Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.

PDZ domains1 are modular protein interaction domains that play a role in protein targeting and protein complex assembly. Once termed Discs-large homology regions (DHRs) or GLGF repeats (after a conserved Gly-Leu-Gly-Phe sequence found within the domain), these domains of ϳ90 amino acids are now primarily known by an acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, the Drosophila septate junction protein Discs-large, and the tight junction protein ZO-1. Since their initial identification, PDZ and PDZ-like domains have been recognized in numerous proteins from organisms as diverse as bacteria, plants, yeast, metazoans, and Drosophila (1). In fact, they are among the commonest protein domains represented in sequenced genomes. Analysis of the human, Drosophila, and Caenorhabditis elegans genomes estimates the presence of 440 PDZ domains in 259 different proteins, 133 PDZ domains in 86 proteins, and 138 PDZ domains in 96 proteins, respectively (2).The structural features of PDZ domains allow them to mediate specific protein-protein interactions that underlie the assembly of large protein complexes involved in signaling or subcellular transport. Not surprisingly, disrupting these interactions can play a role in human diseases. Mutations in a gene encoding harmonin, a PDZ-containing protein, cause Usher syndrome type 1C, an autosomal recessive disorder characterized by congenital sensorineural deafness, vestibular dysfunction, and blindness (3-5). This was the first mutation in a PDZ-encoding gene linked to a human disease. Subsequently, mutations in the periaxin gene, which also encodes a PDZ-containing protein, have been identified as a cause of Dejerine-Sottas neuropathy, a severe demyelinating form of peripheral neuropathy (6, 7). The Structural Basis of PDZ Binding and SpecificityThe notion that PDZ domains serve as protein interaction modules emerged from the finding that the first and second PDZ (PDZ1 and -2) domains of PSD-95 can bind the extreme C-terminal peptide sequence of Shaker-type K ϩ channels (8) and NMDA receptor NR2 subunits (9, 10). PDZ3 of the protein tyrosine phosphatase FAP-1/PTP1E similarly was identified as a binding site for the C terminus of the cell surface receptor Fas (11). These studies further demonstrated that PDZ domains maintained their activity and selectivity when expressed in heterologous proteins, establishing these motifs as modular domains that bind the C termini of target proteins in a sequence-specific manner.The structural basis for PDZ specificity became apparent with the solution of the x-ray crystallographic structure of PDZ domains complexed with their cognate peptide ligands. First solved for PDZ3 of PSD-95 (12, 13), numerous additional PDZ crystal structures have been determined in recent years, including PDZ2 of , the single PDZ domain of CASK (15), syntrophin (16) and neuronal nitric-oxide synthase (nNOS) (17), PDZ2 from human phosphatase hPTP1E (18), PDZ1 of the Na ϩ /H ϩ exchanger regulatory factor (NHERF) (19), a...