Association of solubilized angiotensin II receptors with a 60-kDa protein in murine neuroblastoma N1E-115 cells

Stephanie June Mah, University of Pennsylvania


The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In this study, polyclonal antibodies raised against purified guinea pig uterine PLC-$\alpha$, reacted with a 60 kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-$\alpha$ with cell surface angiotensin II-receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized. The synthetic detergent 3- ((3-cholamidopropyl)dimethylammonio) -1-propanesulfonate (CHAPS) was more effective in solubilizing AngII-Rs than other detergents, such as digitonin, sodium cholate and Triton X-100. Binding of $\sp{125}$I-AngII or the antagonist $\sp{125}$I-Sarc$\sp1$,IIe$\sp8$-AngII (SARILE) to the CHAPS-solubilized membranes was saturable and of high affinity. These solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. CHAPS effectively solubilized an immunoreactive 60 kDa protein and PLC activity, from the N1E-115 membranes. Anti-PLC-$\alpha$ antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. A 60 kDa $\sp{35}$S-Trans S-labeled protein, comigrating with immunoreactive PLC-$\alpha$, was immunoprecipitated from the 1% CHAPS extract by the antisera. Additionally, anti-PLC-$\alpha$ antisera coimmunoprecipitated approximately 20% of solubilized AngII-Rs prebound with $\sp{125}$I-AngII but failed to precipitate receptors prebound with the antagonist $\sp{125}$I-SARILE. The anti-PLC-$\alpha$ antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with $\sp{125}$I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with the 60 kDa protein was not a result of detergent-promoted protein-protein interaction. Monoclonal antibodies against another PLC isozyme, PLC-$\gamma$, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP$\gamma$S, suggesting that the interaction between AngII-Rs and the 60 kDa protein is mediated by a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.

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Recommended Citation

Mah, Stephanie June, "Association of solubilized angiotensin II receptors with a 60-kDa protein in murine neuroblastoma N1E-115 cells" (1994). Dissertations available from ProQuest. AAI9427574.