Utilisation of compounds from venoms in drug discovery.

Difficult drug targets are becoming the normal course of business in drug discovery, sometimes due to large interacting surfaces or only small differences in selectivity regions. For these, a different approach is merited: compounds lying somewhere between the small molecule and the large antibody in terms of many properties including stability, biodistribution and pharmacokinetics. Venoms have evolved over millions of years to be complex mixtures of stable molecules derived from other somatic molecules, the stability comes from the pressure to be ready for delivery at a moment's notice. Snakes, spiders, scorpions, jellyfish, wasps, fish and even mammals have evolved independent venom systems with complex mixtures in their chemical arsenal. These venom-derived molecules have been proven to be useful tools, such as for the development of antihypotensive angiotensin converting enzyme (ACE) inhibitors and have also made successful drugs such as Byetta® (Exenatide), Integrilin® (Eptifibatide) and Echistatin. Only a small percentage of the available chemical space from venoms has been investigated so far and this is growing. In a new era of biological therapeutics, venom peptides present opportunities for larger target engagement surface with greater stability than antibodies or human peptides. There are challenges for oral absorption and target engagement, but there are venom structures that overcome these and thus provide substrate for engineering novel molecules that combine all desired properties. Venom researchers are characterising new venoms, species, and functions all the time, these provide great substrate for solving the challenges presented by today's difficult targets.

REFERENCE INTERVALS FOR PLASMA BIOCHEMISTRY OF HEMOLYMPH IN SUBADULT CHILEAN ROSE TARANTULA (GRAMMASTOLA ROSEA) UNDER CHEMICAL RESTRAINT.

Tarantulas are a commonly kept species that are occasionally presented to veterinarians in exotic practice. A recent study on Grammastola rosea hemolymph biochemistry has been performed with nonanesthetized adult theraphosids. The objective of this study was to produce reference intervals for biochemistry biomarkers in hemolymph of chemically restrained G. rosea for use diagnostically by exotic veterinarians. Cardiac hemolymph collection was performed on 20 subadult tarantulas under general anesthesia with isoflurane. Samples were processed by a commercial laboratory. Statistics performed on the data include outlier exclusion, descriptive statistics, normality tests, and Pearson correlations. Reference intervals were made for total protein, creatine kinase (CK), aspartate aminotransferase (AST), glucose, uric acid (UA), calcium, and phosphorus. No cortisol was detected. The majority of the intervals produced were normally distributed with the exceptions of UA, phosphorus, and CK. Pearson correlation tests found several significant (P = <0.05) correlations between variables. The majority of the data displayed a normal distribution, unlike the previous study, with a greater number of replicates. The total protein, glucose, UA, calcium, and AST values generated were similar to those reported in the previous study. Conversely several variables such as phosphorus, CK, and albumin were not consistent with those previously reported. Evidence is presented for a lack of albumin, CK, and AST in Arachnida and thus previous data for these proteins is likely to be artifactual.

Global patterns of apparent copy number variation in birds revealed by cross-species comparative genomic hybridization.

There is a growing interest in copy number variation (CNV) and the recognition of its importance in phenotype, disease, adaptation and speciation. CNV data is usually ascertained by array-CGH within-species, but similar inter-species comparisons have also been made in primates, mice and domestic mammals. Here, we conducted a broad appraisal of putative cross-species CNVs in birds, 16 species in all, using the standard array-CGH approach. Using a chicken oligonucleotide microarray, we detected 790 apparent CNVs within 135 unique regions and developed a bioinformatic tool 'CNV Analyser' for analysing and visualising cross-species data sets. We successfully addressed four hypotheses as follows: (a) Cross-species CNVs (compared to chicken) are, as suggested from preliminary evidence, smaller and fewer in number than in mammals; this 'dogma' was rejected in the light of the new evidence. (b) CNVs in birds are likely to have a functional effect through an association with genes; a large proportion of detected regions (70 %) were indeed associated with genes (suggesting functional significance), however, not necessarily more so than in mammals. (c) There are more CNVs in birds with more rearranged karyotypes; this hypothesis was rejected. Indeed, Falco species contained fewer than most with relatively standard (chicken-like) karyotypes. (d) There are more CNVs per megabase on micro-chromosomes than macrochromosomes; this hypothesis was accepted. Indeed, in species with rearranged karyotypes characterised by chromosomal fusions, the fused former microchromosomes still 'behaved' as though they were their microchromosomal ancestors. Gene ontology analysis of CNVRs revealed enrichment in immune response and antigen presentation genes and five CNVRs were perfectly correlated with the unique loss of sexual dichromatism in one Galliformes species.

