Purinergic signalling in Dictyostelium discoideum. (360G-Wellcome-081601_Z_06_Z)

Extracellular nucleotides act at ionotropic P2X and metabotropic P2Y receptor families and play a fundamental role in a wide array of physiological processes including neurotransmission, smooth muscle contraction, immune cell function and platelet aggregation. We have recently identified a family of P2X-like proteins and demonstrated a purinergic calcium response in the amoeba Dictyostelium. This opens a unique opportunity to explore the molecular mechanisms governing purinergic receptor functio n in a well established model eukaryotic organism offering a range of powerful molecular genetic techniques and functional screens. In this project we aim to establish Dictyostelium as a model system for purinergic receptor research. We will characterise the functional properties of Dictyostelium P2X-like receptors by electrophysiology in Xenopus oocytes and by recording calcium responses in aequorin expressing Dictyostelium. A genome wide screen of orphan Dictyostelium GPCRs will be conducted b y RNAi to identify P2Y receptors and the molecular identity of the receptors mediating the purinergic calcium response will be determined by analysis of null and overexpressing purinoceptor strains. GFP-tagged receptors will be used to determine subcellular localisations and roles in cellular function will be investigated by studying phenotypic changes due to ablation and over expression of purinergic receptor genes.