Characterization of the Mas-related gene family : structural and functional conservation of human and rhesus MrgX receptors

Recently, a large family of G-protein-coupled receptors called Mas-related genes (Mrgs), which is selectively expressed in small-diameter sensory neurons of dorsal root ganglia, was described. A subgroup of human Mrg receptors (MrgX1–X4) is not found in rodents and this has hampered efforts to define the physiological roles of these receptors. MrgX receptors were cloned from rhesus monkey and functionally characterized alongside their human orthologs. Most of the human and rhesus MrgX receptors displayed high constitutive activity in a cellular proliferation assay. Proliferative responses mediated by human or rhesus MrgX1, or rhesus MrgX2 were partially blocked by pertussis toxin (PTX). Proliferative responses mediated by rhesus MrgX3 and both human and rhesus MrgX4 were PTX insensitive. These results indicate that human and rhesus MrgX1 and MrgX2 receptors activate both Gq- and Gi-regulated pathways, while MrgX3 and MrgX4 receptors primarily stimulate Gq-regulated pathways. Peptides known to activate human MrgX1 and MrgX2 receptors activated the corresponding rhesus receptors in cellular proliferation assays, Ca2+-mobilization assays, and GTP-γS-binding assays. Cortistatin-14 was selective for human and rhesus MrgX2 receptors over human and rhesus MrgX1 receptors. BAM22 and related peptides strongly activated human MrgX1 receptors, but weakly activated rhesus MrgX1, human MrgX2, and rhesus MrgX2 receptors. These data suggest that the rhesus monkey may be a suitable animal model for exploring the physiological roles of the MrgX receptors. Keywords: Nociception, dorsal root ganglia, mas, G-protein-coupled receptor, Bam22, cortistatin-14 Introduction The G-protein-coupled receptor (GPCR) superfamily is the most exploited gene family for drug discovery; yet, over 50% of the GPCR family remains classified as ‘orphan receptors', receptors whose functions and regulatory ligands remain unknown. Given that GPCRs are one of the most targeted gene families for therapeutic intervention, understanding the physiological roles of these genes is of great importance. Recently, the identification of a large family of GPCRs that are related to the MAS oncogene (Young et al., 1986), called Mas-related genes (Mrgs), was reported (Dong et al., 2001). These genes, also known as the sensory neuron-specific receptors (SNSRs) or dorsal root receptors (DRRs), comprise a large family of over 50 rodent and human orphan GPCRs. The restricted expression of many members of the Mrg family to sensory neurons of the dorsal root ganglion (DRG) suggests that these receptors may play a role in nociception; however, this has not yet been conclusively established. These genes were first identified by subtracting cDNAs from neonatal murine wild-type and Neurogenin1 (Ngn1−/−) knockout DRG. Ngn−/− mice do not generate sensory neurons (Ma et al., 1999). In humans, the MrgX subset of Mrg receptors is selectively expressed in DRG (Lembo et al., 2002). Expression of a subset of these genes is primarily restricted to small-diameter sensory neurons in both rodents and in humans (Dong et al., 2001; Lembo et al., 2002; Robas et al., 2003), further implicating these receptors in nociception. Interestingly, the DRG-specific members of this family show discrete patterns of expression that appear to be only partially overlapping within the sensory neurons, implying that these receptors may each play unique roles in pain sensation. A major limitation in developing animal models for understanding the function of the MrgX receptors is that MrgX orthologs do not exist in rodents. Indeed, the genomic organization of Mrg receptors varies quite dramatically even among highly related species. For example, the mouse genome contains 22 MrgA genes and 14 MrgC genes, whereas rat contains only one each, a difference that is thought to exist because of an atypical expansion of this receptor family (Zylka et al., 2003). The expression patterns of Mrg receptors are similar between mouse and rat, with DRG-specific receptors expressed in neurons which are IB4 positive, and express the glial cell line-derived neurotrophic factor (GDNF) receptor c-Ret (Snider & McMahon, 1998; Julius & Basbaum, 2001; Zylka et al., 2003). Despite these similarities, MrgD receptors in rat are coexpressed with the capsaicin receptor VR1, whereas in mouse they are not, and in rat all Mrg-expressing neurons coexpress the purinergic receptor P2X3, whereas in mouse only MrgD is coexpressed with P2X3 (Zhang et al., 2005). However, no orthologs of human MrgX receptors exist in any of the rodent species examined to date. Although limited functional characterization of this family has been described (Dong et al., 2001; Bender et al., 2002; Han et al., 2002; Lembo et al., 2002; Robas et al., 2003), the functional properties and physiological functions of these genes remain largely unknown. It is assumed that rodent Mrg receptors expressed in DRG probably perform similar functions as human receptors expressed in DRG. However, given the differences between human and rodent receptors, and given that each receptor subtype may perform specific functions, extrapolating results from experiments with rodents may not accurately predict the functions of human receptors. Recently, MrgX receptors have been shown to be expressed in macaque (Zhang et al., 2005). As rhesus monkeys are a commonly used animal model for experimental biology and drug discovery, we cloned and pharmacologically analyzed rhesus monkey MrgX receptor counterparts for each of the known human MrgX receptors, and found that they are functionally as well as structurally similar to the known human MrgX receptors. These results indicate that rhesus monkeys may be useful for exploring the functions of these receptors.