Indian Ornamental Tarantula (Poecilotheria regalis) Venom Affects Myoblast Function and Causes Skeletal Muscle Damage.

Envenomation by the Indian ornamental tarantula (Poecilotheria regalis) is medically relevant to humans, both in its native India and worldwide, where they are kept as pets. Muscle-related symptoms such as cramps and pain are commonly reported in humans following envenomation by this species. There is no specific treatment, including antivenom, for its envenomation. Moreover, the scientific knowledge of the impact of this venom on skeletal muscle function is highly limited. Therefore, we carried out this study to better understand the myotoxic properties of Poecilotheria regalis venom by determining its effects in cultured myoblasts and in the tibialis anterior muscle in mice. While there was no effect found on undifferentiated myoblasts, the venom affected differentiated multinucleated myotubes resulting in the reduction of fusion and atrophy of myotubes. Similarly, intramuscular administration of this venom in the tibialis anterior muscle in mice resulted in extensive muscle damage on day 5. However, by day 10, the regeneration was evident, and the regeneration process continued until day 20. Nevertheless, some tissue abnormalities including reduced dystrophin expression and microthrombi presence were observed on day 20. Overall, this study demonstrates the ability of this venom to induce significant muscle damage and affect its regeneration in the early stages. These data provide novel mechanistic insights into this venom-induced muscle damage and guide future studies to isolate and characterise individual toxic component(s) that induce muscle damage and their significance in developing better therapeutics.

Peripheral Arterial Thrombosis following Russell's Viper Bites.

Envenomings by Russell's viper ( Daboia russelii ), a species of high medical importance in India and other Asian countries, commonly result in hemorrhage, coagulopathies, necrosis, and acute kidney injury. Although bleeding complications are frequently reported following viper envenomings, thrombotic events occur rarely (reported only in coronary and carotid arteries) with serious consequences. For the first time, we report three serious cases of peripheral arterial thrombosis following Russell's viper bites and their diagnostic, clinical management, and mechanistic insights. These patients developed occlusive thrombi in their peripheral arteries and symptoms despite antivenom treatment. In addition to clinical features, computed tomography angiography was used to diagnose arterial thrombosis and ascertain its precise locations. They were treated using thrombectomy or amputation in one case that presented with gangrenous digits. Mechanistic insights into the pathology through investigations revealed the procoagulant actions of Russell's viper venom in standard clotting tests as well as in rotational thromboelastometry analysis. Notably, Russell's viper venom inhibited agonist-induced platelet activation. The procoagulant effects of Russell's viper venom were inhibited by a matrix metalloprotease inhibitor, marimastat, although a phospholipase A 2 inhibitor (varespladib) did not show any inhibitory effects. Russell's viper venom induced pulmonary thrombosis when injected intravenously in mice and thrombi in the microvasculature and affected skeletal muscle when administered locally. These data emphasize the significance of peripheral arterial thrombosis in snakebite victims and provide awareness, mechanisms, and robust strategies for clinicians to tackle this issue in patients.

An effective method for terrestrial arthropod euthanasia.

As scientific understanding of invertebrate life increases, so does the concern for how to end that life in an effective way that minimises (potential) suffering and is also safe for those carrying out the procedure. There is increasing debate on the most appropriate euthanasia methods for invertebrates as their use in experimental research and zoological institutions grows. Their popularity as pet species has also led to an increase in the need for greater veterinary understanding. Through the use of a local injection of potassium chloride (KCl) initially developed for use in American lobsters, this paper describes a safe and effective method for euthanasia in terrestrial invertebrates. Initial work focused on empirically determining the dose for cockroaches, which was then extrapolated to other arthropod species. For this method of euthanasia, we propose the term 'targeted hyperkalosis' to describe death through terminal depolarisation of the thoracic ganglia as a result of high potassium concentration.

Rapid development of a salivary calculus in submandibular gland and its potential causes in a young victim following Russell's viper bite.

