[Selective signaling pathway via feeding-related ciliary GPCR, melanin-concentrating hormone receptor 1].

G-protein-coupled receptors (GPCRs), which constitute a highly diverse family of seven transmembrane receptors, respond to external signals and regulate a variety of cellular and physiological processes. GPCRs are encoded by about 800 different genes in human and they represent the largest family of drug targets in clinical trials, which accounts for about 30% of approved drugs acting on 108 unique GPCRs. Signaling through GPCRs can be optimized by enriching receptors, selective binding partners, and downstream effectors in discrete cellular environment. The primary cilium is a ubiquitous organelle that functions as a sensory antenna for surrounding physical and chemical stimuli. Primary cilium's compartment is as little as 1/10,000th of the total cell volume. Therefore, the ciliary membrane is highly enriched for specific signaling molecules, allowing the primary cilium to organize signaling in a highly ordered microenvironment. Recently, a set of non-olfactory GPCRs such as somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1) have been found to be selectively targeted to cilia on several mammalian cell types including neuronal cells both in vitro and in vivo approaches. Moreover, investigations into the pathophysiology have implicated GPCR ciliary signaling in a number of developmental and cellular pathways. Thus, cilia are now considered as an increasingly important connection for GPCR signaling. This review summarizes our current understanding of the signaling pathways though ciliary GPCR, especially feeding- and mood-related GPCR MCHR1, along with specific biological phenomenon as cilia length shortening.

Central melanin-concentrating hormone influences liver and adipose metabolism via specific hypothalamic nuclei and efferent autonomic/JNK1 pathways.

BACKGROUND & AIMS: Specific neuronal circuits modulate autonomic outflow to liver and white adipose tissue. Melanin-concentrating hormone (MCH)-deficient mice are hypophagic, lean, and do not develop hepatosteatosis when fed a high-fat diet. Herein, we sought to investigate the role of MCH, an orexigenic neuropeptide specifically expressed in the lateral hypothalamic area, on hepatic and adipocyte metabolism. METHODS: Chronic central administration of MCH and adenoviral vectors increasing MCH signaling were performed in rats and mice. Vagal denervation was performed to assess its effect on liver metabolism. The peripheral effects on lipid metabolism were assessed by real-time polymerase chain reaction and Western blot. RESULTS: We showed that the activation of MCH receptors promotes nonalcoholic fatty liver disease through the parasympathetic nervous system, whereas it increases fat deposition in white adipose tissue via the suppression of sympathetic traffic. These metabolic actions are independent of parallel changes in food intake and energy expenditure. In the liver, MCH triggers lipid accumulation and lipid uptake, with c-Jun N-terminal kinase being an essential player, whereas in adipocytes MCH induces metabolic pathways that promote lipid storage and decreases lipid mobilization. Genetic activation of MCH receptors or infusion of MCH specifically in the lateral hypothalamic area modulated hepatic lipid metabolism, whereas the specific activation of this receptor in the arcuate nucleus affected adipocyte metabolism. CONCLUSIONS: Our findings show that central MCH directly controls hepatic and adipocyte metabolism through different pathways.

The role of melanin-concentrating hormone in conditioned reward learning.

The orexigenic neuropeptide melanin-concentrating hormone (MCH) is well positioned to play a key role in connecting brain reward and homeostatic systems due to its synthesis in hypothalamic circuitry and receptor expression throughout the cortico-striatal reward circuit. Here we examined whether targeted-deletion of the MCH receptor (MCH-1R) in gene-targeted heterozygote and knockout mice (KO), or systemic treatment with pharmacological agents designed to antagonise MCH-1R in C57BL/6J mice would disrupt two putative consequences of reward learning that rely on different neural circuitries: conditioned reinforcement (CRf) and Pavlovian-instrumental transfer (PIT). Mice were trained to discriminate between presentations of a reward-paired cue (CS+) and an unpaired CS-. Following normal acquisition of the Pavlovian discrimination in all mice, we assessed the capacity for the CS+ to act as a reinforcer for new nose-poke learning (CRf). Pharmacological disruption in control mice and genetic deletion in KO mice impaired CRf test performance, suggesting MCH-1R is necessary for initiating and maintaining behaviors that are under the control of conditioned reinforcers. To examine a dissociable form of reward learning (PIT), a naïve group of mice were trained in separate Pavlovian and instrumental lever training sessions followed by the PIT test. For all mice the CS+ was capable of augmenting ongoing lever responding relative to CS- periods. These results suggest a role for MCH in guiding behavior based on the conditioned reinforcing value of a cue, but not on its incentive motivational value.

