Recombinant Human Polycystic kidney disease 2-like 2 protein (PKD2L2)
Description
Introduction to PKD2L2
Recombinant Human Polycystic Kidney Disease 2-Like 2 Protein (PKD2L2) is a genetically engineered form of the human PKD2L2 protein, a member of the polycystin family. This protein shares structural homology with Polycystin-2 (PKD2), a calcium-permeable cation channel implicated in autosomal dominant polycystic kidney disease (ADPKD) . PKD2L2 is encoded by the PKD2L2 gene (UniProt ID: Q9NZM6) and is expressed in tissues such as kidney, brain, and testis . Recombinant PKD2L2 is produced in E. coli with an N-terminal His tag, enabling purification and functional studies .
Primary Structure
The full-length PKD2L2 protein comprises 624 amino acids, including six transmembrane domains and cytoplasmic N- and C-termini . Key domains include:
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Transmembrane helices (S1–S6): Critical for ion channel formation.
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Extracellular domain: Implicated in channel assembly and ligand interactions .
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EF-hand motifs: Potential calcium-binding regions (absent in some homologs like polycystin-L) .
Table 1: Amino Acid Sequence Highlights
Tertiary and Quaternary Structure
Cryo-EM studies of PKD2 (a homolog) reveal a homotetrameric architecture with a central ion permeation pathway . PKD2L2 likely adopts a similar structure, featuring:
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Two constriction points: A selectivity filter (residues 641–643) and a cytoplasmic gate (residues Leu677–Asn681) .
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Voltage-sensor domain: Retains two gating charges, suggesting regulatory mechanisms .
Ion Channel Activity
PKD2L2 is hypothesized to function as a nonselective cation channel with permeability to Na⁺, K⁺, and Ca²⁺ . Key functional characteristics include:
Pathophysiological Role
While direct mutations in PKD2L2 are not linked to ADPKD, its interaction with PKD1/PKD2 may modulate cystogenesis:
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Compensatory upregulation: Deleting miR-17 motifs in PKD2 enhances PC2 expression and reduces cyst growth in PKD1-mutant models .
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Cellular signaling: Derepression of PKD2L2 correlates with reduced Yap1 and c-Myc activity, pathways implicated in cyst proliferation .
Research Applications
Recombinant PKD2L2 is utilized in diverse experimental contexts:
Physicochemical Properties
Therapeutic Implications
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miR-17 targeting: Anti-miR-17 oligonucleotides (e.g., RGLS4326) upregulate PKD2L2 expression, mitigating cyst growth in murine ADPKD models .
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Compensatory mechanisms: Enhanced PKD2L2 expression restores mitochondrial function and reduces CREB/YAP signaling in PKD1-mutant cells .
Comparative Analysis: PKD2 vs. PKD2L2
Future Directions
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Mechanistic studies: Elucidate PKD2L2’s role in calcium signaling and cyst suppression.
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Therapeutic targeting: Explore small molecules or gene therapies to modulate PKD2L2 expression in polycystic diseases.
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please indicate your preference in the order remarks, and we will fulfill your request.
Note: All protein shipments are standardly packaged with blue ice packs. If dry ice shipping is required, please inform us in advance as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
Basic Research Questions
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What is PKD2L2 and how does it relate to polycystic kidney disease genes?
PKD2L2 (polycystin 2 like 2, transient receptor potential cation channel) is a protein-coding gene that belongs to the polycystin family. It shows significant homology to both polycystin-1 and polycystin-2, which are encoded by PKD1 and PKD2 genes respectively. These genes, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD) . PKD2L2 encodes a protein of 613 amino acids that is approximately 65% similar to polycystin-2 . While PKD2L2 itself has not been directly implicated in renal cystic diseases, its striking homology to PKD1 and PKD2 implies similar roles that may contribute to understanding the function of both polycystin-1 and polycystin-2 .
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Where is PKD2L2 expressed in human tissues?
Expression analysis reveals that PKD2L2 is primarily found in testis . Unlike some other polycystin family members that show broader tissue expression patterns, PKD2L2 appears to have a more restricted expression profile. For comparison, PKDREJ, another polycystin family member, shows additional expression in retina, brain, liver, and spleen as detected by RT-PCR . This tissue-specific expression pattern suggests PKD2L2 may have specialized functions in reproductive biology rather than broad physiological roles across multiple tissues.
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What is the genomic location and structural organization of PKD2L2?
Advanced Research Questions
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What methodologies are recommended for expressing recombinant PKD2L2 protein for functional studies?
For successful expression of recombinant PKD2L2 protein, researchers should consider the following methodological approach:
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Expression system selection: Baculovirus-insect cell expression systems have proven effective for PKD2L2 and related proteins . This approach is particularly valuable as membrane proteins like PKD2L2 often fold incorrectly or form inclusion bodies in bacterial systems.
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Vector design: Incorporate appropriate fusion tags to facilitate purification and detection. For example, N-terminal polyhistidine-tagged GST fusion constructs have been successfully used .
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Protein purification protocol:
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Activity verification: The specific activity can be determined using synthetic peptide substrates. For similar proteins, activity around 30 nmol/min/mg has been reported using synthetic CREBtide peptide (KRREILSRRPSYR) as substrate .
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Quality control: Assess purity (>82% by SDS-PAGE) and verify molecular mass (expected ~120 kDa) .
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How can researchers investigate the ion channel properties of PKD2L2?
Investigation of PKD2L2 ion channel properties requires specialized electrophysiological approaches:
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Lipid bilayer reconstitution: This technique has been successfully used for polycystin-2 from human syncytiotrophoblast apical membranes and could be adapted for PKD2L2. The approach revealed that polycystin-2 functions as a nonselective cation channel with multiple subconductance states and high permselectivity to Ca²⁺ .