Russell's viper bites are known to cause a range of haemotoxic, neurotoxic, myotoxic, cytotoxic and nephrotoxic complications. However, the impact of Russell's viper bites as well as bites from other venomous snakes on sialolithiasis has not been previously reported. Here, we present an interesting case where a Russell's viper bite induced the rapid development of a calculus in submandibular gland in a 10-year-old boy. Upon admission, the victim did not show any symptoms of swelling and/or pain around his oral cavity. He received antivenom treatment to normalise his coagulation parameters, however, on day three he developed swelling and extreme pain around his right mandibular region. An ultrasound investigation revealed the presence of a calculus in his submandibular gland, which was removed using a minor surgical procedure. The histopathological examination revealed this as a poorly calcified salivary calculus, which is composed of cell debris, mucopolysaccharides and lipids. The mechanisms behind its rapid development following a snakebite are unclear although this could be linked to excessive inflammation or modifications to the composition of saliva induced by venom toxins or other unknown factors. This report reveals an unusual complication induced by a Russell's viper bite and alerts clinicians who treat snakebites to be aware of such envenomation effects. Moreover, this will lead to novel research to explore the relationship between venom toxins and functions of salivary glands.

Full Spectrum Lighting Induces Behavioral Changes and Increases Cortisol Immunoreactivity in Captive Arachnids.

The use of full spectrum illumination, including ultraviolet (UV), during captive husbandry of arachnids is common practice. The effect of this on captive arachnids has not been previously investigated. Comparison of key behavioral changes and hemolymph cortisol immunoreactivity was undertaken with and without full spectrum lighting. King baboon spiders, Pelinobius muticus and Indian giant scorpions, Heterometrus swammerdami were selected for the study. Both organisms spent all their time hidden when exposed to full spectrum light compared to low-level ambient light except for one instance. There was no significant difference in burrowing and webbing in P. muticus when exposed to full spectrum lighting. There was a decrease in the number of behaviors or postures expressed in full spectrum lighting compared to ambient light for both species. Cortisol immunoactivity of both species were significantly elevated after exposure to full spectrum lighting. This study provides the first evidence of detectable cortisol immunoactivity in arachnid hemolymph. These levels changed in response to full spectrum illumination and were linked to behavioral changes. This suggests that a common husbandry practice may be detrimental to arachnids.

Priapism following a juvenile Russell's viper bite: An unusual case report.

Following a bite from a juvenile Russell's viper (Daboia russelii), a priapism (painful erection) developed rapidly in a 16-year-old male and only subsided after administration of antivenom 3 hours later. Potential mechanisms for this snakebite-induced priapism are unclear but likely due to venom toxins causing nitric oxide (NO) release and subsequent vasodilation of endothelium in the corpus cavernosum, although the possible involvement of other mechanisms cannot be ruled out. We strongly believe that this unusual case report may lead to further scientific research in order to improve the clinical understanding of the pathophysiology of envenomation due to Russell's viper bites. Although it is too early to speculate, further research may also discover the possibilities of developing venom-based candidate molecules to treat sexual dysfunction in males and females.

Splenic rupture and subsequent splenectomy in a young healthy victim following Russell's viper bite.

Splenic rupture and/or splenectomy is/are not uncommon in clinical arena. Here we present this case of extensive haemorrhage-induced splenic rupture which resulted in splenectomy in a young healthy male (who did not have any previous medical conditions) following a Russell's viper bite. He developed upper abdominal and shoulder pain on his left side along with hypotension and reduced level of haemoglobin on the third day following bite despite antivenom treatment. Following confirmation of splenic rupture and haemoperitoneum by ultrasound and computed tomography scans, an emergency splenectomy was performed using laparotomy. Although Russell's viper bites are known to induce bleeding complications, splenic rupture due to haemorrhage in spleen has not been previously reported. Russell's viper venom toxins such as metalloproteases, serine proteases and phospholipase A2 might have affected the vascular permeability resulting in excessive bleeding and increased pressure in the spleen leading to rupture. Further investigations are required to underpin the impact of snake venom toxins on the architecture and functions of spleen. However, the clinicians who treat snakebites should be aware of this type of rare complications so as to provide appropriate management for such victims.

The Failures of Ethnobotany and Phytomedicine in Delivering Novel Treatments for Snakebite Envenomation.

