Various social behaviours in mice are regulated by chemical signals called pheromones that act through the vomeronasal system. Exocrine gland-secreting peptide 1 (ESP1) is a 7-kDa peptide that is released into male tear fluids and stimulates vomeronasal sensory neurons in female mice. Here, we describe the molecular and neural mechanisms that are involved in the decoding of ESP1 signals in the vomeronasal system, which leads to behavioural output in female mice. ESP1 is recognized by a specific vomeronasal receptor, V2Rp5, and the ligand-receptor interaction results in sex-specific signal transmission to the amygdaloid and hypothalamic nuclei via the accessory olfactory bulb. Consequently, ESP1 enhances female sexual receptive behaviour upon male mounting (lordosis), allowing successful copulation. In V2Rp5-deficient mice, ESP1 induces neither neural activation nor sexual behaviour. These findings show that ESP1 is a crucial male pheromone that regulates female reproductive behaviour through a specific receptor in the mouse vomeronasal system.

The intramembrane cleavage of ␤-amyloid precursor protein by ␥-secretase is the final step in the generation of amyloid ␤-protein. A 59-or 57-residue C-terminal fragment called CTF␥ is produced concomitantly. Putative CTF␥ generated in rat brain membrane preparations was purified and sequenced. Instead of CTF␥, shorter 50-and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of ␤-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to ␥-secretase-like cleavage of Notch 1. Cerebral deposition of amyloid ␤-protein (A␤)1 is an early, invariant, and essential event in the pathogenesis of Alzheimer's disease (AD) (1). A␤ is generated from ␤-amyloid precursor protein (APP) when ␤-secretase cleavage at the extracellular domain produces a 99-residue C-terminal fragment called CTF99 or CTF␤. Subsequent cleavage of CTF␤ in the middle of the transmembrane domain by ␥-secretase primarily produces either a 40-residue protein (A␤40) or a 42-residue protein (A␤42). A␤42 has the higher aggregation potential and is the first species to accumulate in aged and AD brains (2-4). Intramembrane ␥-secretase cleavage of CTF␤ should yield a 59-or 57-residue cytoplasmic C-terminal fragment (CTF41-99 or CTF43-99; APP712-770 or APP714 -770 according to the numbering of the APP770 isoform) called CTF␥. However, CTF␥has not yet been identified in either brain homogenates or cell lysates, presumably because of its instability in vivo. To learn more about the properties of ␥-cleavage, we decided to characterize CTF␥. EXPERIMENTAL PROCEDURES Subcellular Fractionation of Newborn Rat Brains and Chinese Hamster Ovary (CHO) CellsStably Expressing APP751-Fresh newborn (day 1) rat brains were homogenized on ice with 20 strokes in a glassTeflon homogenizer in eight volumes of Buffer H (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, pH 7.4). All subsequent steps were carried out at 4°C unless indicated otherwise. The nuclear fractions were collected by centrifugation of the homogenates at 2,500 ϫ g (brains) or at 800 ϫ g (CHO cells) for 10 min, followed by a brief washing in Buffer H with or without 0.4% Triton X-100. The postnuclear supernatant was separated into cytosolic and membranous fractions by centrifugation at 100,000 ϫ g for 1 h. Each fraction was adjusted to an equal volume for Western blotting.Preparation of Active Membrane Fractions-The postnuclear supernatant from newborn rat brains, as prepared above, was spun at 10,000 ϫ g for 15 min to collect membrane fraction F2P, with additional subsequent spin at 100,000 ϫ g for 1 h to collect F3P. F2P was collected from the CHO cells by centrifugation at 100,000 ϫ g for 1 h. The membrane fractions, after being washed once with the buffer, were incubated at 37°C in Buffer H containing a mixture of protease inhibitors (5 mM EDTA, 5 mM EGTA, 5 mM 1,10-phenanthroline, 10 M bestatin, 10 M amasta...

A novel cleavage of ␤-amyloid precursor protein (APP), referred to as ⑀-cleavage, occurs downstream of the ␥-cleavage and generates predominantly a C-terminal fragment (CTF␥) that begins at Val-50, according to amyloid ␤-protein (A␤) numbering. Whether this cleavage occurs independently of, or is coordinated with, ␥-cleavage is unknown. Using a cell-free system, we show here that, although A␤40 and CTF␥ 50 -99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTF␥ 49 -99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of A␤42. Furthermore, a ␥-secretase inhibitor, which suppresses A␤40 production and paradoxically enhances A␤42 production at low concentrations, caused the proportion of CTF␥ 50 -99 to decrease and that of CTF␥ 49 -99 to increase significantly. These results strongly suggest a link between the production of A␤42 and CTF␥ 49 -99 and provide an important insight into the mechanisms of altered ␥-cleavage caused by mtAPP and mtPS1/2.Senile plaques, one of the neuropathological hallmarks of Alzheimer's disease (AD), 1 are composed primarily of amyloid ␤-protein (A␤) (1). Two major A␤ species consisting of 40 and 42 residues are generated mainly in neurons and constitutively secreted. A shorter one, A␤40, is predominant, and a longer one, A␤42, is a minor species (Ͻ10%) among secreted A␤ species. A␤ is produced from ␤-amyloid precursor protein (APP), through sequential cleavage by proteases referred to as ␤-and ␥-secretases. ␤-Secretase was identified as a type I membrane aspartic protease ␤-site APP-cleaving enzyme (BACE) (2), but the identity of ␥-secretase has remained unknown. ␥-Secretase cleaves APP in the middle of the transmembrane domain, releasing A␤ and its counterpart, C-terminal fragment ␥ of APP (CTF␥). Most recent studies have shown that ␥-secretase forms a large complex composed of presenilin (PS) 1 or 2, nicastrin, PEN-2, and APH-1, and the activity of ␥-secretase is now known to depend on these proteins (3-7).One of the A␤ species, A␤42, has a much higher aggregation potential (8, 9) and is believed to be initially deposited in senile plaques (10). It is reasonable to postulate that A␤42 accumulation in the brain is the very initial event in the development of AD including sporadic AD. Indeed, all mutations of PS1/2 and some mutations of APP that cause familial AD result in increased A␤42 production (11).Recently, we and other groups found that APP is cleaved by PS-dependent ␥-secretase, not only in the middle of the transmembrane domain (␥-cleavage) but also near the cytoplasmic membrane boundary (⑀-cleavage) (12-15). The major product of the latter process is a CTF␥ of APP that begins at Val-50. This cleavage site is a few residues inside the membrane from the cytoplasmic/membrane boundary and is similar to site 3 cleavage of Notch (16). Since production of CTF␥ is inhibited by a dominan...

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.