RAMP3 Human

What is RAMP3 and how does it function in human cells?

RAMP3 is a single transmembrane-spanning protein that serves as a molecular chaperone and allosteric modulator of G-protein-coupled receptors (GPCRs). It belongs to the RAMP family, which includes RAMP1, RAMP2, and RAMP3 . These proteins were initially characterized through their interactions with Family B GPCRs, but evidence suggests a significantly broader role across GPCR families. RAMP3 functions by forming heterodimeric complexes with various receptors, altering their trafficking, ligand binding properties, and signaling outcomes.

Unlike RAMP1 and RAMP2, which primarily localize intracellularly in the absence of interacting GPCRs, RAMP3 exhibits endogenous plasma membrane expression even when expressed alone . This unique characteristic suggests distinct functional properties. Additionally, RAMP3 contains a type I PDZ recognition motif (-DTLL) in its C-terminal tail, which is absent in other RAMP family members and plays a critical role in determining receptor recycling pathways .

Which GPCRs interact with RAMP3 and how are these interactions detected?

RAMP3 interacts with a surprisingly wide array of GPCRs, particularly within the chemokine receptor family. Comprehensive screening has identified robust interactions between RAMP3 and all 24 tested chemokine receptors, with particularly strong interactions with atypical chemokine receptors (ACKRs) .

To detect these interactions, researchers utilize multiple complementary approaches:

• Bioluminescence Resonance Energy Transfer (BRET): By expressing GPCR-rLuc fusion proteins with RAMP-YFP constructs in HEK293T cells and measuring energy transfer as a function of increasing RAMP concentration, researchers can generate saturation curves that indicate direct protein interactions. Interactions are typically classified based on maximum BRET signal (Bmax), curve fitting characteristics, and BRET50 values (concentration of acceptor yielding 50% of maximum BRET) .

• Cell-surface expression assays: Flow cytometry with epitope-tagged RAMPs (typically FLAG or HA tags) can determine whether co-expression with GPCRs alters RAMP localization to the plasma membrane .

• Confocal microscopy: Direct visualization of co-localization between tagged RAMP3 and GPCRs .

• Proximity ligation assay (PLA): Confirms close proximity (≤40 nm) between RAMP3 and interacting receptors, providing evidence of direct molecular interaction .

How does RAMP3 expression differ across human tissues?

While the search results don't provide comprehensive information on tissue-specific RAMP3 expression patterns in humans, research indicates that RAMP3 expression is dynamically regulated in various physiological and pathological contexts. RAMP3 appears to be particularly important in vascular tissues, as evidenced by its role in retinal angiogenesis . The genetic deletion of RAMP3 in mouse models leads to significant defects in guided angiogenesis, demonstrating its importance in vascular development .

RAMP3 expression patterns likely vary across tissues based on the specific GPCR repertoire expressed and the functional requirements for receptor trafficking and recycling within those tissues. For researchers interested in tissue-specific expression, quantitative PCR, western blotting of tissue lysates, and immunohistochemistry would be appropriate methodological approaches.

What is the molecular mechanism by which RAMP3 regulates ACKR3 recycling?

RAMP3 determines the endosomal sorting and recycling fate of ACKR3 through a mechanism dependent on its PDZ recognition motif. When ACKR3 is co-expressed with wild-type RAMP3, ligand stimulation (with either adrenomedullin or SDF-1/CXCL12) results in internalization of the receptor complex followed by rapid recycling through Rab4-positive vesicles (associated with the fast recycling pathway) .

The molecular mechanism involves:

• Formation of a stable ACKR3-RAMP3 complex at the cell surface

• Ligand-induced internalization of this complex into early endosomes

• RAMP3 PDZ motif-dependent sorting into Rab4-positive recycling vesicles

• Rapid return to the plasma membrane (within 30 minutes of ligand stimulation)

When the PDZ motif of RAMP3 is deleted (RAMP3ΔPDZ), this rapid recycling pathway is disrupted, and the receptor complex is instead diverted to Rab11-positive slow-recycling endosomes . This indicates that the PDZ recognition sequence in RAMP3 is a critical molecular determinant of ACKR3 trafficking and recycling fate.

Importantly, this mechanism is ligand-independent, occurring with both adrenomedullin and SDF-1/CXCL12 stimulation, suggesting a general role for RAMP3 in ACKR3 trafficking regardless of the activating ligand .

