Recombinant Paralichthys olivaceus Calcitonin gene-related peptide type 1 receptor (calcrl)

What is Paralichthys olivaceus calcitonin gene-related peptide type 1 receptor?

Paralichthys olivaceus calcitonin gene-related peptide type 1 receptor (calcrl) is a G protein-coupled receptor that functions as a receptor for calcitonin gene-related peptide (CGRP). In flounder, this receptor plays crucial roles in vasodilation and osmotic regulation processes. The receptor requires association with receptor activity-modifying proteins (RAMPs) for functional activity, forming part of the broader CGRP receptor family found throughout various physiological systems . The receptor's molecular characterization has revealed it to be structurally related to the calcitonin receptor family, with specific adaptations for teleost fish physiology.

How does the structure of P. olivaceus calcrl compare to mammalian CALCRL?

P. olivaceus calcrl shares fundamental structural characteristics with mammalian CALCRL while maintaining species-specific adaptations. Both function as G protein-coupled receptors with transmembrane domains, but the flounder variant shows sequence divergence reflecting evolutionary adaptation to aquatic environments. When associated with RAMP1, calcrl forms a CGRP receptor that consists of a heterodimeric structure with multiple hydrophobic and hydrophilic regions throughout its chains . Molecular phylogenetic analysis has confirmed that P. olivaceus CLR candidates (XP_019955157.1 and XP_019966707.1) are authentic members of the CLR family, with distinctive sequence features that differentiate them from CTR (calcitonin receptor) orthologs .

What are the physiological roles of calcrl in Paralichthys olivaceus?

In Paralichthys olivaceus, calcrl plays several key physiological roles:

• Vasodilation: Functions as a vasodilatory neural peptide receptor in the vascular system

• Osmotic regulation: Participates in the mechanisms controlling ionic and water balance, critical for a marine teleost

• Development: Though not fully characterized, evidence suggests a role in embryonic development, as demonstrated by expression analysis in flounder embryos

• Signal transduction: Mediates cellular signaling through G protein coupling, primarily activating adenylate cyclase and resulting in the generation of intracellular cyclic adenosine monophosphate (cAMP)

Unlike mammals, where CALCRL functions have been extensively characterized across multiple systems, the specific functions in non-mammalian vertebrates like flounder continue to be an active area of research.

What receptor complexes does P. olivaceus calcrl form, and how do they differ functionally?

While specific information about P. olivaceus calcrl receptor complexes is limited in the search results, we can infer from homologous systems that calcrl likely forms different receptor complexes through association with various RAMPs, similar to mammalian CALCRL. By extension from what is known about CALCRL receptor complexes:

• Calcrl + RAMP1: Likely forms a CGRP-specific receptor

• Calcrl + RAMP2: Likely forms an adrenomedullin (AM) receptor

• Calcrl + RAMP3: Possibly forms a dual CGRP/AM receptor

These different complexes would have distinct ligand specificities and potentially different downstream signaling pathways, although the specific functions in P. olivaceus require further characterization through direct experimental evidence.

How do genomic variations in P. olivaceus calcrl influence its function?

The search results identify two CLR candidates from P. olivaceus genome (XP_019955157.1 and XP_019966707.1), suggesting potential genomic variations . Through molecular phylogenetic analysis, these candidates were determined to be authentic members of the CLR family, with XP_019955157.1 sharing evolutionary proximity to mefugu CLR1 and XP_019966707.1 to mefugu CLR2 . These variations likely influence:

• Ligand binding specificity

• Signal transduction efficiency

• Receptor coupling with different G proteins

• Tissue-specific expression patterns

While the search results don't provide specific experimental data on how these variations affect function in P. olivaceus, research in other systems indicates that genetic variations in CALCRL can significantly impact cardiovascular function, as evidenced by the influence of regulatory elements carrying coronary artery disease risk SNPs on CALCRL expression in humans .

What are the expression patterns of calcrl during P. olivaceus development?

Researchers have investigated CGRP function related to early development in P. olivaceus through expression analysis of flounder CGRP and CLRs in embryos. The study collected embryos at various developmental stages (immediately post-fertilization through later stages) and employed RT-PCR techniques to detect expression . While the search results don't provide the complete expression profile, they indicate that calcrl expression during development was specifically analyzed to gain insights into CGRP's embryonic functions. The expression analysis employed EF1-α as a control gene, with PCR parameters of 25 cycles at specific temperature conditions (96°C for 0.5 min, 60°C for 1 min, and 72°C for 2 min, followed by a single cycle at 72°C for 15 min) .

How does calcrl signaling contribute to osmotic regulation in teleost fish?

In teleost fish including P. olivaceus, calcrl is associated with osmotic regulation processes, which are critical for maintaining ion and water balance in marine environments . While the exact signaling mechanisms aren't fully detailed in the search results, we can infer that calcrl likely functions through:

• Coupling with G proteins (primarily Gs) to activate adenylate cyclase

• Generation of intracellular cAMP as a second messenger

• Activation of downstream effectors that regulate ion channels and transporters

This signaling pathway would ultimately influence ion transport across gill epithelial cells, which is crucial for maintaining osmotic balance in marine teleosts. The involvement of calcrl in seawater adaptation of flounder has been specifically investigated, with expression analyses of its receptor mRNA in the gill providing evidence for this physiological role .