Snakebite envenomation (SBE) is a high-priority, neglected tropical disease. This devastating occupational health hazard disproportionately affects rural farming communities in tropical countries. This is exacerbated by the distribution and densities of venomous snakes, incidence of encounters, and limited access to advanced healthcare, including antivenom. Before the development of antivenom, desperation and spiritual beliefs led patients to experiment with a wide range of traditional treatments. Many of these treatments still survive today, particularly in regions where access to healthcare is limited. Plants are a major source of bioactive molecules, including several lifesaving medications that are widely used to this day. However, much of the research into the use of traditional plant treatments for SBE are limited to preliminary analysis or have focused on techniques used to confirm antibody efficacy that are not suitable for non-antibody-containing treatments. Modern drugs are developed through a robust pharmaceutical drug discovery and development process, which applies as much to SBE as it does to any other disease. This review discusses specifically why research into ethnobotanical practices has failed to identify or develop a novel treatment for SBE and proposes specific approaches that should be considered in this area of research in the future.

Kinome scale profiling of venom effects on cancer cells reveals potential new venom activities.

The search for novel and relevant cancer therapeutics is continuous and ongoing. Cancer adaptations, resulting in therapeutic treatment failures, fuel this continuous necessity for new drugs to novel targets. Recently, researchers have started to investigate the effect of venoms and venom components on different types of cancer, investigating their mechanisms of action. Receptor tyrosine kinases (RTKs) comprise a family of highly conserved and functionally important druggable targets for cancer therapy. This research exploits the novelty of complex venom mixtures to affect phosphorylation of the epidermal growth factor receptor (EGFR) and related RTK family members, dually identifying new activities and unexplored avenues for future cancer and venom research. Six whole venoms from diverse species taxa, were evaluated for their ability to illicit changes in the phosphorylated expression of a panel of 49 commonly expressed RTKs. The triple negative breast cancer cell line MDA-MB-468 was treated with optimised venom doses, pre-determined by SDS PAGE and Western blot analysis. The phosphorylated expression levels of 49 RTKs in response to the venoms were assessed with the use of Human Phospho-RTK Arrays and analysed using ImageLab 5.2.1 analysis software (BioRad). Inhibition of EGFR phosphorylation occurred with treatment of venom from Acanthoscurria geniculata (Theraphosidae), Heterometrus swammerdami (Scorpionidae), Crotalus durissus vegrandis (Crotalidae) and Naja naja (Elapidae). Western green mamba Dendroaspis viridis venom increased EGFR phosphorylation. Eph, HGFR and HER were the most affected receptor families by venoms. Whilst the importance of these changes in terms of effect on MDA-MB-468 cells' long-term viability and functionality are still unclear, the findings present exciting opportunities for further investigation as potential drug targets in cancer and as tools to understand better how these pathways interact.

Venomous snakebites: Rapid action saves lives-A multifaceted community education programme increases awareness about snakes and snakebites among the rural population of Tamil Nadu, India.

The lack of public awareness surrounding the dangers of snakebite envenomation (SBE) is one of the most critical factors contributing to SBE-induced complications, and subsequently exacerbating the number of deaths and disabilities resulting from SBE. In this study, we deployed a multifaceted community education programme to educate students, healthcare professionals and members of the public in rural areas of Tamil Nadu, India about the dangers of SBE, appropriate first aid measures and the 'do's and don'ts' following a snakebite. An assessment of prior knowledge within these communities identified several misconceptions concerning snakes and SBE. Using a combination of direct engagement (estimated to reach over 200,000 people), information leaflets (200,000 distributed), posters, video documentaries, media and social media (>2.8 million engagements), over the course of one year (January to December 2019) we reached over 3 million people in rural Tamil Nadu (around 8% of population). Evaluation of community-based assemblies indicated that at least 90% of attendees were able to recall the key messages at the end of the events, and at least 85% were able to recall the key messages even after 12 months. Due to high demand, a one-day symposium was organised to provide clinical knowledge and training on SBE to 250 healthcare professionals in rural Tamil Nadu. Notably, an assessment of patient data (291 victims) collected from a snakebite referral hospital over the same 12-month period (2019) indicated that arrival time at hospital following a snakebite was significantly faster and the effective first aid measures were administered to patients who were aware of our activities compared to those that were not. Overall, our approach provides a framework on how to educate rural communities about the dangers of SBE and thereby, mitigate delayed SBE treatment leading to an overall reduction in SBE-induced mortality, morbidity, treatment costs and other socio-economic ramifications.