How does RAMP3 influence chemotactic gradient formation and directed cell migration?

RAMP3 plays a crucial role in chemotactic gradient formation through its regulation of ACKR3 recycling. ACKR3 functions as a scavenger receptor, binding and removing ligands like adrenomedullin (AM) and SDF-1/CXCL12 from the extracellular environment without initiating classical G-protein signaling. This scavenging activity helps establish concentration gradients essential for directed cell migration .

The RAMP3-dependent rapid recycling of ACKR3 enhances this scavenging function by:

• Allowing the receptor to undergo multiple rounds of internalization and recycling

• Increasing the total ligand-binding capacity of each receptor

• Creating sharper concentration gradients through more efficient ligand clearance

• Enabling dynamic spatiotemporal regulation of chemotactic signals

In vivo evidence supports this mechanism. Genetic deletion of either ACKR3 or RAMP3 in mice results in significant defects in retinal angiogenesis, characterized by reduced numbers of endothelial tip cells and impaired filopodia formation . Specifically, researchers observed:

These findings demonstrate that both ACKR3 and RAMP3 are essential for establishing the chemotactic gradients required for proper vascular development .

How do RAMP3-dependent mechanisms differ between ACKR3 and other chemokine receptors?

While RAMP3 interacts with most chemokine receptors, its effects appear to be receptor-specific. The detailed investigation of RAMP3-ACKR3 interactions reveals distinct functional consequences compared to other chemokine receptors:

• Effect on receptor localization: ACKR3 and other atypical chemokine receptors (ACKRs) reduce RAMP3 cell-surface expression when co-expressed, whereas typical chemokine receptors like CCR5 and CXCR2 enhance RAMP3 surface expression . This suggests that ACKR3-RAMP3 complexes primarily localize intracellularly or are targeted for degradation, while typical chemokine receptor-RAMP3 complexes favor plasma membrane localization.

• Impact on signaling: RAMP3 does not enable G-protein coupling for ACKR3, which remains unable to signal through either Gαs or Gαi/o pathways even when complexed with RAMP3 . Similarly, RAMP3 does not alter β-arrestin recruitment to ACKR3 following ligand stimulation. This contrasts with canonical RAMP-GPCR interactions (e.g., CLR-RAMP3), where RAMPs can influence signaling outcomes.

• Receptor trafficking: The most significant RAMP3-dependent effect on ACKR3 is its influence on post-endocytic sorting and recycling. It remains to be determined whether RAMP3 exerts similar trafficking effects on other chemokine receptors .

For researchers studying different chemokine receptors, it is essential to evaluate RAMP3 effects on receptor-specific functions rather than assuming uniform consequences across the receptor family.

What role does the PDZ recognition motif in RAMP3 play in receptor trafficking?

The PDZ recognition motif (-DTLL) at the C-terminal tail of RAMP3 serves as a critical molecular determinant of receptor trafficking. This motif is unique to RAMP3 among the RAMP family members and confers specific protein-protein interaction capabilities that influence receptor sorting .

Experimental evidence demonstrates that:

• When wild-type RAMP3 forms a complex with ACKR3, ligand stimulation leads to receptor internalization followed by rapid recycling through Rab4-positive vesicles .

• Deletion of the PDZ motif (RAMP3ΔPDZ) disrupts this rapid recycling pathway. Instead, the ACKR3-RAMP3ΔPDZ complex is diverted to Rab11-positive endosomes associated with slow recycling .

• This PDZ-dependent sorting mechanism applies regardless of the stimulating ligand (either adrenomedullin or SDF-1/CXCL12), indicating it's a general mechanism for RAMP3-dependent receptor trafficking .

The PDZ motif likely serves as a binding site for PDZ domain-containing proteins involved in endosomal sorting. While the specific PDZ proteins interacting with RAMP3 in the context of ACKR3 trafficking weren't identified in the search results, previous work has established that the RAMP3 PDZ motif plays a similar role in the recycling of other RAMP3-interacting receptors, such as the canonical CLR-RAMP3-NSF complex .

What techniques are most effective for studying RAMP3-GPCR interactions?

