Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and beta2-microglobulin (beta2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, beta2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.     

A recent class of chemosensory neurons developed in mouse and rat

In most animal species, the vomeronasal organ ensures the individual recognition of conspecifics, a prerequisite for a successful reproduction. The vomeronasal organ expresses several receptors for pheromone detection. Mouse vomeronasal type-2 receptors (V2Rs) are restricted to the basal neurons of this organ and organized in four families. Family-A, B and D (family ABD) V2Rs are expressed monogenically (one receptor per neuron) and coexpress with either Vmn2r1 or Vmn2r2, two members of family-C V2Rs. Thus, basal neurons are characterized by specific combinations of two V2Rs. To investigate this issue, we raised antibodies against all family-C V2Rs and analyzed their expression pattern. We found that six out of seven family-C V2Rs (Vmn2r2-7) largely coexpressed and that none of the anti-Vmn2r2-7 antibodies significantly stained Vmn2r1 positive neurons. Thus, basal neurons are divided into two complementary subsets. The first subset (Vmn2r1-positive) preferentially coexpresses a distinct group of family-ABD V2Rs, whereas the second subset (Vmn2r2-7-positive) coexpresses the remaining group of V2Rs. Phylogenetic reconstruction and the analysis of genetic loci in various species reveal that receptors expressed by this second neuronal subset are recent branches of the V2R tree exclusively present in mouse and rat. Conversely, V2Rs expressed in Vmn2r1 positive neurons, are phylogenetically ancient and found in most vertebrates including rodents. Noticeably, the more recent neuronal subset expresses a type of Major Histocompatibility Complex genes only found in murine species. These results indicate that the expansion of the V2R repertoire in a murine ancestor occurred with the establishment of a new population of vomeronasal neurons in which coexists the polygenic expression of a recent group of family-C V2Rs (Vmn2r2-7) and the monogenic expression of a recent group of family-ABD V2Rs. This evolutionary innovation could provide a molecular rationale for the exquisite ability in individual recognition and mate choice of murine species.     

A multireceptor genetic approach uncovers an ordered integration of VNO sensory inputs in the accessory olfactory bulb

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.     

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems

In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes metabotropic glutamate receptors, calcium-sensing receptors, fish olfactory receptors, and taste receptors for sweet molecules and amino acids. The large extracellular region of these receptors is folded as a dimer and, in this form, binds agonists that in many cases are amino acids. It has recently been reported that V2Rs must be physically associated with specific major histocompatibility complex class Ib molecules (MHC) for their expression in both mouse vomeronasal neurons and heterologous cell lines. Here, we show that in contrast to the other V2Rs, V2R2, an atypical member of this receptor family, can be successfully and abundantly expressed by insect cells without the requirement of escort molecules like MHC. Moreover, the extracellular binding domain of V2R2, secreted as a soluble product, forms dimers via cysteine-mediated sulphur bridges. Overall, the data presented in this paper confirm that V2R2 diverges from the other members of the V2R family and suggest a different role for this receptor in pheromonal communication.     

ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes,Recognizes 4-Hydroxyphenylacetic Acid,a Putative Reproductive Pheromone

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.     

T2Rs function as bitter taste receptors

Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds.     

Comparative genomics of odorant and pheromone receptor genes in rodents

We applied a comprehensive data-mining strategy to examine the repertoires of rat and mouse odorant receptors (ORs) and type 1 pheromone receptors (V1Rs) using the mm5 (mouse) and rn3 (rat) genomes. We identified 1576 rat OR genes, including 292 pseudogenes. The rat V1R repertoire is composed of 115 intact genes and 72 pseudogenes. The mouse OR and V1R databases were updated using the new assembly mm5, from which 1375 mouse ORs and 308 V1Rs were identified, with more than 100 putative pseudogenes from mm2 now identified as intact because of the higher sequence quality. With these new data we have conducted a series of genomic analyses of the OR and V1R genes from mouse and rat. Orthologous OR clusters were identified in mouse and rat and comparison analysis was performed at three incremental levels: families, coding sequences, and motifs. At the family level, we found that V1R genes have more species-specific families than OR genes. About 20% of intact V1R genes have no orthologous counterpart in the same family, whereas less than 1% of intact ORs are similarly isolated. At the coding sequence level, OR genes are more conserved between mouse and rat than V1R genes. OR genes share greater similarity with their orthologous counterparts than with their closest neighbor, whereas V1R genes show the opposite tendency. Motifs were identified to obtain biological insights. Motifs specific for species or families were found in OR and V1R genes, which may result in the differential pheromone-dependent behaviors and perception of odors between mouse and rat.     

Adaptive Diversification of Vomeronasal Receptor 1 Genes in Rodents

The vomeronasal receptor 1 (V1R) are believed to be pheromone receptors in rodents. Here we used computational methods to identify 95 and 62 new putative V1R genes from the draft rat and mouse genome sequence, respectively. The rat V1R repertoire consists of 11 subfamilies, 10 of which are shared with the mouse, while rat appears to lack the H and I subfamilies found in mouse and possesses one unique subfamily (M). The estimations of the relative divergence times suggest that many subfamilies originated after the split of rodents and primates. The analysis also reveals that these clusters underwent an expansion very close to the split of mouse and rat. In addition, maximum likelihood analysis showed that the nonsynonymous and synonymous rate ratio for most of these clusters was much higher than one, suggesting the role of positive selection in the diversification of these duplicated V1R genes. Because V1R are thought to mediate the process of signal transduction in response to pheromone detection, we speculate that the V1R genes have evolved under positive Darwinian selection to maintain the ability to discriminate between large and complex pheromonal mixtures.Reviewing Editor: Dr. Rasmus Nielsen     

Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity

Taste represents a major form of sensory input in the animal kingdom. In mammals, taste perception begins with the recognition of tastant molecules by unknown membrane receptors localized on the apical surface of receptor cells of the tongue and palate epithelium. We report the cloning and characterization of two novel seven-transmembrane domain proteins expressed in topographically distinct subpopulations of taste receptor cells and taste buds. These proteins are specifically localized to the taste pore and are members of a new group of G protein-coupled receptors distantly related to putative mammalian pheromone receptors. We propose that these genes encode taste receptors.     

The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the alpha and beta subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.     

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.     

Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs.     

Transcriptional regulation of the mouse calbindin-D9k gene by the ovarian sex hormone

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gene is expressed under the control of a PRE.     

Site-directed mutations in the third intracellular loop of the serotonin 5-HT(1A) receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.