Cloning, phylogenetic analysis and expression of somatolactin and its receptor in Cichlasoma dimerus: their role in long-term background color acclimation.

Somatolactin (SL) and SL receptor (SLR) belong to the growth hormone and cytokine type I receptor superfamilies, respectively. However, further research is required to define the duplications and functions of SL and its receptors in basal vertebrates including environmental background color adaptation in fish. In the present study, we cloned and sequenced SL and its putative receptor (SLR), classified and compared the sequences phylogenetically, and determined SL and SLR mRNA expression levels during long-term background color exposure in Cichlasoma dimerus, a freshwater South American cichlid. Our results show that C. dimerus SL and SLR share high sequence similarity with homologous from other perciform fish. Phylogenetic analysis indicates that C. dimerus SL belongs to the SLα clade sub-group. C. dimerus SLR is clearly a member of the GHR1 receptor subgroup, which includes the experimentally validated SLR from salmonids. Higher transcript levels of SLα in the pituitary and SLR in the epidermis and dermis cells of fish scales were observed in fish following long-term black background color exposure compared to those exposed to a white background. A higher number of melanophores was also observed in fish exposed for 10days to a black background compared to those exposed to a white background. These changes were concomitant to differences in SL or SLR transcript levels found in fish exposed to these two different background colors. Our results suggest, for the first time, that SLR is expressed in fish scales, and that there is an increase in SL in the pituitary and the putative SLR in likely target cells, i.e., melanophores, in long-term black background exposure in C. dimerus.

A preliminary investigation of the role of melanin-concentrating hormone (MCH) and its receptors in appetite regulation of winter flounder (Pseudopleuronectes americanus).

In order to better understand the role of melanin-concentrating hormone (MCH) in the regulation of appetite in fish, the mRNAs of two forms of MCH, prepro-MCH and MCH2, and two forms of MCH receptors, MCH-R1 and MCH-R2, were isolated from winter flounder (Pseudopleuronectes americanus). In addition, the mRNA expressions of these peptides and their receptors were determined under fed and fasted conditions. Both MCHs are expressed in forebrain and midbrain, as well as peripheral tissues including gut and gonads. Both MCH-Rs are ubiquitously expressed in the brain and periphery. Fasting induced an increase in the expression levels of MCH and MCH-R1 mRNAs in optic tectum/thalamus and hypothalamus but had no effect on either MCH2 or MCH-R2 mRNA expressions. Our results suggest that MCH and MCH-R1, but not MCH2 and MCH-R2 might have a role in the regulation of appetite in flounder.

Ayurvedic genomics, constitutional psychology, and endocrinology: the missing connection.

