Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells

Namyr A Martinez,Alondra M Ayala,Magdiel Martinez,Freddyson J Martinez-Rivera,Jorge D Miranda,Walter I Silva

doi:10.1074/jbc.m116.730226

Namyr A Martinez, Alondra M Ayala + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.730226

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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.

Highlights

Neurodegenerative conditions are among the leading causes of death and disability in the United States and have dramatically increased in incidence during the last decade [1, 2]
The hHAP2Y2R Localization in Cav-1 Membrane Raft Microdomains Is Modulated by ATP in Human 1321N1 Astrocytoma Cells—Demonstration of membrane raft residence demands the use of a series of complementary experimental approaches; subcellular co-fractionation, confocal imaging co-localization, and co-immunoprecipitation
Immunofluorescence microscopy showed that Cav-1 co-localizes with the hHAP2Y2R under basal conditions (Fig. 1E) and addition of 100 ␮M ATP decreases hHAP2Y2R/Cav-1 colocalization (Fig. 1F). These findings strongly suggest that hHAP2Y2R interacts directly with Cav-1 under basal conditions and that activation of hHAP2Y2R causes its dissociation from Cav-1 and plasma membrane rafts (MRs) in 1321N1 astrocytoma cells

Summary

The abbreviations used are

P2Y2R, P2Y2 nucleotide receptor; MRs, membrane rafts; Cav-1, caveolin-1; SCRAM, scrambled; KD, knockdown; PCRA, primary cultures of rat astrocytes; IP, immunoprecipitation; ANOVA, analysis of variance. Caveolin-1 and the P2Y2R Signaling Regulation regulates P2Y2R signal transduction by extracellular ATP, including intracellular calcium mobilization and Akt and ERK1/2 activities. Our results suggest that the interaction between P2Y2R and Cav-1 in raft microdomains is a key factor mediating nucleotide signaling in astrocytic cells regulating their protective, trophic, and degenerative functions in the CNS

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