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 µM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

The Urgent Need to Develop Novel Strategies for the Diagnosis and Treatment of Snakebites.

Snakebite envenoming (SBE) is a priority neglected tropical disease, which kills in excess of 100,000 people per year. Additionally, many millions of survivors also suffer through disabilities and long-term health consequences. The only treatment for SBE, antivenom, has a number of major associated problems, not least, adverse reactions and limited availability. This emphasises the necessity for urgent improvements to the management of this disease. Administration of antivenom is too frequently based on symptomatology, which results in wasting crucial time. The majority of SBE-affected regions rely on broad-spectrum polyvalent antivenoms that have a low content of case-specific efficacious immunoglobulins. Research into small molecular therapeutics such as varespladib/methyl-varespladib (PLA2 inhibitors) and batimastat/marimastat (metalloprotease inhibitors) suggest that such adjunctive treatments could be hugely beneficial to victims. Progress into toxin-specific monoclonal antibodies as well as alternative binding scaffolds such as aptamers hold much promise for future treatment strategies. SBE is not implicit during snakebite, due to venom metering. Thus, the delay between bite and symptom presentation is critical and when symptoms appear it may often already be too late to effectively treat SBE. The development of reliable diagnostical tools could therefore initiate a paradigm shift in the treatment of SBE. While the complete eradication of SBE is an impossibility, mitigation is in the pipeline, with new treatments and diagnostics rapidly emerging. Here we critically review the urgent necessity for the development of diagnostic tools and improved therapeutics to mitigate the deaths and disabilities caused by SBE.

The emerging field of venom-microbiomics for exploring venom as a microenvironment, and the corresponding Initiative for Venom Associated Microbes and Parasites (iVAMP).

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date with the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remaining largely unknown. We highlight the potential for the discovery of venom microbiomes within the adaptive landscape of venom systems. The considerable number of convergently evolved venomous animals, juxtaposed with the comparatively few known studies to identify microbial communities in venom, provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics (i.e., venom-microbiomics) and introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing collaborative consortium. We express commitment to the diversity, inclusion and scientific collaboration among researchers interested in this emerging subdiscipline through expansion of the iVAMP consortium.

The effect of deletion of the orphan G - protein coupled receptor (GPCR) gene MrgE on pain-like behaviours in mice.

BACKGROUND: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated. RESULTS: The absence of MrgE had no effect on the development of pain responses to a noxious chemical stimulus or an acute thermal stimulus. However, in contrast, the development but not the maintenance of neuropathic pain was affected by deletion of MrgE. The expression of Mrg genes was not significantly affected in the MrgE knockout (KO) mice with the sole exception of MrgF. In addition, the expression of 77 of 84 genes involved in sensory neuron development and function was also unaffected by deletion of MrgE. Of the 7 genes affected by MrgE deletion, 4 have previously been implicated in nociception. CONCLUSION: The data suggests that MrgE may play a role in selective pain behavioural responses in mice.

Impact of Naja nigricollis Venom on the Production of Methaemoglobin.

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin.

Venom: the sharp end of pain therapeutics.

Adequate pain control is still a significant challenge and largely unmet medical need in the 21st century. With many small molecules failing to reach required levels of potency and selectivity, drug discovery is once again turning to nature to replenish pain therapeutic pipelines. Venomous animals are frequently stereotyped as inflictors of pain and distress and have historically been vilified by mankind. Yet, ironically, the very venoms that cause pain when directly injected by the host animal may actually turn out to contain the next generation of analgesics when injected by the clinician. The last 12 months have seen dramatic discoveries of analgesic tools within venoms. Spiders, snakes and even centipedes are yielding peptides with immense therapeutic potential. Significant advances are also taking place in delivery methods that can improve bioavailability and pharmacokinetics of these exciting natural resources. Turning proteinaceous venom into pharmaceutical liquid gold is the goal of venomics and the focus of this article.

Interactions between the Mas-related receptors MrgD and MrgE alter signalling and trafficking of MrgD.

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to beta-alanine but not L-alanine by elevating intracellular [Ca(2+)], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to beta-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of beta-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of beta-alanine to elevate intracellular [Ca(2+)], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted beta-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.