Recombinant Human Fibroblast growth factor receptor-like 1 (FGFRL1), partial (Active)

What is the basic structure of FGFRL1 and how does it differ from conventional FGF receptors?

FGFRL1 maintains significant structural homology with classical FGF receptors (FGFR1-FGFR4) in its extracellular domain, sharing up to 50% amino acid similarity . The protein contains three extracellular Ig-like domains, a single transmembrane domain, and a relatively short intracellular domain . The critical structural distinction lies in FGFRL1's lack of the intracellular tyrosine kinase domain that is essential for signal transduction in conventional FGFRs . This fundamental difference suggests that despite its ability to bind FGF ligands, FGFRL1 cannot mediate FGF signaling independently through transphosphorylation . The domain structure of FGFRL1 more closely resembles cell adhesion proteins from the immunoglobulin superfamily, particularly nectins and nectin-like molecules .

What ligands does FGFRL1 bind and with what relative affinities?

FGFRL1 demonstrates selective binding to multiple FGF ligands with high affinity. Both the membrane-bound receptor and its soluble ectodomain can interact with several FGF family members including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22 . These interactions have been verified through multiple methodologies including ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis . Additionally, FGFRL1 forms heterophilic interactions with heparan sulfate proteoglycans at the cell surface of neighboring cells, particularly with glypican-4 and glypican-6, which have been identified as specific interaction partners through tandem LC mass spectrometry . The binding of FGFRL1 to both FGF ligands and cell surface proteoglycans supports its dual functionality in both growth factor signaling modulation and cell-cell adhesion.

What developmental processes require FGFRL1 expression?

FGFRL1 plays crucial roles in multiple developmental processes, as evidenced by knockout studies in mice. During embryonic development, FGFRL1 is expressed in cartilaginous bone precursors, diaphragm and tongue muscles, heart, aorta, lung, pancreas, and kidney . Mice with targeted disruption of the FGFRL1 gene display severe developmental abnormalities. Most notably, they lack metanephric kidneys and possess a hypoplastic diaphragm that cannot properly inflate the lungs, resulting in neonatal lethality immediately after birth . These phenotypes highlight the essential role of FGFRL1 in proper formation of the skeletal system, particularly the diaphragm muscle, and metanephric kidney development. The expression pattern of FGFRL1 has been found to form part of a synexpression group with FGF8, suggesting it likely modulates FGF8-mediated activation of one or more FGFRs during development .

How can recombinant FGFRL1 be effectively expressed and purified for functional studies?

For functional studies of FGFRL1, researchers commonly employ inducible gene expression systems, particularly the tetracycline-inducible (Tet-On) system that allows controlled expression of the protein . When expressing recombinant FGFRL1, considerations must be made regarding whether to express the full-length protein or specific domains. Many studies utilize FGFRL1ΔC constructs that contain the extracellular domain and transmembrane helix but lack the intracellular portion . This approach helps isolate effects attributable to the extracellular and transmembrane domains.

For purification of soluble FGFRL1 ectodomain, researchers can take advantage of the natural shedding process that occurs in cell culture systems. The ectodomain is cleaved from the cell membrane by an as-yet-unidentified protease that cuts the receptor in the membrane-proximal region . Alternatively, expression constructs can be designed to produce only the soluble extracellular portion. Purification typically employs affinity chromatography, using tags incorporated into the recombinant protein or exploiting FGFRL1's natural affinity for heparin. When designing experiments, researchers should account for potential post-translational modifications to ensure proper protein folding and function.

What cell models are most suitable for studying FGFRL1 function?

Several cell models have proven effective for investigating FGFRL1 functions. HEK 293 Tet-On cells represent a widely used model that allows inducible expression of FGFRL1 through doxycycline administration . These cells are particularly valuable for adhesion studies and xenograft tumor models. When HEK 293 Tet-On cells are induced to express FGFRL1ΔC, they form large aggregates and clusters, demonstrating the protein's role in cell adhesion .

