GRO g Human
Description
Introduction and Nomenclature
Growth Regulated Oncogene gamma (GRO gamma) is a member of the CXC chemokine family and is officially designated as Chemokine (C-X-C motif) ligand 3 (CXCL3). This cytokine is also known by several alternative names including GRO3 oncogene (GRO3), GRO protein gamma (GROg), and macrophage inflammatory protein-2-beta (MIP2b) . CXCL3 belongs to a family of three closely related human GRO genes, with GRO alpha (CXCL1) and GRO beta (CXCL2) sharing significant sequence homology .
The identification of these three related human GRO genes revealed their inflammatory and growth-regulatory properties. Initial studies demonstrated that GRO beta and GRO gamma share 90% and 86% identity at the deduced amino acid level with the original GRO alpha isolate . These proteins play crucial roles in various physiological and pathological processes, including inflammation, immune response, and cancer progression.
Protein Structure and Properties
GRO gamma/CXCL3 is a single, non-glycosylated polypeptide chain containing 73 amino acids with a molecular mass of approximately 7902 Dalton . The protein is typically produced as a recombinant in Escherichia coli expression systems for research and analytical purposes . Structurally, the human CXCL3 protein spans residues Ala35 to Asn107 (Accession # P19876), often with an N-terminal His-Tag for purification purposes .
Key physical and chemical properties of GRO gamma/CXCL3 include:
| Property | Characteristic |
|---|---|
| Molecular Weight | 7902 Dalton |
| Amino Acid Length | 73 amino acids |
| Isoelectric Point | 9.6 |
| Subcellular Location | Secreted |
| Protein Purity | >95% (in recombinant form) |
The significant differences in the 3' untranslated region of the GRO genes, including different numbers of ATTTA repeats associated with mRNA instability, contribute to their differential regulation and expression patterns . Additionally, one amino acid substitution of proline in GRO alpha by leucine in GRO beta and GRO gamma leads to a large predicted change in protein conformation, potentially affecting their biological activities .
Genetic Information
The gene encoding CXCL3 is located on chromosome 4 at position 4q21 in a cluster with other CXC chemokines . DNA hybridization with oligonucleotide probes and partial sequence analysis of genomic clones have confirmed that the three GRO forms are derived from related but different genes . Interestingly, the GRO alpha cDNA clone hybridizes to all three genes, indicating their close evolutionary relationship .
The CXCL3 gene contains a 122-base-pair conserved region in the 3' untranslated region that is shared among all three GRO genes, suggesting an important regulatory role . Part of this region is also conserved in the Chinese hamster genome, further supporting its functional significance .
Receptor Interactions and Signaling
CXCL3 primarily mediates its effects through interaction with a cell surface chemokine receptor called CXCR2 . This interaction triggers various intracellular signaling cascades that regulate cellular responses. In certain contexts, CXCL3 has been shown to induce Erk1/2 and ETS1 phosphorylation, promoting expression of other genes such as CD133 .
Immunological Functions
GRO gamma exhibits significant immunological functions, particularly:
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Chemotactic activity for neutrophils, attracting these immune cells to sites of inflammation
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Control of migration and adhesion of monocytes, key players in the inflammatory response
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Potential role in inflammation through interaction with immune cells and endothelial tissues
In the inflammatory response, CXCL3 may exert its effects on endothelial cells in an autocrine fashion, contributing to the recruitment of immune cells to affected tissues . The processed form GRO-gamma(5-73) shows a fivefold higher chemotactic activity for neutrophilic granulocytes compared to the unprocessed form .
Developmental Functions
Beyond its immunological roles, CXCL3 has been implicated in developmental processes, particularly in the central nervous system. Research has shown that CXCL3 regulates the migration of precursors of cerebellar granule neurons toward the internal layers of the cerebellum during morphogenesis . This finding suggests broader physiological roles for CXCL3 beyond inflammation and immune response.
Tissue-Specific Expression
Expression studies have revealed interesting patterns of CXCL3 distribution across tissues. In human colonic tissues, GRO gamma shows constitutive expression in both normal and neoplastic mucosa, whereas GRO alpha expression is differentially elevated in colon carcinoma specimens compared to normal tissue . Expression of GRO beta appears minimal in colonic tissues, suggesting distinct regulatory mechanisms for each GRO family member .