A recent methodological approach for human classification, diagnosis, and therapeutics through the combination of current Western constitutional psychology somatotypes and traditional Indian medicine (prakriti) body types and mind (manas) is herein presented. The striking similarities between psychologic somatotypes and Indian medicine body types permits proposal of a finite genopsycho-somatotyping of humans. Genopsycho-somatotyping of humans consists of a set of common physiologic, physical, and psychologic attributes related to a common basic birth constitution that remains somewhat permanent during human lifetime, since it is proposed that this birth constitution is programmed in the person's DNA (genes). This mainly provides a tool for classifying the human population based on broad and finite phenotype clusters across different ethnicity, languages, geographical location, or self-reported ancestry. In spite of any social or environmental traumatic event, I propose for males that every basic constitution has an associated identification organ, a measured property or marker, a soma, and some psyche general tendencies suggesting specific behavior or recurrent conduct. Three (3) basic extreme genopsycho-somatotypes or birth constitutions are enunciated: mesomorphic or andrus (Pitta), endomorphic or thymus (Khapa), and ectomorphic or thyrus (Vata). The method further predicts that male andrus constitution across races shares similarities in androgen (An) nuclear receptor behavior, whereas thymus constitutions are mainly regulated by T-cells (Tc) nuclear receptor behavior. Moreover, it suggests that thyrus constitutions share similarities in thyroxine (Th) nuclear receptor behavior. These proposed nuclear receptors are expected to regulate the expression of specific genes, thereby controlling the embryonic development, adult homeostasis, and metabolism of the human organism in a very profound way. The method finally predicts small differences in measured property (An, Tc, and Th nuclear receptors behavior) within a birth constitution across different races to be expected by modulation effects in melanocyte-stimulating hormone receptor behavior.

The hinge region: an important receptor component for GPHR function.

Glycoprotein hormone receptors (GPHRs) are members of the seven-transmembrane-spanning receptor family characterized by a large ectodomain. The hinge region belongs to a part of the GPHR ectodomain for which the three-dimensional structure has not yet been deciphered, leaving important questions unanswered concerning ligand binding and GPHR activation. Recent publications indicate that specific residues of the hinge region mediate hormone binding, receptor activation and/or intramolecular signaling for the three GPHRs, emphasizing the importance of this region. Based on these findings, the hinge region is involved at least in part in hormone binding and receptor activation. This review summarizes functional data regarding the hinge region, demonstrating that this receptor portion represents a link between ligand binding and subsequent GPHR activation.

Electrophysiological effects of MCH on neurons in the hypothalamus.

Melanin concentrating hormone (MCH) has been implicated in many brain functions and behaviors essential to the survival of animals. The hypothalamus is one of the primary targets where MCH-containing nerve fibers and MCH receptors are extensively expressed and its actions in the brain are exerted. Since the identification of MCH receptors as orphan G protein coupled receptors, the cellular effects of MCH have been revealed in many non-neuronal expression systems (including Xenopus oocytes and cell lines), however, the mechanism by which MCH modulates the activity in the neuronal circuitry of the brain is still under investigation. This review summarizes our current knowledge of electrophysiological effects of MCH on neurons in the hypothalamus, particularly in the lateral hypothalamus. Generally, MCH exerts inhibitory effects on neurons in this structure and may serve as a homeostatic regulator in the lateral hypothalamic area. Given the contrast between the limited data on cellular functions of MCH in the hypothalamus versus a fast growing body of evidence on the vital role of MCH in animal behavior, further investigations of the former are warranted.

The effects of stressful stimuli and hypothalamic-pituitary-adrenal axis activation are reversed by the melanin-concentrating hormone 1 receptor antagonist SNAP 94847 in rodents.

Melanin-concentrating hormone (MCH) is an orexigenic and dipsogenic neuropeptide that has been reported to mediate acute behavioral and neuroendocrine stress-related responses via MCH(1) receptor activation in rodents. The purpose of the present investigation was to use the MCH(1) receptor antagonist SNAP 94847 (N-(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperidin-4-yl}-4-methyl-phenyl)-isobutyramide) to determine the effects of MCH(1) receptor blockade on MCH-evoked adrenocorticotropic hormone (ACTH) release, chronic mild stress-induced anhedonia, stress-induced hyperthermia and forced swim stress-induced immobility. The appropriate dose range for testing SNAP 94847 was determined by measuring MCH-evoked water drinking. The corresponding occupancy of MCH(1) receptors in rat striatum was also measured across a broad dose range. Orally administered (p.o.) SNAP 94847 (1-10 mg/kg) corresponds to 30-60% occupancy at MCH(1) receptors and significantly blocks water drinking induced by the intracerebroventricular (i.c.v.) injection of MCH. MCH (i.c.v.) significantly elevates plasma levels of ACTH in rats, and SNAP 94847 (2.5 mg/kg, p.o.) blocks MCH-evoked ACTH release. Using the chronic mild stress paradigm, we show that repeated daily exposure to environmental stressors for 5 weeks significantly suppresses sucrose intake in rats, and that SNAP 94847 (1 mg/kg, BID) for 1-5 weeks restores baseline sucrose intake. Moreover, a single administration of SNAP 94847 attenuates stress-induced hyperthermia and the behavioral effects of forced swim stress with minimal effective doses of 2.5 and 30 mg/kg (p.o.), respectively. The regulation of ACTH release and reversal of the effects of chronic and acute stress by SNAP 94847 are suggestive of a role for MCH(1) receptor blockade in the treatment of disorders characterized by high allostatic load.

