4's divergent effects in different tissues

  • For cancer studies, compare effects across multiple cancer types due to documented opposite effects

  • Include appropriate controls for both membrane-bound and soluble forms

How reliable is serum GPC4 as a biomarker for kidney function?

Serum GPC4 shows significant promise as a kidney function biomarker:

What is the current understanding of GPC4's role in cancer progression?

GPC4's role in cancer demonstrates remarkable context dependency:

• Cancer type specificity:

  • Functional genomics assays reveal divergent effects on proliferation between glioblastoma and non-small cell lung cancer

  • TCGA survival analysis shows variable prognostic significance across cancer types

• Proposed mechanisms:

  • Modulation of growth factor signaling, particularly through FGF pathway interactions

  • Potential involvement in Wnt signaling, though this needs further characterization

  • Possible immunomodulatory functions in the tumor microenvironment

• Therapeutic implications:

  • Potential as a targeted therapy, but requires cancer-type specific approaches

  • Possible biomarker for stratifying patients for certain treatment modalities

  • May serve as a companion diagnostic to predict treatment responses
    Researchers should approach GPC4-targeted cancer therapies with awareness of its potentially opposing functions in different cancer contexts.

How does GPC4 contribute to metabolic regulation and insulin resistance?

GPC4's metabolic functions center on adipose tissue biology and insulin signaling:

• Adipokine functions:

  • Acts as an insulin-sensitizing adipokine released by adipocytes

  • Shows differential expression patterns between various adipose tissue depots

  • Circulating levels are elevated in obese patients with insulin resistance

• Insulin signaling effects:

  • Soluble GPC4 binds to and enhances signaling through the insulin receptor

  • Supports adipocyte differentiation, potentially influencing fat distribution patterns

  • May represent a compensatory mechanism in insulin resistance states

• Therapeutic potential:

  • Possible target for metabolic disorders including type 2 diabetes

  • Potential biomarker for metabolic health beyond traditional measures

  • May help explain differential metabolic risk associated with specific fat distribution patterns These multifaceted roles place GPC4 at the intersection of metabolism, obesity research, and precision medicine approaches to metabolic disorders.