Regulatory Mechanisms
The expression of GRO genes, including CXCL3, is subject to regulation by various cytokines and inflammatory mediators. Specific inducing agents that regulate CXCL3 expression include:
| Inducing Agent | Effect on CXCL3 Expression |
|---|---|
| Interleukin 1 (IL-1) | Upregulation |
| Tumor Necrosis Factor (TNF) | Upregulation |
| Phorbol 12-myristate 13-acetate | Upregulation |
| Lipopolysaccharide | Upregulation |
In colon carcinoma cell lines such as HT29, both GRO alpha and IL-8 are inducible by IL-1 beta and TNF alpha, highlighting the cytokine-responsive nature of these chemokines .
5.1.1 Colorectal Cancer
Studies examining CXCL3 expression in human colon carcinoma have revealed intriguing associations with cancer progression. Non-metastatic and low metastatic colon carcinoma cells express lower levels of CXCL1 and CXCR2 mRNA and protein compared to highly metastatic colon carcinoma cells . The constitutive expression of CXCL1 and its receptor CXCR2 appears to be associated with metastatic potential and modulates colon cancer cell proliferation and invasive phenotype .
Exogenous addition of recombinant CXCL1 significantly enhances the proliferation and invasiveness of colon carcinoma cells, while neutralizing antibodies to CXCL1 and CXCR2 inhibit cell proliferation in highly metastatic cell lines . These findings suggest that the CXCL3/CXCR2 axis may represent a potential therapeutic target in colorectal cancer.
5.1.2 Hepatocellular Carcinoma (HCC)
Research on hepatocellular carcinoma has shown that CXCL3 contributes to CD133+ cancer stem cell (CSC) maintenance and forms a positive feedback loop with CD133 . In HCC patients, serum CXCL3 levels are higher than in healthy individuals, and higher CXCL3 expression is associated with poor prognosis .
Significantly, high levels of CXCL3 correlate with vascular invasion and tumor capsule formation in HCC . Knockdown of CXCL3 inhibits CD133+ HCC CSCs' self-renewal and tumorigenesis, suggesting its role in cancer stem cell maintenance . Mechanistically, exogenous CXCL3 induces Erk1/2 and ETS1 phosphorylation and promotes CD133 expression, indicating a positive feedback loop between CXCL3 and CD133 gene expression in HCC cells via Erk1/2 activation .
Therapeutic Potential
Given its involvement in cancer progression and inflammatory processes, CXCL3 represents a potential therapeutic target for various conditions:
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In HCC, targeting the CXCL3-CD133 feedback loop might disrupt cancer stem cell maintenance and inhibit tumor growth
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In colorectal cancer, neutralizing CXCL3 or its receptor CXCR2 could potentially reduce cancer cell proliferation and invasion
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In inflammatory conditions, modulating CXCL3 activity might help control neutrophil recruitment and activation
Recent Research Developments
Recent research continues to expand our understanding of CXCL3's functions and potential applications. Ongoing studies are exploring its role in various cancers beyond those already mentioned, as well as its involvement in other inflammatory and immune-related conditions.
The commercially available recombinant Human CXCL3/GRO gamma protein has been shown to chemoattract BaF3 mouse pro B cells transfected with human CXCR2, with an ED50 (effective dose for 50% response) of 0.4-2.4 ng/mL . This finding further supports the chemotactic role of CXCL3 and provides a quantitative measure of its potency.
Product Specs
Q&A
What is GRO gamma and how does it relate to the GRO family of proteins?
GRO gamma (CXCL3) is a small cytokine belonging to the CXC chemokine family. It is one of three related human GRO genes (along with GRO alpha and GRO beta) that share high sequence homology. GRO gamma shares 86% identity at the amino acid level with GRO alpha, while GRO beta shares 90% identity. These proteins have inflammatory and growth-regulatory properties. A critical distinction between these proteins is that GRO gamma contains a leucine substitution in place of the proline found in GRO alpha, which results in significant predicted conformational differences in the protein structure .
What are the alternative nomenclatures for GRO gamma in scientific literature?
GRO gamma is also known by several alternative names in scientific literature including:
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Chemokine (C-X-C motif) ligand 3 (CXCL3)
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GRO3 oncogene (GRO3)
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GRO protein gamma (GROg)
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Macrophage inflammatory protein-2-beta (MIP2b)
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Dendritic Cell Inflammatory Protein 1 (DCIP-1)
What is the genomic location and organization of human GRO gamma?
The human GRO genes, including GRO gamma, have been mapped to chromosomal locus 4q21. This was determined using a GRO alpha cDNA clone that hybridized to all three GRO genes. The three forms are derived from related but different genes, as confirmed by DNA hybridization with oligonucleotide probes and partial sequence analysis of genomic clones .