Based on the search results, several complementary techniques have proven effective for studying RAMP3-GPCR interactions, each with specific advantages:

• Bioluminescence Resonance Energy Transfer (BRET) provides a quantitative measure of protein-protein interactions in living cells. For RAMP-GPCR interactions:

  • Express GPCR-rLuc fusion proteins with RAMP-YFP constructs in HEK293T cells

  • Measure energy transfer as a function of increasing RAMP concentration

  • Analyze saturation curves using multivariate criteria:

    • Bmax > 0.100 (minimum signal threshold)

    • Hyperbolic rather than linear curve fitting

    • BRET50 values (concentration of acceptor yielding 50% of maximum BRET)

• Cell-surface expression assays using flow cytometry determine whether co-expression with GPCRs alters RAMP localization:

  • Express epitope-tagged RAMPs (FLAG or HA) with and without GPCRs

  • Use antibodies against extracellular epitope tags to measure surface expression

  • Quantify changes in RAMP surface expression as an indicator of interaction

• Confocal microscopy visualizes co-localization:

  • Express differentially tagged RAMP3 and GPCRs (e.g., Myc-ACKR3 and HA-RAMP3)

  • Perform immunofluorescence staining of non-permeabilized cells to examine membrane co-localization

  • Use permeabilized cells to assess intracellular distribution

• Proximity ligation assay (PLA) confirms close molecular proximity:

  • This technique detects proteins within ≤40 nm of each other

  • Produces punctate fluorescent signals at interaction sites

  • Particularly useful for confirming cytoplasmic interactions

The most robust approach involves combining multiple techniques, as each provides complementary information. While BRET measures total protein-protein interactions regardless of cellular location, FACS and confocal microscopy provide spatial information about interaction consequences .

How can researchers effectively measure RAMP3's influence on ligand scavenging?

To measure RAMP3's influence on ligand scavenging, the following methodological approaches are effective:

• Co-culture scavenging assays:

  • Generate "reporter" cells expressing a signaling receptor (e.g., CLR-RAMP3) and a cAMP biosensor (EPAC)

  • Co-culture these cells with "scavenger" cells expressing ACKR3 with or without RAMP3

  • Stimulate with increasing ligand concentrations and measure cAMP production in reporter cells

  • Compare ligand potency (EC50) between conditions to detect scavenging activity

• Direct ligand binding and internalization assays:

  • Use fluorescently labeled ligands to measure binding and uptake

  • Compare cells expressing ACKR3 alone versus ACKR3 with RAMP3

  • Assess rate and extent of ligand internalization using flow cytometry or confocal microscopy

• Resensitization assays to measure functional consequences of receptor recycling:

  • Pre-treat cells with ligand to induce desensitization

  • Wash out ligand and allow varying recovery periods

  • Challenge with fresh ligand and measure response

  • Compare recovery kinetics between ACKR3 alone versus ACKR3-RAMP3

One study demonstrated that when CLR-RAMP3-EPAC reporter cells were co-cultured with ACKR3-RAMP3-expressing cells, there was a significant decrease in adrenomedullin potency compared to co-culture with non-scavenging control cells (EC50 shifted from 0.52 nM to 1.3 nM), indicating effective ligand scavenging by the ACKR3-RAMP3 complex .

What approaches can distinguish between RAMP3-dependent and independent receptor recycling?

To distinguish between RAMP3-dependent and independent receptor recycling pathways, researchers can employ several complementary approaches:

• Endosomal marker co-localization studies:

  • Express fluorescently tagged ACKR3 with and without RAMP3

  • Stimulate with ligand and track co-localization with different endosomal markers:

    • Rab4 (fast recycling endosomes)

    • Rab11 (slow recycling endosomes)

    • Rab7 (late endosomes/degradation pathway)

  • Quantify co-localization over time to determine receptor trafficking fate

• RAMP3 mutant studies:

  • Compare trafficking of receptors when co-expressed with wild-type RAMP3 versus RAMP3ΔPDZ (lacking the PDZ recognition motif)

  • This approach isolates the specific contribution of the RAMP3 PDZ motif to receptor sorting and recycling

• Receptor recycling kinetics measurements:

  • Use antibody feeding assays or reversible biotinylation techniques

  • Quantify the rate of receptor return to the plasma membrane after ligand-induced internalization

  • Compare kinetics between RAMP3-expressing and non-expressing cells

• Pharmacological and genetic inhibitors:

  • Use inhibitors of different recycling pathways (e.g., Rab4 or Rab11 dominant negatives)

  • Employ siRNA knockdown of components of recycling machinery

  • Determine whether RAMP3-dependent recycling uses distinct molecular machinery

Research has shown that ACKR3 expressed alone or with RAMP3ΔPDZ primarily traffics through Rab11-positive slow recycling endosomes, while ACKR3 co-expressed with wild-type RAMP3 preferentially sorts to Rab4-positive fast recycling endosomes . This distinction provides a clear marker for RAMP3-dependent versus independent recycling pathways.