What signaling pathways are activated by P. olivaceus calcrl?

Based on the available information and homology to other species, P. olivaceus calcrl likely activates several signaling pathways:

• cAMP pathway: Through coupling with Gs proteins, activating adenylate cyclase and increasing intracellular cAMP levels

• Possibly calcium signaling: Through coupling with Gq proteins

• Potential inhibitory pathways: Through coupling with Gi proteins

By extension from studies on CALCRL in other systems, we can hypothesize that P. olivaceus calcrl may influence pathways similar to those mediated by CALCRL in other contexts, including:

• eNOS (endothelial nitric oxide synthase) signaling

• APLN (apelin) signaling

• Angiopoietin pathways

• Prostaglandin production

• EDN1 (endothelin-1) signaling

What are the optimal conditions for expression and purification of recombinant P. olivaceus calcrl?

Based on the commercially available recombinant P. olivaceus calcrl protein information, the following conditions are recommended:

Expression System:

• Host: E. coli

• Construct: Full-length protein (amino acids 21-465) with N-terminal His-tag

Purification Method:

• Affinity chromatography using the His-tag

• SDS-PAGE to confirm purity (>90%)

Storage Conditions:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use

• Avoid repeated freeze-thaw cycles

• Lyophilized powder form can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

What techniques are most effective for studying calcrl-RAMP interactions in fish models?

While the search results don't specifically address techniques for studying calcrl-RAMP interactions in fish, we can infer from general research practices in this field:

• Co-immunoprecipitation (Co-IP): To detect physical interactions between calcrl and different RAMPs

• Fluorescence Resonance Energy Transfer (FRET): To study real-time protein-protein interactions in living cells

• Surface Plasmon Resonance (SPR): To measure binding kinetics between calcrl and RAMPs

• Yeast Two-Hybrid assays: To identify potential interaction partners

• Functional assays: Such as cAMP accumulation assays to assess the functional consequences of different calcrl-RAMP combinations

Molecular phylogenetic analysis has been specifically mentioned as a technique used to classify CLR and CTR from the flounder genome, which helps in understanding the evolutionary relationships and functional characteristics of these receptor systems .

How can researchers effectively detect calcrl expression in different tissues of P. olivaceus?

Based on the research methodologies described in the search results, effective techniques for detecting calcrl expression include:

• RT-PCR:

  • RNA extraction from different tissues

  • cDNA synthesis

  • PCR amplification with calcrl-specific primers

  • Analysis on agarose gel (2.5% NuSieve GTG agarose gel) stained with ethidium bromide

• Quantitative PCR (qPCR):

  • For more quantitative assessment of expression levels

  • Requires reference genes (such as EF1-α as used in the referenced study)

• In situ hybridization:

  • For spatial localization of calcrl mRNA in tissue sections

  • Particularly useful for developmental studies

• Immunohistochemistry:

  • Using antibodies against calcrl protein

  • Requires validation of antibody specificity for P. olivaceus calcrl

The PCR parameters used in previous studies (25 cycles at 96°C for 0.5 min, 60°C for 1 min, and 72°C for 2 min, followed by a single cycle at 72°C for 15 min) provide a starting point for optimization .

How can CRISPR-Cas9 be applied to study calcrl function in P. olivaceus?

CRISPR-Cas9 technology can be powerful for studying calcrl function in P. olivaceus through several approaches:

• Gene Knockout:

  • Design sgRNAs targeting conserved regions of calcrl

  • Create complete knockout models to study loss-of-function phenotypes

  • Analyze effects on vasodilation, osmotic regulation, and development

• Enhancer Modification:

  • Target regulatory regions controlling calcrl expression

  • Based on studies in other systems, enhancer deletion can significantly affect calcrl expression levels

  • For example, CRISPR deletion of an enhancer region in human CALCRL resulted in downregulation of expression

• Domain-Specific Modifications:

  • Create precise mutations in domains responsible for RAMP interaction

  • Modify G protein coupling domains to alter signaling specificity

  • Engineer tagged versions for in vivo tracking

• Knock-in Strategies:

  • Introduce reporter genes (GFP, luciferase) to monitor expression patterns

  • Create humanized versions to study species-specific differences

The approach would require optimization of microinjection techniques for P. olivaceus embryos and validation of editing efficiency in this species.

What challenges exist in translating findings from P. olivaceus calcrl to mammalian CALCRL systems?