What structural elements distinguish the untranslated regions of GRO gamma?
The GRO genes exhibit significant differences in their 3' untranslated regions, including different numbers of ATTTA repeats associated with mRNA instability. Interestingly, a 122-base-pair region in the 3' region is conserved among the three GRO genes, and part of this region is also conserved in the Chinese hamster genome, suggesting an important regulatory role. These structural differences may contribute to differential expression patterns and mRNA stability among the GRO protein family members .
What is the active form of recombinant human GRO gamma?
The active form of recombinant human GRO gamma typically comprises amino acids Thr39-Asn107 of the full protein. This E. coli-derived human CXCL3/GRO gamma protein represents the mature, biologically active form of the cytokine. Additional N-terminal processing of mature CXCL3 by the removal of amino acids 35-38 increases its chemotactic activity by several fold, demonstrating the importance of proper processing for optimal biological function .
How is GRO gamma activity quantitatively measured in research settings?
The chemotactic activity of GRO gamma can be quantitatively assessed using cell migration assays. For example, recombinant Human CXCL3/GRO gamma chemoattracts BaF3 mouse pro-B cells transfected with human CXCR2. The ED50 (effective dose for 50% response) for this chemoattractant effect ranges from 0.4-2.4 ng/mL. This standardized assay provides a reliable method for measuring the biological activity of GRO gamma preparations in research settings .
What are the primary biological functions of GRO gamma?
GRO gamma serves several important biological functions:
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Controls migration and adhesion of monocytes
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Acts as a chemoattractant for neutrophils and endothelial cells
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Regulates the migration of precursors of cerebellar granule neurons toward the internal layers of the cerebellum during morphogenesis
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Mediates inflammatory responses
How does GRO gamma mediate its cellular effects?
GRO gamma mediates its effects on target cells primarily by interacting with the cell surface chemokine receptor CXCR2. Upon binding to CXCR2, GRO gamma triggers a cascade of intracellular signaling events that ultimately result in chemotaxis (directed cell migration) and other cellular responses. The specificity of this interaction allows for precise control of cellular movement during both normal physiological processes and pathological conditions .
What factors regulate GRO gamma expression?
Expression studies have revealed that GRO gamma is subject to both tissue-specific regulation and regulation by specific inducing agents. These inducing agents include:
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Interleukin 1 (IL-1)
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Tumor necrosis factor (TNF)
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Phorbol 12-myristate 13-acetate (PMA)
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Lipopolysaccharide (LPS)
This complex regulation allows for context-dependent expression of GRO gamma in response to various inflammatory and immunological stimuli .
What expression systems are optimal for producing recombinant GRO gamma?
E. coli expression systems are commonly used for the production of recombinant human GRO gamma protein. These systems typically express the mature form of the protein (amino acids 39-107), which retains biological activity. When designing expression constructs, researchers should consider that additional N-terminal processing (removal of amino acids 35-38) can significantly enhance the chemotactic activity of the protein. Proper folding and disulfide bond formation are critical for ensuring the biological activity of the recombinant protein .
How can researchers design functional assays to evaluate GRO gamma activity?
Researchers can employ several methodologies to evaluate GRO gamma activity:
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Chemotaxis assays: Using BaF3 cells transfected with human CXCR2 to measure dose-dependent cell migration. The ED50 typically ranges from 0.4-2.4 ng/mL.
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Calcium flux assays: Measuring intracellular calcium mobilization in CXCR2-expressing cells upon GRO gamma stimulation.
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Binding assays: Using radiolabeled or fluorescently labeled GRO gamma to determine binding kinetics with CXCR2.
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Monocyte adhesion assays: Evaluating the ability of GRO gamma to promote monocyte adhesion to endothelial cells or extracellular matrix components.
These functional assays provide complementary information about different aspects of GRO gamma biology .
What approaches can be used to study GRO gamma regulation at the genomic level?
To study GRO gamma regulation at the genomic level, researchers can employ several approaches:
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Promoter analysis: Cloning the GRO gamma promoter region into reporter constructs to identify regulatory elements.
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Chromatin immunoprecipitation (ChIP): Identifying transcription factors that bind to the GRO gamma promoter.
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3' UTR analysis: Investigating the role of the conserved 122-base-pair region in the 3' untranslated region and ATTTA repeats in regulating mRNA stability.
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Expression profiling: Analyzing GRO gamma expression in response to various stimuli (IL-1, TNF, PMA, LPS) to elucidate regulatory pathways.
These approaches can help uncover the complex regulatory mechanisms controlling GRO gamma expression in different cellular contexts .