How should researchers design experiments to detect functional consequences of RAMP3-GPCR interactions?

When designing experiments to detect functional consequences of RAMP3-GPCR interactions, researchers should consider multiple aspects of receptor biology:

• Receptor trafficking and localization:

  • Design pulse-chase experiments to track receptor movement after ligand stimulation

  • Use surface biotinylation or antibody feeding assays to specifically monitor internalization and recycling

  • Include appropriate temporal resolution (minutes to hours) to capture both rapid and slow trafficking events

• Ligand binding and scavenging:

  • Include co-culture systems that can detect changes in ligand availability

  • Design competitive binding assays to determine if RAMP3 alters receptor affinity for different ligands

  • Use gradient formation assays to assess functional consequences in cell migration models

• Signaling pathway analysis:

  • Test multiple signaling pathways (G-protein subtypes, β-arrestin recruitment, etc.)

  • Use biosensors (e.g., EPAC for cAMP) for real-time measurements

  • Consider that RAMP3 may affect signaling duration rather than amplitude through its effects on receptor recycling

• Controls and comparative analysis:

  • Include RAMP3ΔPDZ mutants to isolate PDZ-dependent effects

  • Compare RAMP3 effects to other RAMP family members (RAMP1, RAMP2)

  • Include receptor mutants that disrupt RAMP3 binding to confirm specificity

• Physiological relevance:

  • Validate findings in primary cells expressing endogenous levels of receptors and RAMPs

  • Design in vivo experiments that can detect consequences of RAMP3-GPCR interactions (e.g., angiogenesis assays)

Critical experimental parameters include ligand concentration (use dose-response curves rather than single concentrations), temporal dynamics (include multiple time points), and cell type (results may differ between overexpression systems and endogenous settings).

What considerations are important when comparing in vitro versus in vivo RAMP3 data?

When comparing in vitro and in vivo RAMP3 data, researchers should consider several important factors:

• Expression level differences:

  • In vitro overexpression systems often have much higher RAMP3 and receptor levels than physiological contexts

  • Quantify relative expression levels when possible and consider dose-dependent effects

  • Use inducible expression systems to test different RAMP3:receptor ratios

• Cellular context:

  • In vivo, multiple RAMP-interacting GPCRs may be expressed simultaneously in the same cell

  • Competition between receptors for limited RAMP3 could affect outcomes

  • Tissue-specific factors might influence RAMP3 function or localization

• Physiological ligand concentrations and gradients:

  • In vitro studies often use bolus ligand addition at concentrations higher than physiological levels

  • In vivo, cells experience shallow gradients of multiple ligands simultaneously

  • Microfluidic systems that generate defined gradients may better approximate in vivo conditions

• Relevant physiological endpoints:

  • Molecular mechanisms observed in vitro should be connected to physiological outcomes

  • The retinal angiogenesis defects observed in RAMP3-/- mice provide a key in vivo validation of mechanistic findings from cell culture experiments

  • Genetic models should include both global and conditional knockouts to distinguish cell-autonomous effects

• Translational considerations:

  • Differences between model organisms and humans in RAMP3 sequence, expression, and function

  • Species-specific differences in PDZ-domain interactions might affect trafficking outcomes

  • Pharmacological interventions targeting RAMP3-GPCR interfaces should be validated across species

The research on RAMP3 and ACKR3 provides a good example of effective translation between in vitro and in vivo approaches. The molecular mechanisms of RAMP3-dependent ACKR3 recycling identified in cell culture systems were validated through corresponding angiogenesis phenotypes in genetic mouse models .

How should experiments be designed to account for cell-type specific RAMP3 effects?