C2C12 myoblasts have also proven valuable for studying FGFRL1, particularly in the context of differentiation and ectodomain shedding . These cells naturally express FGFRL1 and demonstrate enhanced shedding of the receptor's ectodomain during differentiation . For developmental studies, Xenopus embryos provide an excellent model, as ectopic expression of FGFRL1 in these embryos antagonizes FGFR signaling during early development .

When selecting cell models, researchers should consider the specific aspect of FGFRL1 function under investigation, whether it be cell adhesion, decoy receptor activity, tumor suppression, or developmental processes.

What imaging techniques best visualize FGFRL1 localization and trafficking?

Immunofluorescence microscopy represents the primary approach for visualizing FGFRL1 localization in cellular contexts. Using monoclonal antibodies against FGFRL1, researchers can detect the protein predominantly at the cell membrane in expressing cells . Higher-resolution imaging reveals interesting subcellular distribution patterns, including FGFRL1 localization to filopodia. By adjusting the microscope focus to the plane of the glass slide rather than the site of strongest fluorescence, researchers can observe FGFRL1 signals at numerous spike-like protrusions emerging from the plasma membrane .

For co-localization studies, dual fluorescent labeling can be employed. For instance, combining FGFRL1 antibody detection with phalloidin staining for filamentous actin has revealed partial co-localization of FGFRL1 with actin in filopodia . Scanning electron microscopy provides complementary ultra-structural information, demonstrating that FGFRL1-induced protrusions have an average diameter of 180±28 nm, consistent with filopodia .

For trafficking studies, live-cell imaging with fluorescently tagged FGFRL1 constructs would be optimal, particularly when investigating the shedding process of the ectodomain from the cell surface.

How does FGFRL1 mediate cell-cell adhesion at the molecular level?

FGFRL1 functions as a bona fide cell-cell adhesion protein through its extracellular domain interactions. The extracellular portion of FGFRL1 forms heterophilic interactions with heparan sulfate proteoglycans, particularly glypican-4 and glypican-6, at the surface of neighboring cells . These interactions promote cell adhesion in vitro, as demonstrated by experiments with recombinant polypeptides corresponding to the extracellular domain . Importantly, when mutations are introduced into the polypeptide chain, this adhesive activity is completely lost, confirming the specificity of the interaction .

When overexpressed in cell lines, FGFRL1 protein accumulates at intersections where two cells make contact . This localization pattern is consistent with its role in facilitating cell-cell adhesion. In HEK 293 Tet-On cells, overexpression leads to pronounced cell aggregation and the formation of large multicellular clusters . At the subcellular level, FGFRL1 expression stimulates the formation of filopodia, which are actin-rich membrane protrusions that facilitate cell-cell contacts . These multiple lines of evidence collectively establish FGFRL1 as a functional cell adhesion molecule that mediates intercellular interactions through its extracellular domain.

How can FGFRL1-mediated cell adhesion be quantitatively measured?

Several experimental approaches can be employed to quantitatively assess FGFRL1-mediated cell adhesion. In vitro adhesion assays using recombinant FGFRL1 protein coated on culture dishes provide a direct measure of cells' ability to adhere to FGFRL1 . The number of attached cells can be quantified after washing steps to remove non-adherent cells, providing a quantitative metric of adhesion strength.

For cell-cell adhesion studies, researchers can utilize doxycycline-inducible expression systems to control FGFRL1 levels and measure the resulting cell clustering . Quantitative analysis can involve measuring cluster size, number of cells per cluster, or the percentage of cells engaged in cluster formation. Time-lapse microscopy would allow tracking of the kinetics of cell aggregation following FGFRL1 induction.

For more detailed molecular analysis, co-immunoprecipitation studies can quantify FGFRL1 interactions with binding partners such as glypicans . Additionally, atomic force microscopy could provide direct measurements of the forces involved in FGFRL1-mediated adhesion between cells or between cells and substrates coated with FGFRL1 binding partners.