Several significant challenges exist when attempting to translate findings between fish and mammalian CALCRL systems:

• Evolutionary Divergence:

  • Despite conservation of core functions, significant sequence divergence exists

  • Different regulatory mechanisms and expression patterns

• Physiological Context Differences:

  • Fish osmoregulation vs. mammalian systems

  • Different vascular system organization and regulation

  • Species-specific developmental programs

• RAMP Association Variations:

  • Potential differences in RAMP preferences and binding kinetics

  • Differential expression of RAMPs across tissues in different species

• Environmental Adaptations:

  • P. olivaceus is adapted to marine environments with specific physiological demands

  • Temperature-dependent effects on receptor function and signaling

• Technical Limitations:

  • Different experimental models and techniques optimized for each system

  • Antibody cross-reactivity issues

  • Different pharmacological profiles of agonists and antagonists

Despite these challenges, comparative studies can provide valuable insights into conserved mechanisms and species-specific adaptations of CALCRL signaling.

How might transcriptional regulation of calcrl differ between teleost fish and mammals?

Transcriptional regulation of calcrl likely differs significantly between teleost fish and mammals due to evolutionary divergence:

• Enhancer Elements:

  • In humans, CALCRL expression is regulated by an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer

  • Fish-specific enhancers might respond to different environmental conditions, particularly osmotic stress

• Transcription Factor Binding:

  • While HSF1 regulates CALCRL expression in human endothelial cells, different transcription factors may predominate in fish

  • Temperature-dependent regulation may be more prominent in poikilothermic fish

  • Shear stress response elements may differ between fish and mammals

• Alternative Splicing:

  • Different patterns of alternative splicing may generate species-specific isoforms

  • The flounder genome contains multiple CLR candidates that may be regulated differently

• Epigenetic Regulation:

  • Different patterns of DNA methylation and histone modifications

  • Potential for environment-induced epigenetic changes, particularly in response to salinity changes

Molecular characterization studies have identified two distinct CLR candidates in flounder (XP_019955157.1 and XP_019966707.1), suggesting potentially more complex regulation than in some mammalian systems .

How has calcrl evolved across different fish species and other vertebrates?

The evolutionary history of calcrl reflects adaptive diversification across vertebrate lineages:

• Phylogenetic Relationships:

  • Molecular phylogenetic analysis has shown that CLR-type and CTR-type genes are vertebrate orthologs

  • The flounder CLR candidates (XP_019955157.1 and XP_019966707.1) show evolutionary relationships with mefugu CLR1 and CLR2, respectively

• Functional Conservation:

  • Core functions in vasodilation appear conserved from fish to mammals

  • The CGRP receptor system is present throughout vertebrates, suggesting ancient evolutionary origins

• Adaptive Divergence:

  • Species-specific adaptations reflect environmental pressures

  • Marine teleosts may have evolved specialized functions related to osmoregulation

  • Mammalian systems show adaptations for homeothermy and terrestrial physiology

• Receptor-Ligand Co-evolution:

  • Co-evolution of calcrl with its ligands (CGRP, adrenomedullin)

  • Differential selection pressures on receptor-ligand pairs across lineages

The presence of multiple CLR candidates in the flounder genome suggests potential gene duplication events during teleost evolution, consistent with the teleost-specific genome duplication hypothesis .

What functional differences exist between the two identified CLR candidates in P. olivaceus?

The search results identify two CLR candidates in P. olivaceus (XP_019955157.1 and XP_019966707.1), which show phylogenetic relationships with mefugu CLR1 and CLR2, respectively . While detailed functional characterization is not provided in the search results, we can infer potential differences:

• Ligand Specificity:

  • Different binding affinities for CGRP vs. adrenomedullin

  • Potential differences in RAMP association preferences

• Expression Patterns:

  • Likely differential tissue distribution

  • Potentially different developmental expression profiles

• Signaling Properties:

  • May couple preferentially to different G protein subtypes

  • Different signaling efficacies and downstream pathway activation

• Physiological Roles:

  • One variant may be more specialized for osmoregulation

  • The other might have stronger roles in vascular function or development

Further experimental characterization would be required to definitively establish the functional differences between these two receptor variants.

How do environmental factors influence calcrl expression and function in P. olivaceus?

Environmental factors likely play significant roles in regulating calcrl expression and function in P. olivaceus, given its aquatic habitat:

• Salinity Effects:

  • Previous studies have investigated the potential involvement of CGRP in seawater adaptation of flounder through expression analysis of its receptor mRNA in the gill

  • Osmotic stress may regulate calcrl expression as part of adaptive responses

• Temperature Influence:

  • As an ectothermic organism, temperature likely affects:

    • Receptor conformation and binding kinetics

    • Expression levels through temperature-sensitive transcription factors

    • Signaling efficacy and duration

• Hypoxia Response:

  • Low oxygen conditions may modulate calcrl expression

  • Potential role in vascular adaptation to hypoxic stress

• Developmental Plasticity:

  • Environmental conditions during early development may program calcrl expression patterns

  • Studies have examined expression during embryonic stages maintained in specific water conditions (17 ± 1°C)

By analogy with mammalian systems, mechanical forces such as fluid shear stress may also regulate calcrl expression, as CALCRL has been identified as an important mediator of the endothelial fluid shear stress response in humans .