Cells were labeled for 10 min with [35S]methionine and then chased with normal medium. by proteasome inhibitors, suggesting that MOCA directs nascent APP to proteasomes for destruction. It is concluded that MOCA plays a major role in APP metabolism and that the effect of MOCA on APP secretion and cell adhesion is usually a downstream consequence of MOCA-directed APP catabolism. This is a new mechanism by which the expression of APP is usually regulated. = 3) and presented as the percentage of B103 APP695 (100%). (E) Expression of N-cadherin, E-cadherin, and APLP2 in B103 and HEK-293 cells, and L-NIL the secretion of APLP2 in B103 cells. Actin served as a loading control. Open in a separate window Physique 2. PS1 and APP secretion. (A) The expression of the endogenous PS1 in B103 cells was determined by Western blot analysis. (B) Effects of -secretase inhibitors II (g-II) (50 M), III (g-III) (50 M), IV (g-IV) (5 M), and V (g-V) (10 M) on APP secretion and intracellular expression of COOH-terminal stubs in B103(APP695) and B103(APP695/MOCA) cells are shown. Effects of Calp III (100 M), MG132 (10 M), and lactacystin (Lact) (20 M) were tested as well. Cells were treated Epha5 in the presence of various inhibitors for 16 h. (C) The secretion of APP in APP and MOCA-containing cells stably transfected with either wild-type human PS1 (clones 5, 6, 11, 13) or mutant form (L392V) (clones 1, A1, A12, A13). (D) Summary of the effect of MOCA on APPs secretion. The secretion of APPs was reduced by MOCA with further reduction of APPs secretion in cells coexpressing MOCA and elevated PS1. The secretion level of sAPP in cells coexpressing both MOCA and mutant PS1 (L392V) was lower than that in cells only transfected with mutant PS1 (L392V). Human PS1 holoproteins (hPS1FL) and the NH2-terminal fragment (hPS1NTF) were detected using monoclonal antibody recognizing PS1 (Borchelt et al., 1996). The overexpression of DOCK180 protein in B103 cells is also indicated. Because PS1 is usually associated with -secretase cleavage of APP (Wolfe and Haass, 2001) and in situ hybridization shows that MOCA and PS1 L-NIL are expressed in overlapping areas of the brain and are colocalized on cells expressing both proteins (Kashiwa et al., 2000), it was asked if the effect of MOCA on APP secretion is usually altered by PS1 expression or activity. The endogenous expression of PS1 was detected in B103 cells using an antibody specific to rat PS1. Fig. 2 A shows the abundant processed NH2-terminal fragments of PS1 (mol wt 19C21 kd). A very weak band with a mol wt 90 kd was also observed, which may be a complexed form of PS1 (unpublished data). To block PS1-related -secretase activities, several inhibitors, which are specific for -secretase, were used. All of these brokers have been shown to decrease A production with concomitant increases in the levels of the corresponding COOH-terminal fragments at the concentrations we used. -Secretase inhibitor II is usually a transition-state analogue, which selectively inhibits the -secretase cleavage of APP and Notch-1 (Wolfe et al., 1998, 1999; De Strooper et al., 1999; Berezovska et al., 2000). Several dipeptidyl aldehydes (-secretase inhibitor III [Z-LL-CHO], -secretase inhibitor IV [2-naphthoyl-VF-CHO], -secretase inhibitor V [Z-LF-CHO], and calpain inhibitor III [Z-VL-CHO, Calp III]) were also tested (Higaki et al., 1995; Figueiredo-Pereira et al., 1999; Sinha and Lieberburg, 1999). No effect of -secretase inhibitors II and V on APP secretion was found in B103 cells expressing MOCA. APP secretion was partially restored by -secretase inhibitors III and IV and by Calp III in B103 cells expressing MOCA and wild-type cells. As controls, MG132 and lactacystin dramatically affected APP secretion and intracellular expression (see the following). All of the secretase inhibitors were functional because they all increased the accumulation of COOH-terminal L-NIL stubs (Fig. 2 B). There was much less accumulation of these molecules in MOCA-expressing cells, probably because of the limited substrate (APP) availability. Comparable results were also observed in HEK293 cells stably transfected with MOCA (unpublished data). In addition, the effect of the overexpression of human PS1 on APP secretion was studied (Fig. 2 C). The full-length wild-type or a mutant PS1 gene was stably transfected into the B103 (APP695/MOCA) cells, and APPs assayed. Fig. 2 C shows that APPs release was further reduced L-NIL relative to cells expressing MOCA.