Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells

Abstract

P2X₄ and P2X₇ receptors are abundantly expressed in alveolar epithelial cells, and are thought to play a role in regulating fluid haemostasis. Here, we analyzed the expression and localization of the P2X₄R, and characterized the interaction between Cav-1 and both P2X₄R and P2X₇R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X₄R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X₄R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mβ-CD displaced Cav-1 and P2X₄R from the low-density to the high-density fractions. Suppression of Cav-1 protein expression using short hairpin RNAs resulted in a large reduction in P2X₄R levels. Double immunofluorescence showed that P2X₄R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X₄R and P2X₇R. Co-immunoprecipitation experiments confirmed the interaction between P2X₇R and Cav-1. ATP stimulation increased the level of P2X₄R in the lipid raft/caveolae fraction, whereas Cav-1 content remained constant. Our results support recent evidence that P2X receptors are present in both raft and non-raft compartments of the plasma membrane and thus exhibit variable ATP sensitivity.

Introduction

P2 purinergic receptors are found in the cell membrane of many tissues (Burnstock and Knight, 2004). P2Y receptors are G-protein-coupled receptors, whereas P2X receptors are ligand-gated cation channels that are activated by extracellular nucleotides, particularly ATP (Khakh and North, 2006). ATP is an important signaling molecule that is released by different mechanisms into the extracellular compartment in response to cues such as mechanical stimulation and fluid flow (Schwiebert and Zsembery, 2003). Shear stress, in particular, is known to be a potent physiological stimulus of nucleotide release in pulmonary epithelium (Watt et al., 1998). These extracellular nucleotides further mediate physiological responses, including transmembrane ionic conductance and fluid dynamics, via membrane-bound purinergic receptors (Sawynok, 2007). Stimulation of purinergic receptors, in turn, influences cytokine release and induces apoptosis (reviewed in (Zsembery et al., 2003, Taylor et al., 1999, Burnstock, 2006). Recent studies have indicated that alveolar epithelial type I cells express P2X₄ receptor (P2X₄R) and P2X₇ receptor (P2X₇R) (Qiao et al., 2003, Chen et al., 2004). Both receptors are highly specific for alveolar epithelial type I cells. For example, mRNA and protein of the P2X₄ receptor were not present in freshly isolated alveolar epithelial type II cells, but appeared after transdifferentiation toward the alveolar epithelial type I cell type between Day 1 and Day 8 in culture (Qiao et al., 2003). The presence of P2X receptors in pulmonary epithelial cells has been confirmed by electrophysiological and molecular biological studies (Zsembery et al., 2003, Taylor et al., 1999, Chen et al., 2004, Ma et al., 2006, Qiao et al., 2003, Barth et al., 2007), and there is some evidence that activation of P2X receptors in the lung regulates alveolar fluid clearance (Taylor et al., 1999). Other physiological functions of P2X receptors in the alveolar epithelium are not known.

Growing evidence indicates that P2X family members are present in lipid rafts (Vacca et al., 2004, Garcia-Marcos et al., 2006, Barth et al., 2007) and that the integrity of caveolae is important for proper ion channel function (Sampson et al., 2004). Lipid rafts are cholesterol/sphingolipid-rich membrane domains that are involved in a number of cellular processes, including transmembrane receptor signaling, vesicle traffic and protein sorting (Parton and Simons, 2007). Caveolae are plasma invaginations, 50–100 nm in diameter, that are found in different cell types and represent specific lipid microdomains whose main component is Caveolin-1 (Cav-1), a 21–24 kDa protein that inserts into the caveolar membrane via an intramembrane hairpin loop (Razani et al., 2002). Importantly, Cav-1 modulates the activity and distribution of P2X receptors (Garcia-Marcos et al., 2006, Barth et al., 2007). Alveolar epithelial type I cells are particular rich in caveolae (Newman et al., 1999).

To further define the combined role of Cav-1 and P2X receptors in alveolar epithelial lung biology, we investigated the potential interaction between Cav-1 and both P2X₄R and P2X₇R. This in vitro interaction between Cav-1 and both P2X₄R and P2X₇R as shown with GST pull-down experiments may have specific consequences for proper P2X receptor function in lung epithelial cells. For the first time we could confirm the interaction between P2X₇R and Cav-1 in co-immunoprecipitation experiments. We also found that P2X₄R is present inside and outside of caveolin-rich membrane fractions of E10 mouse epithelial lung cells. In adddition, Cav-1 knock-down significantly altered P2X₄R expression and subcellular distribution, similar to previous findings with P2X₇R (Barth et al., 2007).