X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of ␤-amyloid peptides (A␤) that are associated with Alzheimer's disease. Whereas X11␣ interacts with APP via its phosphotyrosine-binding domain, recent reports indicate that additional regulatory interactions involve the N terminus of X11. Here we report that the syntaxin-1a-binding protein Munc18a, which interacts with the Munc18a-interacting domain (MID) at the N terminus of X11, strongly regulates the actions of X11 on APP metabolism. When co-expressed with X11␣, Munc18a potentiated the retention of APP and suppression of A␤ secretion by X11␣. As a result, the constitutive release of A␤40 was nearly abolished. Experiments using N terminus deletion mutants of X11␣/␤ and the MID-deficient X11␥ revealed that the majority of the regulatory effect by Munc18a occurred independent of a direct interaction of Munc18a with X11, although the presence of X11 was required. Munc18a expression induced a small increase in ␤-secretase activity, whereas it also intensified the reduction in A␤40 secretion by X11␣. These data indicate that Munc18a in concert with X11 acts to suppress ␥-secretase processing. We conclude that Munc18a acts through direct and indirect interactions with X11 proteins and powerfully regulates APP metabolism and A␤ secretion.The two major pathological features in the brains of patients with Alzheimer's disease are the presence of ␤-amyloid peptide (␤-AP or A␤) 1 containing senile plaques and neurofibrillary tangles (1). A␤ peptides were derived from proteolytic processing of a precursor protein, namely amyloid precursor protein (APP). The cleavage sites and extent of processing depend on its trafficking pathways as different APP derivatives have been mapped to distinct compartments of the cell, presumably where specific APP processing enzymes, or secretases, reside (2-6). A major pathway for A␤ production involves internalization of APP, as directed by an ENPTY sequence at the C terminus of APP, following its delivery to the plasma membrane (7). This internalization motif has been found to interact with several phosphotyrosine-binding domain (PTB) containing proteins such as Fe65 and X11, although phosphorylation within this motif is not required for binding (8 -12). The interaction between the PTB domains of Fe65 or X11 proteins and the C terminus of APP has been shown to effect the distribution and turnover of APP, and the secretion of A␤ (13-16). Mammalian X11 proteins (X11␣, -␤, and -␥) are homologues of lin-10 in Caenorhabditis elegans. In conjunction with lin-2 and lin-7, lin-10 has been shown to be required for the precise targeting and localization of certain membrane proteins, such as the GLR-1 glutamate receptor (17, 18). Similar to lin-10, X11 proteins possess multiple protein interacting domains (see Fig. 1A) and have been ascribed to function as adaptor proteins that were critical for protein trafficking (19 -23). It has therefore been postulated that X11 may modulate APP ...

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1‐V12, Rac1‐L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin‐permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1‐N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21‐activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1‐V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1‐V12‐mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin‐A inhibited secretion and eliminated the secretory effects of Rac1‐V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.