Vomeronasal Receptors Type 1

V1Rs, like V2Rs and ORs, are 7TM receptors belonging to the GPCR su-perfamily. They share no sequence homology with V2Rs and ORs, and their closest, although very distant, relatives are the taste receptor (T2Rs). V1Rs were first described in the rat (Dulac and Axel, 1995) and later in other mammals (Rodriguez, 2005), teleosts (Pfister et al., 2007) and amphibians (Shi and Zhang, 2007). In the mouse, they form the second largest protein-coding gene superfamily, comprising more than 150 genes and about the same number of pseudogenes (Zhang et al., 2004a). This repertoire has been sub-divided into 12 families, with members of the same family sharing at least 40% of identity at the amino acid level (Rodriguezet al., 2002). V1R families are usually organized in clusters on several chromosomes, with members of two families intermingled if present in the same cluster (Del Punta et al., 2000; Zhang et al., 2007).

While rodents share similar V1R repertoires (although species-specific families exist) (Grus and Zhang, 2004), other phylogenetic groups exhibit more divergent V1R gene families. The V1R repertoire thus varies a lot in numbers, from 1–5 in teleosts (Pfister et al., 2007) or 2–3 in humans (Rodriguez and Mombaerts, 2002) to 270 in the platypus (Grus et al., 2007) (Figure 3.6 on page 28). It seems that a large V1R repertoire does correlate with a well developed VNO (Grus and Zhang, 2006). Expression of V1Rs in the VNO is not exclusive, since in humans and in goats, V1Rs are expressed in the MOE (Rodriguez et al., 2000; Wakabayashi et al., 2002).

A single V1R receptor/ligand couple has been identified so far in rodents (Boschatet al., 2002). This finding resulted from experiments based on elec-trophysiological recordings and calcium imaging techniques that showed that the mouse pheromone 2-heptanone activates the V1RB2 receptor. Found in both female and male adult urine, 2-heptanone is known to act on female mice triggering the extension of the estrous cycle (Schwendeet al., 1984; An-dreoliniet al., 1987; Jemioloet al., 1989). The V1RB2 receptor does not react to molecules highly-related to 2-heptanone at the chemical level, suggesting

0.1

Figure 3.6: Phylogenetic tree of the mouse, rat and dog V1R gene repertoires. This tree was obtained by maximum likelihood analysis with 1000 bootstraps (the most significant values are shown). Families h and i are specific to the mouse, while family m is exclusively present in the rat. All the other members are present in both species.

that V1Rs may function as very narrowly tuned pheromone receptors.

V1Rs have intronless coding sequences with usually only one 5⁰ untrans-lated exon. Their putative promoters share remarkably conserved sequences (Lane et al., 2002; Stewart and Lane, 2007). The level of promoter conser-vation is similar to that of the coding sequence, at least between promoters of a given family, since interfamily homologies of promoters are restricted to few domains only (Lane et al., 2002; Stewart and Lane, 2007).

V1R Gene Transcription

As mentioned earlier, V1Rs are transcribed by VSNs located in the api-cal part of the VNE. Apart from being members of the GPCR superfamily, V1Rs share another feature with ORs: they are expressed in a monogenic and monoallelic fashion (Matsunami and Buck, 1997; Rodriguez et al., 1999;

Del Punta et al., 2002b). Indeed, members of different families are tran-scribed in different subsets of neurons (Dulac and Axel, 1995; Pantages and Dulac, 2000; Rodriguez et al., 2002; Roppolo et al., 2007) and in compound heterozygous mice carrying a different marker for each parental allele of the V1rb2 gene, VSNs never coexpress both alleles (Rodriguez et al., 1999; Bel-luscio et al., 1999).

While it is still unclear how this peculiar transcription is achieved, a study brought more insights into V1R gene regulation. In transgenic mice containing a V1R null allele, the coding sequence of the V1rb2 gene was replaced with the one of the GFP marker. Transcription of such a V1R null allele did not produce the V1RB2 protein. VSNs expressing the deletion allele did coexpress another random V1R (Roppolo et al., 2007), suggesting, as what is observed in OR regulation, a mechanism of negative feedback mediated by the first expressed receptor.

A second level of regulation was identified. This one is not V1R-wide but acts at the cluster level. It has been termed “gene cluster lock” (Roppolo et al., 2007).

V1R Transduction Pathway

V1Rs are likely coupled with the G_αi2 subunit of the heterotrimeric G-protein. Upon V1R binding, the G-protein activates a phospholipase C (PLC2)-dependent cascade, where the secondary messenger is not cAMP but inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). In this case, the Ca²⁺-permeable channel is not CNGA2, but instead TRPC2, whose aper-ture allows an influx of Ca²⁺ and thus membrane depolarization (Dulac and Torello, 2003; Lucas et al., 2003) (Figure 3.7 on page 30). The signaling cascade in the VNO is thus different from the one used by ORs.

Knock-out mice have been generated for most of the genes involved in the VNO transduction cascade: the behavioral deficiencies corresponding to these mice are remarkable. We will quickly review a few of them.

The deletion of TRPC2 leads to a very surprising phenotype. Mutant

Pheromone

Figure 3.7: Vomeronasal receptor transduction cascade. Modified from Rodriguez, 2003.

males act normally towards females, but instead of showing aggressiveness towards other males, they try to mount them (Leypold et al., 2002; Stowers et al., 2002). Female mutant mice are also affected and show typical male be-haviors, such as investigation of the anogenital area, mount attempts, pelvic thrusts and ultrasound vocalizations (Kimchi et al., 2007). These effects are caused by a loss of gender preference, given that in TRPC2 knock-out animals the frequency of mounting is the same for both genders. It has been suggested that in TRPC2 knock-out mice a male sexual circuit is ac-tivated/derepressed. Other effects are present in lactating mutant females:

reduced aggressiveness and larger sexual receptiveness toward intruders (Ley-pold et al., 2002; Kimchi et al., 2007) as well as loss of responses to urine and 2-heptanone (Leypold et al., 2002; Stowers et al., 2002; Kelliher et al., 2006).

A less dramatic but more targeted deletion leads to different effects. The lack of the G_αi2 gene has strong effects on the AOB: the number of api-cal neurons coming from V1R-expressing neurons is reduced and null mice show deficiencies in pheromone-dependent activity (Norlinet al., 2003). Male knock-out mice, while normal for mating motivation and gender choice, tend to be more aggressive towards intruder males. On the contrary, female knock-out mice do not show any aggressive behavior towards intruders, a situation which resembles the V1Rab knock-out animals, which we will discuss in the next paragraph.

Mutants have also been generated upstream of the transduction machin-ery,i.e. at the level of the chemoreceptors. The deletion of about 10% of the V1R repertoire (all V1Ra and V1Rb genes) leads to deficiencies to respond to specific molecules. Mice lacking the V1Rab cluster do not respond any-more to at least three pheromones (Del Punta et al., 2002a). Moreover, they show altered behaviors compared to wild type animals: a) lactating females are less aggressive towards intruders; b) males are sexually less interested in females and c) experienced males are sexually less active.