title: Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells.
abstract: Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca(2+) concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca(2+) responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.
authors.affiliation: From the Departments of Physiology and Biophysics.
authors.forename: Namyr A
authors.identifier: 0000-0002-2286-3750
authors.initials: NA
authors.lastname: Martinez
authors.affiliation: Psychiatry, and.
authors.forename: Alondra M
authors.initials: AM
authors.lastname: Ayala
authors.affiliation: From the Departments of Physiology and Biophysics.
authors.forename: Magdiel
authors.initials: M
authors.lastname: Martinez
authors.affiliation: Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936.
authors.forename: Freddyson J
authors.initials: FJ
authors.lastname: Martinez-Rivera
authors.affiliation: From the Departments of Physiology and Biophysics.
authors.forename: Jorge D
authors.initials: JD
authors.lastname: Miranda
authors.affiliation: From the Departments of Physiology and Biophysics, walter.silva@upr.edu.
authors.forename: Walter I
authors.initials: WI
authors.lastname: Silva
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D058473:Receptors, Purinergic P2Y2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D051242:Caveolin 1; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium
keywords: ATP; astrocyte; caveolae; caveolin; cell signaling; lipid raft; purinergic receptor