What is the relationship between FGFRL1-mediated adhesion and contact inhibition?

FGFRL1-mediated cell adhesion appears to play a crucial role in maintaining contact inhibition, a fundamental property of normal cells that limits proliferation upon reaching confluence. In xenograft tumor models, forced expression of FGFRL1 in HEK 293 Tet-On cells completely inhibits tumor growth in nude mice . This tumor-suppressive effect is attributed to the restoration of contact inhibition through FGFRL1-mediated cell adhesion .

The molecular mechanism governing how FGFRL1 inhibits tumor growth involves complex signaling pathways. Similar to other cell adhesion proteins like CADM1, it appears that FGFRL1-mediated intercellular interactions may activate signaling cascades that regulate the actin cytoskeleton and cell proliferation . In the case of CADM1, homophilic interactions at the surface of adjacent cells activate the phosphatidylinositol 3-kinase (PI3K) pathway and lead to actin cytoskeleton reorganization .

For FGFRL1, the effect is exerted through the extracellular domain's heterophilic interactions with heparan sulfate-containing proteins on neighboring cells . These interactions likely trigger signaling events that restore normal contact inhibition in cells that have lost this property, such as repeatedly subcultured HEK 293 Tet-On cells or cancer cells .

What evidence supports FGFRL1's function as a decoy receptor for FGF ligands?

Multiple lines of evidence support FGFRL1's role as a decoy receptor for FGF ligands. First, structural analysis reveals that FGFRL1 possesses the extracellular domains necessary for FGF binding but lacks the intracellular tyrosine kinase domain required for signal transduction . This structural arrangement is characteristic of decoy receptors, which can bind ligands without initiating downstream signaling.

Second, binding studies using various methodologies (ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance) demonstrate that both membrane-bound FGFRL1 and its soluble ectodomain can bind multiple FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22 . This binding capability allows FGFRL1 to potentially sequester these growth factors away from signaling-competent FGFRs.

Third, functional studies in Xenopus embryos show that ectopic expression of FGFRL1 antagonizes FGFR signaling during early development . Additionally, overexpression of FGFRL1 inhibits the expression of an FGF-inducible reporter gene construct in cultured cells . These functional effects are consistent with FGFRL1 acting as a negative regulator of FGF signaling by competing for ligand binding.

Finally, the shedding of FGFRL1's ectodomain from cell membranes generates soluble receptors capable of ligand scavenging in the extracellular environment, further supporting its decoy receptor function.

How does the FGFRL1 ectodomain shedding process occur, and how can it be modulated experimentally?

The FGFRL1 ectodomain is shed from cell membranes by an as-yet-unidentified protease that cleaves the receptor in its membrane-proximal region . This shedding process has been observed in various cell types, including differentiating C2C12 myoblasts and HEK293 cells . The release of the soluble ectodomain generates receptor fragments capable of binding FGF ligands in the extracellular space, potentially extending the decoy receptor function beyond the cell surface.

To experimentally modulate FGFRL1 shedding, researchers can employ various protease inhibitors. In shedding inhibition experiments, FGFRL1-overexpressing HEK293 cells can be treated with inhibitors such as pepstatin A, leupeptin, GM6001, furin inhibitor Dec-RVKR-CMK, Bace Inhibitor II (Z-VLL-CBO), or phorbol 12-myristate 13-acetate . By analyzing the levels of soluble FGFRL1 in the culture medium after inhibitor treatment, researchers can determine which class of proteases is responsible for the shedding.

For studying the biological significance of the shedding process, researchers can generate non-cleavable FGFRL1 mutants by modifying the putative cleavage sites. Comparison of the cellular effects of wild-type FGFRL1 versus non-cleavable mutants would provide insights into the relative importance of membrane-bound versus soluble forms of the receptor in modulating FGF signaling.

How can FGFRL1 be utilized in cancer research models?