A sea lamprey glycoprotein hormone receptor similar with gnathostome thyrotropin hormone receptor.

The specificity of the vertebrate hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes is explained by the evolutionary refinement of the specificity of expression and selectivity of interaction between the glycoprotein hormones GpH (FSH, LH, and TSH) and their cognate receptors GpH-R (FSH-R, LH-R, and TSH-R). These two finely tuned signaling pathways evolved by gene duplication and functional divergence from an ancestral GpH/GpH-R pair. Comparative analysis of the protochordate and gnathostome endocrine systems suggests that this process took place prior or concomitantly with the emergence of the gnathostome lineage. Here, we report identification and characterization of a novel glycoprotein hormone receptor (lGpH-R II) in the Agnathan sea lamprey. This 781 residue protein was found approximately 43% identical with mammalian TSH-R and FSH-R representative sequences, and similarly with these two classes of mammalian receptors it is assembled from ten exons. A synthetic ligand containing the lamprey glycoprotein hormone beta-chain tethered upstream of a mammalian alpha-chain activated the lGpH-R II expressed in COS-7 cells but in a lesser extent than lGpH-R I. Molecular phylogenetic analysis of vertebrate GpH-R protein sequences suggests a closer relationship between lGpH-R II and gnathostome thyrotropin receptors. Overall, the presence and characteristics of the lamprey glycoprotein hormone receptors suggest existence of a primitive functionally overlapping glycoprotein hormone/glycoprotein hormone receptor system in this animal.

Physiological arousal: a role for hypothalamic systems.

The lateral hypothalamus (LH) has long been known as a homeostasis center of the brain that modulates feeding behavior, arousal and reward. The hypocretins (Hcrts, also called orexins) and melanin-concentrating hormone (MCH) are neuropeptides produced in two intermingled populations of a few thousand neurons in the LH. The Hcrts have a prominent role in regulating the stability of arousal, since Hcrt system deficiency leads to narcolepsy. MCH is an important modulator of energy balance, as MCH system deficiency in mice leads to leanness and increased metabolism. Recently, MCH has been proposed to modulate rapid eye movement sleep in rodents. In this review, we propose a working model of the cross-talk between Hcrt and MCH circuits that may provide an arousal balance system to regulate complex goal-oriented behaviors.

Retention of genes involved in the adenohypophysis-mediated endocrine system in early vertebrates.

The adenohypophysis of vertebrates receives peptide hormones from the hypothalamus and secretes hormones that regulate diverse physiologic processes in peripheral organs. The adenohypophysis-mediated endocrine system is widely conserved across vertebrates but not invertebrates. Phylogenetic analysis indicates that the emergence of this system coincided with two rounds of whole-genome duplication (2R-WGD) in early vertebrates, but direct evidence linking these events has been unavailable. We detected all human paralogons (series of paralogous regions) formed in early vertebrates as traces of 2R-WGD, and examined the relationship between 2R-WGD and the evolution of genes essential to the adenohypophysis-mediated endocrine system. Regarding genes encoding transcription factors (TFs) involved in the terminal differentiation into hormone-secreting cells in adenohypophyseal development, we showed that most pairs of these genes and their paralogs were part of paralogons. In addition, our analysis also indicated that most of the paralog pairs in families of adenohypophyseal hormones and their receptors were part of paralogons. These results suggest that 2R-WGD played an important role in generating genes encoding adenohypophyseal TFs, hormones, and their receptors for increasing the diversification of hormone repertoire in the adenohypophysis-mediated endocrine system of vertebrates.

