Control of feeding behavior inC. elegansby human G protein-coupled receptors permits screening for agonist-expressing bacteria

Abstract

G protein-coupled receptors (GPCRs) have a key role in many biological processes and are important drug targets for many human diseases. Therefore, understanding the molecular interactions between GPCRs and their ligands would improve drug design. Here, we describe an approach that allows the rapid identification of functional agonists expressed in bacteria. Transgenic Caenorhabditis elegans expressing the human chemokine receptor 5 (CCR5) in nociceptive neurons show avoidance behavior on encounter with the ligand MIP-1␣ and avoid feeding on Escherichia coli expressing MIP-1␣ compared with control bacteria. This system allows a simple activity screen, based on the distribution of transgenic worms in a binary food-choice assay, without a requirement for protein purification or tagging. By using this approach, a library of 68 MIP-1␣ variants was screened, and 13 critical agonist residues involved in CCR5 activation were identified, four of which (T8, A9, N22, and A25) have not been described previously, to our knowledge. Identified residues were subsequently validated in receptor binding assays and by calcium flux assays in mammalian cells. This approach serves not only for structure/function studies as demonstrated, but may be used to facilitate the discovery of agonists within bacterial libraries. CCR5 ͉ MIP-1␣G PCRs constitute the largest family of mammalian cell surface proteins. They have central roles in physiological processes and represent major targets for current pharmaceutical therapies. GPCRs bind a diverse set of ligands including volatile odorants, hormones, and protein ligands such as chemokines and cytokines, which represent a major medically relevant class of GPCR ligands. Caenorhabditis elegans is a bacteria-feeding soil nematode whose strategies for detecting food sources and environmental compounds include using GPCRs expressed in gustatory neurons driving ''hard-wired'' repulsive or attractive responses. Thus, C. elegans provides a potential means of studying GPCR activation. We have previously shown that heterologous expression of GPCRs in this system permit changes in specificity of the avoidance response. Heterologous expression of Somatostatin Receptor 2 and CCR5 in the chemosensory nociceptive neurons ASH and ADL of C. elegans resulted in avoidance behavior when the transgenic worms were exposed to somatostatin and MIP-1␣, respectively (1).The work described above used purified soluble compounds placed in the path of the worm to elicit an avoidance response. Because C. elegans feeds on bacteria including Escherichia coli, and because gustatory neurons have an important role in taste perception and food preference, we reasoned that expression of functional MIP-1␣ in bacteria (2) could drive an avoidance response in the transgenic animals, leading to reduced feeding on bacteria expressing the agonist. Here, we demonstrate that CCR5 transgenic C. elegans exhibit avoidance feeding behavior toward bacteria expressing functional MIP-1␣ compared with bacteria expressing an unrelated protein in a...

Pharmacological Profiling of a Brugia malayi Muscarinic Acetylcholine Receptor as a Putative Antiparasitic Target

“…Pharmacological profiling of parasite GPCRs in this heterologous system requires that the receptors are properly folded and exported to the membrane, that they signal through endogenous G proteins, and that their activation in response to exogenous ligands can be measured through convenient phenotypic endpoints. Building on previous work leveraging C. elegans as a heterologous expression platform for the expression of human GPCRs ( 79 , 80 ) and anthelmintic targets ( 35 , 61 – 63 ), we first established transgenic lines expressing Bma- GAR-3 in the C. elegans ASH sensory amphid neuron and the body wall muscle. These parasitized C. elegans strains were used to develop and optimize tissue-specific assays to measure receptor activation.…”

Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme

“…The four IDE cleavage sites (between residues 18-19, 45-46, 46-47 and 48-49) reside at the two structurally adjacent loops and they overlap with residues that are indispensable for the biological functions of MIP-1a (Figure 7D). When residues R18, Q19, K45, R46 and R48 were mutated into alanines, MIP-1a-binding affinity to its cognate receptor CCR5 was greatly attenuated (Teng et al, 2008). Interestingly, residues of MIP-1a crucial for receptor binding all map to the same side of the IDE activity on substrate V was measured at 371C in the presence of indicated concentrations of MIP-1a.…”

Feeding and the Rhodopsin Family G-Protein Coupled Receptors in Nematodes and Arthropods

“…Much less is known about their homologs and cognate activating peptides in non-vertebrates. However, comparative sequence approaches and functional studies suggest that the involvement of GPCRs in metazoa feeding behavior emerged early and has been maintained during the species radiation (Brody and Cravchik, 2000; Hewes and Taghert, 2001; Fredriksson and Schioth, 2005; Teng et al, 2008). GPCRs have emerged via gene or genome duplication events followed by selection of the gene duplicates.…”