Designing experiments to account for cell-type specific RAMP3 effects requires careful consideration of multiple factors:

• Comprehensive cell type selection:

  • Include multiple relevant cell types (e.g., endothelial cells, immune cells, neurons)

  • Compare immortalized cell lines with primary cells derived from tissues of interest

  • Consider using patient-derived cells to capture human variation

• Endogenous expression profiling:

  • Quantify baseline expression of RAMP3 and potential interacting GPCRs in each cell type

  • Use qPCR, western blotting, and immunofluorescence to assess expression patterns

  • Consider single-cell RNA sequencing to identify cell populations with co-expression

• Manipulation strategies:

  • Use both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches

  • Employ CRISPR-Cas9 for genetic manipulation of endogenous loci

  • Consider inducible systems to control timing and level of expression

• Context-dependent signaling:

  • Test multiple functional readouts appropriate for each cell type

  • For endothelial cells, include migration, tube formation, and sprouting assays

  • For immune cells, assess chemotaxis and activation markers

  • For neurons, evaluate growth cone guidance and morphology

• Tissue microenvironment factors:

  • Consider culturing cells on different extracellular matrix components

  • Test effects of hypoxia, inflammatory mediators, and growth factors that may regulate RAMP3

  • Use co-culture systems to capture cell-cell interactions

In the context of RAMP3-ACKR3 interactions, research has demonstrated important cell-type specific functions in vascular endothelial cells during retinal angiogenesis . Similar approaches could be applied to investigate potential roles in other cell types where ACKR3 has established functions, such as cortical interneurons, immune cells, and cancer cells .

What are the implications of RAMP3 research for potential therapeutic development?

The expanding understanding of RAMP3 biology has several important implications for therapeutic development:

• RAMP3-GPCR interface as a drug target:

  • The molecular interface between RAMP3 and chemokine receptors could be targeted by small molecules or biologics

  • This approach could provide greater specificity than targeting the receptors directly

  • Precedent exists in the form of erenumab, an anti-CGRP migraine drug that targets the RAMP1-CLR interface

• Potential therapeutic areas:

  • Cancer: Targeting RAMP3-ACKR3 interactions could modulate CXCR4/CXCL12 signaling involved in cancer metastasis

  • Inflammation: Modulating chemokine receptor function through RAMP3 could provide novel anti-inflammatory approaches

  • Vascular biology: Based on the role in angiogenesis, RAMP3-targeted therapeutics might have applications in diseases with pathological vascularization

• Therapeutic modalities:

  • Small molecule inhibitors of RAMP3-GPCR interactions

  • Peptide or antibody-based biologics targeting the interface

  • Gene therapy approaches to modulate RAMP3 expression in specific tissues

• Pharmacological considerations:

  • Targeting receptor trafficking rather than signaling represents a novel therapeutic approach

  • Modulating RAMP3-dependent recycling could alter receptor availability and function without directly affecting ligand binding or signaling

  • This might allow for more subtle modulation of receptor systems compared to antagonists

• Challenges and considerations:

  • The widespread interactions of RAMP3 with chemokine receptors may present selectivity challenges

  • Tissue-specific expression and function of RAMP3 should inform therapeutic strategies

  • Species differences in RAMP3 sequence and function must be considered in translational research

The identification of RAMP3 interactions with 23 of 24 described chemokine receptors opens numerous potential therapeutic avenues . The authors note that "These findings identify unique and pharmacologically tractable avenues for the modulation of chemokine function in a wide range of physiological processes" .

What are the key unanswered questions in RAMP3 biology?

Despite significant advances in understanding RAMP3 function, several critical questions remain unanswered:

• Structural basis of RAMP3-GPCR interactions:

  • What structural features determine RAMP3 binding to different GPCRs?

  • How does RAMP3 binding alter receptor conformation and function?

  • Are there common structural motifs that mediate RAMP3 interactions across different GPCR families?

• Regulation of RAMP3 expression and function:

  • What factors regulate RAMP3 expression in different tissues?

  • Does RAMP3 undergo post-translational modifications that affect its function?

  • How is RAMP3 function regulated under different physiological and pathological conditions?

• Comprehensive interactome mapping:

  • Which PDZ domain-containing proteins interact with RAMP3?

  • Are there additional non-GPCR binding partners for RAMP3?

