Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells.
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 the present study, polyclonal antibodies raised against a PLC isoenzyme, 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 AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. 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. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.
Mah
S J
SJ
Department of Animal Biology, University of Pennsylvania, Philadelphia 19104.
Ades
A M
AM
Mir
R
R
Siemens
I R
IR
Williamson
J R
JR
Fluharty
S J
SJ
D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism*; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002793:Cholic Acids; D002850:Chromatography, Gel; D007457:Iodine Radioisotopes; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D011233:Precipitin Tests; D011945:Receptors, Angiotensin / Q000378:metabolism* / Q000502:physiology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D057911:Angiotensin Receptor Antagonists; D000911:Antibodies, Monoclonal; D002793:Cholic Acids; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D011945:Receptors, Angiotensin; D000804:Angiotensin II; C014622:angiotensin II, Ile(8)-; D010738:Type C Phospholipases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate