A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion.
ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100-200 microM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.
Pellegatti
Patrizia
P
Department of Experimental and Diagnostic Medicine, Section of General Pathology and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara 44100, Italy.
Falzoni
Simonetta
S
Pinton
Paolo
P
Rizzuto
Rosario
R
Di Virgilio
Francesco
F
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D007249:Inflammation; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; C546468:P2RX7 protein, human; D011956:Receptors, Cell Surface; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases