Molecular diversity of a family of pain receptors
- Maria Östergård
© BioMed Central Ltd 2001
Received: 25 September 2001
Published: 28 November 2001
A large family of G-protein-coupled receptors, with sequence homology to the oncoprotein MAS1, has been shown to be specifically expressed in nociceptive sensory neurons.
Significance and context
Vertebrate peripheral chemosensory neurons express large families of G-protein-coupled receptors (GPCRs), reflecting the diversity of ligands that these sensory systems detect. In contrast, peripheral somatosensory neurons within the body are thought not to discriminate specifically between different chemical ligands, but rather to respond to polymodal stimuli using broadly tuned receptors such as the vanilloid receptor I (VRI). Dorsal root ganglia (DRGs) contain diverse subpopulations of primary sensory neurons. One category comprises the nociceptors, which respond to a variety of noxious thermal, mechanical and chemical stimuli that cause acute pain. These receptors also mediate the chronic pain associated with inflammatory responses or nerve injury (neuropathic pain). Dong et al. describe a gene family consisting of nearly 50 MAS1-related GPCR genes - Mas-related genes (mrgs) - whose expression indicates an unanticipated degree of molecular diversity among DRG sensory neurons.
The authors used a subtractive cDNA method to clone genes expressed specifically in nociceptive neurons. The material used was cDNA from wild-type mice and the Ngn1 knockout mice. The Ngn1 knockout mice lack a transcription factor (neurogenin 1) needed for the generation of a subclass (TrkA+) of nociceptive neurons. Therefore, genes that are only found in the wild-type mouse cDNA are thought to be specifically expressed in nociceptive neurons.The screen identified several previously unknown genes, among which was one - mrgA1 - encoding a GPCR with the highest homology to MAS1. Further screening of murine cDNA and bacterial artificial chromosome (BAC) clones, as well as computer searches, revealed a family of close to 50 related GPCRs. The mrg genes are divided into three families, mrgA, mrgB and mrgC, plus some additional Mas1-related genes, referred to as mrgD-mrgG. Database searches also identified four human mrg genes (Hs.mrgX). Curiously, several of the identified mrggenes are pseudogenes.
Using in situ hybridization, Dong et al. showed that the mrgA genes were all expressed in sensory neurons in wild-type neonatal DRGs. They were virtually absent from DRGs of Ngn1 -/- mice, consistent with the results of the subtractive hybridization screen. The broadest expression pattern was that of mrgA1, and the rarer mrgA genes (mrgA2-mrgA8) were shown to be expressed in a subset of those neurons that express mrgA1. The expression of mrgA genes in sensory neurons appears highly specific, in that in situhybridization signals have not been detected in any other tissue except for trigeminal ganglia.
Using double-labeled in situ hybridization, Dong et al. were able to pinpoint the exact subpopulation of sensory neurons expressing mrgA1 and mrgD. Expression of these mrg genes was shown to be restricted to nonpeptidergic nociceptive neurons that are positive for isolectin B4 and negative for the vanilloid receptor. As confirmation, the cell-body diameters of the neurons expressing the mrg genes were measured and shown to fall within the size range characteristic of small-diameter nociceptive sensory neurons. The localization of mrgexpression led the authors to conclude that the receptors they encode may be involved in pain perception or pain regulation.
To determine whether mrgAs can function as receptors for neuropeptides, mrgA1 and mrgA4 were cloned into a eukaryotic expression vector and transfected into human embryonic kidney (HEK) cells. Candidate peptides (45) were screened for their ability to activate the mrgAs using an intracellular Ca2+ -release assay. Several neuropeptides, including adrenocorticotropin (ACTH), CGRP-I and II, NPY and somatostatin, produced some level of activation at 1 μM concentration. The most efficient response was elicited by RFamide peptides. Some RFamides could activate the receptors at nanomolar concentrations, ranging from 20 to 200 nM. RFamides have been shown to be involved in nociception, in accord with the suggested action of mrgAs in pain perception and pain regulation. The fact that ACTH activates MRGs as effectively as do the RFamides raises the question of whether the authentic ligand is an RFamide peptide or some unrelated peptide.
Dong et al. have combined a set of straightforward methods in an interesting way to obtain a wide variety of information about a gene family. The same strategy could easily be adapted to other fields of molecular biology in order to identify important genes.