title: RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense.
abstract: RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA(-). The difference between wild type and rpkA(-) was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA(-) we could not detect a difference in endosomal pH but rpkA(-) showed depletion of phosphoinositides (PIP and PIP(2)) when we compared metabolically labeled phosphoinositides from wild type and rpkA(-). Furthermore rpkA(-) exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.
authors.affiliation: Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Köln, Germany.
authors.forename: Tanja Y
authors.initials: TY
authors.lastname: Riyahi
authors.forename: Frederike
authors.initials: F
authors.lastname: Frese
authors.forename: Michael
authors.initials: M
authors.lastname: Steinert
authors.forename: Napoleon N
authors.initials: NN
authors.lastname: Omosigho
authors.forename: Gernot
authors.initials: G
authors.lastname: Glöckner
authors.forename: Ludwig
authors.initials: L
authors.lastname: Eichinger
authors.forename: Benoit
authors.initials: B
authors.lastname: Orabi
authors.forename: Robin S B
authors.initials: RS
authors.lastname: Williams
authors.forename: Angelika A
authors.initials: AA
authors.lastname: Noegel
mesh_terms: D016952:Legionella pneumophila / Q000276:immunology; D010587:Phagocytosis / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000502:physiology*
chemical_list: D043562:Receptors, G-Protein-Coupled
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