How might G-quadruplex structures relate to GRO gene regulation?
While the search results don't directly link G-quadruplexes to GRO gamma regulation, recent research has shown that G-quadruplexes can form in guanine-rich regions of oncogene promoters and influence gene expression. G-quadruplexes are non-canonical secondary structures formed in DNA sequences containing consecutive runs of guanines.
Given that GRO gamma (CXCL3) has been described as the GRO3 oncogene, it is possible that G-quadruplex structures might play a role in regulating its expression. This represents an interesting avenue for future research, particularly since G-quadruplex-interactive compounds have emerged as potential anticancer drugs. Researchers could investigate whether the GRO gamma promoter contains guanine-rich sequences capable of forming G-quadruplexes and whether these structures influence its transcriptional regulation .
What are the potential therapeutic implications of targeting GRO gamma pathways?
Given GRO gamma's role in inflammation and cell migration, targeting its signaling pathways could have therapeutic implications for various conditions:
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Inflammatory disorders: Inhibiting GRO gamma/CXCR2 signaling might reduce inflammatory cell recruitment and ameliorate inflammation.
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Cancer: Since GRO gamma is also known as GRO3 oncogene, targeting its expression or function might have anti-tumor effects in certain malignancies.
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Neurological disorders: Given GRO gamma's role in regulating cerebellar granule neuron migration, modulating its function might influence neurological development or repair.
Advanced research is needed to develop selective modulators of GRO gamma activity and validate these therapeutic approaches in appropriate disease models .
How can emerging technologies advance GRO gamma research?
Several emerging technologies could significantly advance GRO gamma research:
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CRISPR-Cas9 genome editing: Creating precise modifications in the GRO gamma gene to study structure-function relationships.
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Single-cell RNA sequencing: Analyzing GRO gamma expression at the single-cell level to uncover cellular heterogeneity in its expression and response.
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Cryo-electron microscopy: Determining the high-resolution structure of GRO gamma in complex with CXCR2.
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Chemogenetics and optogenetics: Developing tools to precisely control GRO gamma signaling in specific cell populations.
These technologies could provide unprecedented insights into GRO gamma biology and facilitate the development of novel therapeutic approaches .
How can researchers address challenges in differentiating between GRO family members?
Due to the high sequence homology between GRO alpha, beta, and gamma (86-90% at the amino acid level), researchers face challenges in specifically detecting and measuring individual GRO proteins. To address this:
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Design highly specific antibodies: Targeting the regions with the greatest sequence divergence.
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Develop isoform-specific qPCR assays: Designing primers and probes that target unique regions in the three GRO mRNAs.
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Use mass spectrometry: Employing high-resolution mass spectrometry to distinguish between the slightly different peptide fragments of the three proteins.
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Consider the 3' UTR differences: Utilizing the significant differences in the 3' untranslated regions for specific detection of GRO gamma mRNA .
What are the critical considerations for experimental design when studying GRO gamma?
When designing experiments to study GRO gamma, researchers should consider:
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Specificity of detection: Ensuring that methods can distinguish GRO gamma from other GRO family members.
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Post-translational processing: Accounting for the effect of N-terminal processing on activity.
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Receptor expression: Verifying CXCR2 expression in target cells when studying GRO gamma function.
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Dosage effects: Using appropriate concentration ranges (ED50 of 0.4-2.4 ng/mL for chemotaxis) to capture the full dose-response relationship.
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Context-dependent regulation: Including relevant inducing agents (IL-1, TNF, PMA, LPS) when studying GRO gamma expression.
These considerations are essential for designing robust experiments that yield reliable and reproducible results .
Product Science Overview
CXCL3 plays a significant role in the immune system by acting as a potent chemoattractant and activator of neutrophils. Neutrophils are a type of white blood cell essential for the body’s defense against infections. CXCL3 promotes the chemotaxis (movement) and degranulation (release of granules) of neutrophils and basophils, which are other types of white blood cells .
Recombinant human CXCL3 is typically produced in Escherichia coli (E. coli) expression systems. The recombinant protein is purified to high levels of purity, often exceeding 95% as determined by SDS-PAGE with silver staining . The endotoxin levels are kept very low to ensure the protein’s safety and efficacy in research and therapeutic applications .
Recombinant CXCL3 is widely used in research to study its role in immune responses and inflammation. It is also utilized in various assays to investigate its chemoattractant properties and its ability to induce the release of myeloperoxidase from neutrophils . Additionally, CXCL3 is used in cell culture studies to understand its effects on different cell types and its potential therapeutic applications.