FGFRL1 offers multiple applications in cancer research based on its tumor-suppressive properties. Xenograft models using inducible FGFRL1 expression systems have demonstrated the protein's ability to completely inhibit tumor growth in nude mice . This model can be adapted to study various cancer types and investigate the molecular mechanisms underlying FGFRL1's tumor-suppressive effects.

For translational research, analysis of FGFRL1 expression in human tumor samples has revealed significant alterations in various cancer types. Screening of 241 human tumor samples showed major changes in FGFRL1 expression in ovarian tumors, with most samples showing decreased expression relative to matched control tissue . Similar expression changes have been observed in head and neck tumors, bladder cancers, and colorectal cancer cell lines . These findings suggest FGFRL1 could serve as a biomarker for cancer diagnosis or prognosis.

In therapeutic development, strategies targeting FGFRL1 expression might offer novel approaches to cancer treatment. For instance, FGFRL1 mRNA levels are regulated by microRNA-120, which specifically interacts with the 3' end of FGFRL1 mRNA and leads to its degradation . Downregulating microRNA-120 in tumor tissue could potentially increase endogenous FGFRL1 levels, restore contact inhibition, and suppress tumor growth .

What are the technical challenges in studying FGFRL1 signaling interactions?

Investigating FGFRL1 signaling interactions presents several technical challenges. First, as FGFRL1 lacks a tyrosine kinase domain, it cannot directly transduce signals like conventional FGFRs . Therefore, researchers must develop specialized approaches to detect indirect signaling effects mediated through FGFRL1's interactions with other membrane proteins or through its competition with signaling-competent FGFRs.

Second, FGFRL1 functions through multiple mechanisms, including cell adhesion and decoy receptor activity . Distinguishing between these functions experimentally requires careful design of mutants that selectively disrupt specific activities while preserving others. For example, researchers might generate mutants that retain FGF binding but lose adhesion capabilities, or vice versa.

Third, the shedding of FGFRL1's ectodomain adds complexity to signaling studies . Researchers must account for both membrane-bound and soluble forms of the receptor, which may have distinct signaling effects. Developing tools to specifically detect and quantify each form is essential for comprehensive signaling analyses.

Finally, FGFRL1's interactions with the complex heparan sulfate proteoglycan network at cell surfaces introduces additional variables that must be controlled in signaling studies. Researchers might need to manipulate proteoglycan expression or structure to fully understand FGFRL1's signaling capabilities.

How does FGFRL1 interact with the broader FGF/FGFR signaling network?

FGFRL1 functions as an important regulatory component within the broader FGF/FGFR signaling network. As a decoy receptor, FGFRL1 can bind and sequester several FGF ligands, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22 , potentially limiting their availability to signaling-competent FGFRs. This competitive binding represents a mechanism for fine-tuning FGF signaling intensity and duration.

Expression pattern analyses suggest that FGFRL1 is part of a synexpression group with FGF8, indicating it likely modulates FGF8-mediated activation of one or more FGFRs . This co-expression pattern suggests coordinated regulation and potential functional interactions between FGFRL1 and specific FGF ligands during development.

FGFRL1 could potentially form heterodimers with conventional FGFRs through ligand-mediated dimerization . Such heterodimers would be signaling-incompetent due to FGFRL1's lack of a tyrosine kinase domain, effectively preventing transphosphorylation and subsequent signal transduction . This dominant-negative mechanism would represent an additional layer of FGF/FGFR signaling regulation.

The shedding of FGFRL1's ectodomain extends its regulatory influence beyond the cell surface, allowing it to modulate FGF signaling in the extracellular environment . This soluble form might have distinct effects on FGF signaling compared to the membrane-bound receptor, potentially affecting signaling in cells that do not themselves express FGFRL1.

What developmental defects are observed in FGFRL1 knockout models?

