Ikezu T, Trapp BD, Song KS, Schlegel A, Lisanti MP, Okamoto T. This inhibition of Go GTPase activity by either 22C11 or wild-type APP cytoplasmic domain suggests that intracellular interactions between APP and Go could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Go and APP in CSEM. We show that inhibition of basal Go GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Go and APP. The regulation of Go GTPase activity by APP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of APP. or E19 embryos] used in this study, the 22C11 antibody and the polyclonal antibody from Dr P. Frey specifically recognize VCL APP and not APLP2 (Fig. ?(Fig.11). Open in a separate window Fig. 1. Western blotting of APP and APLP2. Extracts from 106 E16 rat cortical neurons cultured for 5 d were loaded on 7% SDS-PAGE and immunoblotted using either 22C11 or CT15, two antibodies recognizing APP or D2C2, an antibody specific to APLP2. The protein bands revealed with 22C11 and CT15 are very similar and differ from that reacting with D2C2. Immunocytochemistry on primary corticostriatal rat cultures was performed as described previously (Allinquant et al., 1994). For immunoprecipitation, 40 g of Triton X-100-insoluble membranes in 500 l GTPase buffer (see below) was adjusted to 100 mMgSO4, 100 nm GTP, and 150 mm NaCl, and the Go iCRT 14 antibody was added overnight at 4C before solubilization by 2%Carbonate step iCRT 14 gradients were performed according to Song et al. (1996). In brief, E19 brain tissues were homogenized with a Dounce homogenizer in 500 mm sodium carbonate, pH 11.0, sonicated, made 45% in sucrose, and placed at the bottom of a iCRT 14 5C35% discontinuous sucrose gradient in 25 mm MES, pH 6.5, and 0.15m NaCl (MBS) containing 250 mm sodium carbonate. After centrifugation in a Beckman SW41 rotor (150,000 Membranes isolated on a Percoll (Pharmacia) step gradient (Smart et al., 1995) were sonicated and loaded at the bottom of a linear 10C20% OptiPrep (Nycomed Pharma, Oslo, Norway) gradient. After centrifugation at 52,000 for 90 min, 4C (SW41 rotor, Beckman), the top five fractions (5 ml) were made 25% in OptiPrep (9 ml total), placed under 2 ml OptiPrep 5%, and centrifuged (52,000 According toSargiacomo et al. (1993), tissues homogenized in MBS plus 1% Triton X-100 were adjusted to 40% sucrose, placed at the bottom of a continuous 5C30% sucrose gradient in MBS, and centrifuged (150,000 for 10 min. Radioactivity in the supernatant was measured by scintillation counting. High-affinity GTPase activity was calculated by substracting the radioactivity released in the presence of 100 nm and 20 m GTP, and the results were expressed in femtomoles of inorganic phosphate released per milligram of protein per minute. When indicated, the membranes were first incubated for 1 hr at 37C with 7 m recombinant peptides in 10 mm Tris, 100 m EDTA, 200 mmNaCl, and protease inhibitors, centrifuged, and resuspended in GTPase buffer. All results presented in this study correspond to high-affinity GTPase activity. All reagents used in the GTPase experiments are from Sigma (St. Louis, MO) and Boehringer Mannheim. ADP?ribosylation ADP ribosylation with pertussis toxin (PTX) or C3 (gift from Dr. P. Boquet, Institute National de la Sant et de la Recherche Mdicale, Nice, France) was as described in Brabet et al. (1990). Membranes (25C50 g) were incubated (1 hr, 37C) in 100 l containing 70 mm Tris-HCl, pH 7.5, 25 mmdithiothreitol,.