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Patch-clamp experiments: Heterologous expression systems such as Sf9 insect cells have been used to confirm channel function of polycystin family members . For PKD2L2 specifically, researchers should note that it "exhibits a lower single conductance but no spontaneous channel activity" compared to other family members .
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Pharmacological profiling: Channel modulators such as Ca²⁺, La³⁺, Gd³⁺, and amiloride, which inhibit polycystin-2, can be tested on PKD2L2 to compare pharmacological profiles .
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Calcium imaging: Since polycystin channels are involved in calcium signaling, calcium flux assays using fluorescent indicators can provide functional insights.
| Channel Property | PKD2 | PKD2L2 | Method of Detection |
|---|---|---|---|
| Single channel conductance | Higher | Lower | Patch-clamp |
| Spontaneous activity | Present | Absent | Patch-clamp |
| Ca²⁺ permeability | High | Likely present | Ion substitution experiments |
| Subconductance states | Multiple | Not fully characterized | Single channel recordings |
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What approaches can be used to study the role of PKD2L2 in polycystic kidney disease models?
While PKD2L2 has not been directly implicated in ADPKD, its homology to PKD2 makes it relevant for comparative studies. Researchers can employ these approaches:
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Gene editing techniques: CRISPR/Cas9 methodology has been successfully used to modify 3'-UTR regions of PKD genes to study their regulation . Similar approaches could target PKD2L2 to examine its potential compensatory role.
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Cellular models: Both 2D and 3D culture systems are valuable for PKD research, including:
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Expression modulation studies: Recent research has shown that preventing cis-inhibition of PKD2 expression can mitigate cystic phenotypes in PKD1-mutant models . Similar approaches could examine whether PKD2L2 expression affects disease progression.
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Co-expression analysis: Since PKD1 and PKD2 proteins interact and are co-expressed in multiple subcellular locations, investigating potential interactions between PKD2L2 and other polycystins could reveal compensatory or regulatory mechanisms .
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mRNA translation regulation: Recent findings demonstrate that PKD1 and PKD2 mRNAs are repressed via their 3′-UTR miR-17 binding elements. Similar regulatory mechanisms could be investigated for PKD2L2 .
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How does PKD2L2 structurally and functionally compare to other polycystin family members?
PKD2L2 shares significant structural and functional similarities with other polycystin family members, but with notable differences:
| Feature | PKD2 (Polycystin-2) | PKD2L2 | PKD2L | PKDREJ |
|---|---|---|---|---|
| Amino acid length | 968 | 613 | 805 | 2253 |
| Similarity to PKD2 | - | ~65% | ~65% | ~35% to both PKD1/PKD2 |
| Primary expression | Kidney, widespread | Testis | Testis | Testis, retina, brain, liver, spleen |
| Chromosome location | 4q22.1 | 10q31 | 5q31 | 22q13.3 |
| Channel activity | Nonselective cation channel | Lower conductance, no spontaneous activity | Not fully characterized | Not fully characterized |
| Disease association | ADPKD | Not associated with ADPKD | Not linked to ADPKD | Not linked to ADPKD |
The structure-function relationship analyses reveal that while PKD2L2 shares significant homology with polycystin-2, its distinct expression pattern and lower channel conductance without spontaneous activity suggest specialized physiological roles that differ from those of PKD2 .
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What are the current methodological approaches for investigating PKD2L2 interactions with other proteins?
To investigate PKD2L2 protein interactions, researchers should consider:
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Co-immunoprecipitation studies: Using specific antibodies against PKD2L2, such as anti-PKD2L2 antibody (HPA043634) , to pull down protein complexes from tissue or cell lysates.
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Proximity labeling approaches: BioID or APEX2-based methods can identify proteins in close proximity to PKD2L2 in living cells.
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Yeast two-hybrid screening: Can be employed to identify potential interaction partners, though validation in mammalian systems is essential.
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Fluorescence resonance energy transfer (FRET): To study dynamic interactions in living cells.
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Cryo-EM structural studies: Recent advances in cryo-EM have enabled structural characterization of TRP channel family members, including polycystin-2-L1 . Similar approaches could be applied to PKD2L2 to understand its structure and potential interaction interfaces.
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Functional co-expression studies: Given that polycystin-1 and polycystin-2 are known to interact at their carboxy termini , similar interaction studies between PKD2L2 and other polycystins could reveal important functional relationships.
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How can researchers distinguish between functions of PKD2L2 and other polycystin proteins in experimental systems?
Distinguishing between polycystin family members in experimental systems requires multiple complementary approaches:
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Gene-specific knockdown or knockout: Using siRNA, shRNA, or CRISPR-Cas9 to specifically target PKD2L2 while monitoring other family members' expression levels.
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Isoform-specific antibodies: Employing validated antibodies with confirmed specificity for Western blotting, immunoprecipitation, and immunohistochemistry .
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Electrophysiological fingerprinting: Since PKD2L2 exhibits "lower single conductance but no spontaneous channel activity" compared to other family members, patch-clamp recordings can help distinguish its contribution to cellular ion channel activity.
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Tissue-specific expression analysis: Since PKD2L2 shows restricted expression primarily in testis , tissue-specific studies can help isolate its functions.
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Domain-specific mutations: Introducing mutations in regions unique to PKD2L2 can help determine which functions are specific to this isoform versus shared with other polycystins.
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Rescue experiments: In knockdown/knockout models, selective re-expression of PKD2L2 versus other family members can reveal which functions are specifically attributable to PKD2L2.