  • How does RAMP3 integrate into larger signaling complexes?

• Functional significance beyond receptor trafficking:

  • Does RAMP3 influence non-canonical signaling pathways?

  • How does RAMP3 affect receptor dimerization or oligomerization?

  • What is the evolutionary advantage of RAMP3's unique properties compared to other RAMP family members?

• Translational aspects:

  • What is the role of RAMP3 in human diseases?

  • Are there genetic variants of RAMP3 associated with disease susceptibility?

  • How can RAMP3-dependent mechanisms be targeted therapeutically?

The authors of the primary research suggest that "additional studies that employ a wide range of biochemical, pharmacological, and cellular assays to elucidate the effects that each RAMP has on the ligand binding, biased functional selectivity, or trafficking of other chemokine GPCRs should provide valuable future exploration and opportunity" .

How can advanced technologies advance RAMP3 research methodology?

Emerging technologies offer exciting opportunities to address complex questions in RAMP3 biology:

• Cryo-electron microscopy and structural biology:

  • Determine high-resolution structures of RAMP3-GPCR complexes

  • Characterize conformational changes induced by RAMP3 binding

  • Identify critical interaction interfaces for drug development

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize RAMP3-receptor complexes in nanoscale detail

  • Single-molecule tracking to monitor real-time dynamics of RAMP3-receptor interactions

  • Intravital microscopy to observe RAMP3-dependent processes in living organisms

• Proteomics and interactomics:

  • Proximity labeling approaches (BioID, APEX) to identify RAMP3 interaction partners

  • Temporal proteomics to track dynamic changes in RAMP3 complexes after stimulation

  • Cross-linking mass spectrometry to map interaction interfaces

• Genetic engineering approaches:

  • CRISPR-Cas9 genome editing to create endogenous tagged RAMP3 variants

  • Conditional and cell-type specific knockout models

  • Humanized mouse models expressing human RAMP3 variants

• Systems biology and computational approaches:

  • Mathematical modeling of RAMP3-dependent receptor trafficking

  • Integration of multi-omics data to understand RAMP3 function in complex biological systems

  • Virtual screening and molecular dynamics simulations to identify potential RAMP3-targeting compounds

The combination of these advanced technologies with established methods like BRET, confocal microscopy, and proximity ligation assays would provide unprecedented insights into RAMP3 biology and facilitate translation of basic research findings into therapeutic applications.

How might RAMP3 research inform our understanding of other receptor trafficking systems?

RAMP3 research provides important paradigms that could inform our understanding of other receptor trafficking systems:

• Molecular determinants of receptor fate:

  • The RAMP3 PDZ motif exemplifies how small molecular determinants can dramatically alter receptor trafficking fate

  • Similar sorting motifs might exist in other accessory proteins or receptors themselves

  • Understanding these determinants could allow precise manipulation of receptor trafficking

• Accessory protein-dependent trafficking:

  • RAMP3 represents a class of accessory proteins that modify receptor trafficking without altering signaling

  • This paradigm suggests that signaling and trafficking can be independently regulated

  • Similar accessory proteins might exist for other receptor families

• Context-dependent receptor recycling:

  • RAMP3 demonstrates how the same receptor (ACKR3) can follow different recycling pathways depending on its protein interaction partners

  • This suggests greater plasticity in receptor trafficking than previously appreciated

  • Similar context-dependent trafficking might occur with other receptor systems

• Physiological consequences of altered trafficking:

  • The defects in retinal angiogenesis observed in RAMP3-/- mice demonstrate how trafficking abnormalities translate to developmental phenotypes

  • Similar connections between trafficking and physiology likely exist for other receptor systems

  • This underscores the importance of studying receptor trafficking in physiologically relevant contexts

• Therapeutic targeting strategies:

  • The RAMP3-GPCR interface represents a novel target for modulating receptor function

  • Similar interfaces between receptors and trafficking regulators might be targetable

  • This expands the repertoire of potential drug targets beyond traditional binding sites

The study of RAMP3-dependent trafficking of ACKR3 provides a model for how accessory proteins can determine receptor fate and ultimately influence complex physiological processes like directed cell migration and tissue development .

Human RAMP3 research thus provides valuable insights that extend beyond its specific molecular interactions, offering broader lessons about receptor biology, trafficking mechanisms, and their physiological significance.