FGFRL1 knockout mice exhibit severe developmental abnormalities affecting multiple organ systems. The most striking defects include:

• Complete lack of metanephric kidneys: FGFRL1-deficient mice fail to develop proper metanephric kidneys, highlighting the essential role of this protein in renal development .

• Hypoplastic diaphragm: The diaphragm muscle in knockout mice is weak and malformed, unable to inflate the lungs after birth . This defect is lethal, causing death immediately after birth due to respiratory failure .

• Skeletal abnormalities: Given FGFRL1's expression in cartilaginous bone precursors during development , knockout models likely exhibit skeletal malformations, though specific defects would need further characterization.

• Potential cardiac defects: FGFRL1 is expressed in the developing heart and aorta , suggesting possible cardiovascular abnormalities in knockout models that warrant detailed investigation.

These severe developmental phenotypes underscore FGFRL1's critical importance in organogenesis, particularly in the formation of the urinary and respiratory systems. The lethality of the knockout emphasizes that FGFRL1 is an essential gene with non-redundant functions in mammalian development.

How can FGFRL1 function be studied in early embryonic development?

To study FGFRL1 function in early embryonic development, researchers can employ several model systems and approaches:

Xenopus embryos offer an excellent model for investigating FGFRL1's role in early development. Ectopic expression of FGFRL1 in Xenopus embryos has been shown to antagonize FGFR signaling during early development . This system allows for microinjection of FGFRL1 mRNA or morpholinos at specific developmental stages and in targeted embryonic regions, enabling precise spatial and temporal manipulation of FGFRL1 activity.

For mammalian studies, conditional knockout mouse models would be valuable for bypassing the embryonic lethality associated with complete FGFRL1 deletion . Using tissue-specific promoters to drive Cre recombinase expression, researchers could delete FGFRL1 in specific tissues or at defined developmental stages, allowing investigation of its tissue-specific functions.

Embryonic stem cell (ESC) differentiation models can also provide insights into FGFRL1's role in early lineage specification. By manipulating FGFRL1 expression in ESCs and directing their differentiation toward specific lineages (particularly kidney, diaphragm, or skeletal precursors), researchers can analyze how FGFRL1 affects cell fate decisions and morphogenesis.

Finally, CRISPR/Cas9 genome editing enables precise modification of the FGFRL1 gene to create specific mutations found in human conditions or to generate reporter lines that allow visualization of FGFRL1 expression dynamics during development.

What human pathologies are associated with FGFRL1 dysfunction?

While direct associations between FGFRL1 mutations and human disorders are still being established, several pathological conditions show altered FGFRL1 expression or function:

Cancer: Significant changes in FGFRL1 expression have been observed in multiple cancer types. Ovarian tumors frequently show decreased FGFRL1 expression compared to matched control tissue, though some samples exhibit dramatic overexpression (up to 25-fold) . Head and neck tumors show FGFRL1 overexpression that correlates with tumor growth and metastasis . Bladder tumors demonstrate significantly decreased FGFRL1 protein expression, often associated with heterozygous deletions at chromosomal region 4p16.3, which includes the FGFRL1 gene . Colorectal cancer cell lines frequently harbor mutations affecting the FGFRL1 reading frame, particularly in the intracellular domain .

Developmental disorders: Given FGFRL1's essential role in kidney development and diaphragm formation in mice , human congenital anomalies affecting these organs may potentially involve FGFRL1 dysfunction. This could include certain forms of renal agenesis or hypoplasia, as well as congenital diaphragmatic defects.

The chromosome region containing FGFRL1 (4p16.3) is affected in Wolf-Hirschhorn syndrome, a condition characterized by growth restriction, intellectual disability, distinct facial features, and various congenital anomalies including heart and kidney defects. While multiple genes are affected in this deletion syndrome, FGFRL1 loss may contribute to some aspects of the phenotype, particularly the renal abnormalities.

What therapeutic potential does FGFRL1 research offer for cancer treatment?