Microinjections of melanin concentrating hormone into the nucleus tractus solitarius of the rat elicit depressor and bradycardic responses.

The presence of melanin-concentrating hormone (MCH) containing processes, projecting from the lateral hypothalamus to the medial nucleus tractus solitarius (mNTS), has been reported in the rat. It was hypothesized that MCH acting within the mNTS may modulate the central regulation of cardiovascular function. This hypothesis was tested in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of MCH (0.25, 0.5, 0.75, and 1 mM) into the mNTS of anesthetized rats elicited decreases in mean arterial pressure (20.4+/-1.6, 50.7+/-3.3, 35.7+/-2.8 and 30.0+/-2.6 mm Hg, respectively). The decreases in heart rate in response to these concentrations of MCH were 40.0+/-8.7, 90.0+/-13.0, 48.0+/-7.3 and 48.0+/-8.0 beats/min, respectively. Maximum cardiovascular responses were elicited by a 0.5 mM concentration of MCH. Cardiovascular responses to MCH were similar in unanesthetized mid-collicular decerebrate rats. Control microinjections of normal saline (100 nl) did not elicit any cardiovascular response. Ipsilateral or bilateral vagotomy significantly attenuated MCH-induced bradycardia. Prior microinjections of PMC-3881-PI (2 mM; MCH-1 receptor antagonist) into the mNTS blocked the cardiovascular responses to microinjections of MCH. Microinjection of MCH (0.5 mM) into the mNTS decreased efferent greater splanchnic nerve activity. Direct application of MCH (0.5 mM; 4 nl) to barosensitive nucleus tractus solitarius (NTS) neurons increased their firing rate. These results indicate that: 1) MCH microinjections into the mNTS activate MCH-1 receptors and excite barosensitive NTS neurons, causing a decrease in efferent sympathetic activity and blood pressure, and 2) MCH-induced bradycardia is mediated via the activation of the vagus nerves.

The melanin-concentrating hormone receptor 2 (MCH-R2) mediates the effect of MCH to control body color for background adaptation in the barfin flounder.

Melanin-concentrating hormone (MCH) is a neuropeptide generated in neurons originating in the hypothalamus, from which axons project to the entire brain and neurohypophysis in fish. MCH has both central and peripheral roles such as food intake and body color change. Here we cloned two MCH receptors (MCH-R) from the barfin flounder, Verasper moseri, Pleuronectiformes. The phylogenetic analysis shows that these are orthologues to the mammalian MCH-R1 and MCH-R2 showing 49 and 30% amino acid sequence identity to the corresponding human receptors while they have 31% amino acid sequence identify between them. Essential amino acid residues for ligand binding, signal transduction and receptor conformation, which have been shown in mammalian MCH-R, are well conserved in the flounder MCH-Rs. MCH-R1 has one intron in the extracellular N-terminal region and MCH-R2 has one intron in the DRY motif, which is a homologous position to one of the five introns of human MCH-R2. Orthologues of MCH-R1 and MCH-R2 may have appeared by gene duplication of the ancestry of MCH-Rs having at least two introns, and then MCH-R1 and MCH-R2 inherited different introns in flounder strains. We also determined their tissue distribution and functional role in rearing condition. Reverse transcription PCR revealed that the expression of MCH-R1 is confined to the brain of the barfin flounder, while transcripts of MCH-R2 were detected in the brain, pituitary, eyeball, gill, atrium, ventricle, head kidney, body kidney, spleen, intestine, inclinator, skeletal muscle testis, ovary, eyed-side skin, and non-eyed-side skin. The expression of MCH-R2 in eyed-side skin was higher in fish reared in a black tank (121 days) than in a white tank while the expression levels of MCH in the brain were significantly greater in the group reared with the white background suggesting down-regulation of this receptor gene with increased levels of MCH. The results suggest that the MCH-R2 mediates the effect of MCH to control body color for background adaptation in the eyed-side skin of the barfin flounder.

