Control of feeding behavior in C. elegans by human G protein-coupled receptors permits screening for agonist-expressing bacteria.
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-1alpha and avoid feeding on Escherichia coli expressing MIP-1alpha 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-1alpha 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.
Teng
Michelle S
MS
Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1HH, United Kingdom.
Shadbolt
Paul
P
Fraser
Andrew G
AG
Jansen
Gert
G
McCafferty
John
J
D000818:Animals; D030801:Animals, Genetically Modified; D065100:CCR5 Receptor Antagonists; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D054405:Chemokine CCL3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005247:Feeding Behavior; D015723:Gene Library; D006801:Humans; D008024:Ligands; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D019713:Receptors, CCR5 / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics; D019076:Transgenes
D065100:CCR5 Receptor Antagonists; D054405:Chemokine CCL3; D008024:Ligands; D019713:Receptors, CCR5; D043562:Receptors, G-Protein-Coupled