FGFRL1 research presents several promising avenues for cancer therapeutics:

Biomarker potential: FGFRL1 expression levels could serve as diagnostic or prognostic markers for certain cancer types . Decreased FGFRL1 expression might identify aggressive tumors that have lost contact inhibition , while altered expression patterns could help classify tumors for targeted treatment approaches.

MicroRNA-based therapies: FGFRL1 mRNA levels are regulated by microRNA-120, which specifically interacts with the 3' end of the FGFRL1 transcript and leads to its degradation . Therapeutic approaches targeting microRNA-120 in tumor tissue could potentially increase endogenous FGFRL1 levels, restore contact inhibition, and suppress tumor growth . This represents a novel potential treatment strategy that warrants further investigation.

Recombinant FGFRL1 as a therapeutic agent: Given FGFRL1's tumor-suppressive effects in xenograft models , administration of recombinant FGFRL1 protein or gene therapy approaches to deliver FGFRL1 to tumor tissues might inhibit cancer growth by restoring contact inhibition.

FGF pathway modulation: As a decoy receptor for multiple FGF ligands , FGFRL1-based therapeutics could potentially modulate FGF signaling in cancers where this pathway is dysregulated. This approach might be particularly relevant for tumors dependent on FGF2, FGF3, FGF4, FGF8, FGF10, or FGF22 signaling .

What are the most pressing unanswered questions about FGFRL1 biology?

Despite significant advances in FGFRL1 research, several critical questions remain unresolved:

Protease identification: The protease responsible for shedding the FGFRL1 ectodomain from cell membranes remains unidentified . Characterizing this enzyme would provide insights into the regulation of FGFRL1 shedding and potential therapeutic targets for modulating FGFRL1 function.

Signaling mechanisms: While FGFRL1 lacks a tyrosine kinase domain, it might still influence intracellular signaling through its short cytoplasmic domain or through interactions with other membrane proteins. The precise molecular mechanisms by which FGFRL1 transmits signals, particularly in the context of contact inhibition, require further elucidation .

Developmental roles: The severe phenotypes in FGFRL1 knockout mice indicate essential developmental functions , but the molecular pathways through which FGFRL1 regulates kidney development and diaphragm formation remain incompletely understood.

Human disease associations: Direct links between FGFRL1 mutations and human congenital disorders have not been firmly established. Comprehensive genetic screening of patients with relevant phenotypes (renal agenesis, diaphragmatic defects) could reveal pathogenic FGFRL1 variants.

Cellular binding partners: While interactions with heparan sulfate proteoglycans and specific glypicans have been identified , the complete interactome of FGFRL1 remains to be characterized, particularly regarding potential protein partners for its short intracellular domain.

What novel methodologies could advance FGFRL1 research?

Several emerging technologies and approaches could significantly advance FGFRL1 research:

Single-cell technologies: Single-cell RNA sequencing and spatial transcriptomics could provide unprecedented insights into FGFRL1 expression patterns during development and disease. These approaches would reveal cell type-specific expression and potential co-expression networks, illuminating FGFRL1's function in various cellular contexts.

CRISPR-based approaches: Beyond simple gene knockout, CRISPR technologies enable precise genome editing to create specific mutations, tagged endogenous proteins, or conditional alleles. CRISPR screens could identify genes that modify FGFRL1 function, revealing new components of its signaling network.

Organoid models: Kidney and diaphragm organoids would provide valuable in vitro systems for studying FGFRL1's role in these tissues' development. Comparing organoid formation with and without FGFRL1 could reveal detailed mechanisms of its developmental functions.

Protein structural studies: High-resolution structural analysis of FGFRL1, particularly in complex with its various FGF ligands and proteoglycan partners, would provide molecular insights into binding specificity and potential for therapeutic targeting.

Systems biology approaches: Integration of transcriptomic, proteomic, and metabolomic data from FGFRL1-manipulated systems could reveal broader networks affected by this protein, potentially uncovering unexpected functions and regulatory relationships.