Adrenocorticotropin hormone stimulates interleukin-18 expression in human HaCaT keratinocytes.

The hypothalamic-pituitary-adrenal (HPA) axis is activated by stress. This involves the production of corticotropin releasing hormone (CRH) with the subsequent release of proopiomelanocortin (POMC) peptides, of which adrenocorticotropin hormone (ACTH) is most important. Although the skin has the capacity to produce CRH and POMC peptides, the immunomodulatory roles of ACTH in skin are yet unknown. IL-18 has been known to affect cells involved in the inflammatory response. In this study, we aimed to identify the regulatory effect of ACTH on IL-18 expression of skin keratinocytes. Exposure of HaCaT cells to ACTH stimulated formation of IL-18 mRNA transcript and its protein products in a dose-dependent manner. Furthermore, we suggest that ACTH-induced IL-18 production is via the caspase-1 activation pathway, as IL-18 production induced by ACTH could be suppressed by caspase-1 inhibitor, and ACTH could increase caspase-1 activity. The effect of ACTH on IL-18 production was blocked by specific inhibitors of p38 kinase (SB203580) or extracellular signal-regulated kinase (ERK) (PD98059). In addition, ACTH-induced rapid phosphorylation of p38 kinase and ERK, and ACTH signaling occurred via melanocortin receptor 1 (MC1R) and receptor 2 (MC2R). These results suggest that ACTH stimulates IL-18 expression in human keratinocytes, which provides an insight into the interaction between ACTH and inflammatory mediators.

Identification of melanin-concentrating hormone receptor and its impact on drug discovery.

The neuropeptide melanin-concentrating hormone (MCH) was originally isolated from the pituitary of salmon, in which it causes skin paling. MCH is also found abundantly in mammalian neurons, and has been detected in the lateral hypothalamus and zona incerta, brain regions that are at the center of feeding behavior. Acute central administration of MCH leads to a rapid and significant increase in food intake, while MCH expression changes in states of altered energy balance, such as fasting and obesity. Furthermore, MCH knockout mice tend toward hypophagia and leanness. In 1999, we and four other groups identified an orphan G-protein-coupled receptor (GPCR) as a specific receptor for MCH (MCH-1 receptor). Although a second MCH receptor (MCH-2 receptor) was isolated in humans, it was found to be non-functional or encode a non-functional pseudogene in non-human species, including rodents. The discovery of these MCH receptors permitted the launch of a broad array of drug screening efforts and three MCH-1 receptor antagonists were identified to reduce food intake and body weight. Interestingly, some antagonists unexpectedly produced evidence that blockade of these receptors has antidepressant and anxiolytic activities. The expressions of the MCH receptors, which have been implicated in regulating emotion, stress and motivation, make MCH an excellent candidate for integrating the various homeostatic stimuli necessary for maintaining the proper conditions of energy metabolism and other physiological functions. Finally, the speed at which MCH receptor studies have been undertaken exemplifies the impact that this deorphanized GPCR will have on setting the stage for more detailed physiological studies.

Functions of melanin-concentrating hormone in fish.

Melanin-concentrating hormone (MCH) was originally discovered in fish, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. MCH is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. Mammalian MCH plays an important role as a neurotransmitter or neuromodulator in regulating food intake and energy homeostasis. MCH signaling system may involve in regulating food intake also in fish. This neuropeptide binds to G-protein-coupled seven transmembrane receptor[s] to mediate its functions. This article reviews MCH and MCH receptor signaling systems in body color change and food intake in fish.

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines.

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.

Melanin-concentrating hormone signaling systems in fish.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. This neuropeptide binds to G-protein-coupled seven transmembrane receptor(s) to mediate its function. MCH was named after its function in teleosts, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. The function of central MCH that has attracted most attention is its involvement in regulating food intake and energy homeostasis in mammals, a role confirmed through a series of experiments, including central administration of MCH or MCH receptor blockers, and genetic manipulation of MCH and its receptors. The aim of this article is to review the recent data on MCH and MCH receptor signaling systems in fish.