A detergent-insoluble membrane compartment contains A beta in vivo.

Ordered assembly of the amyloid-beta protein (A beta) into amyloid fibrils is a critical step in Alzheimer's disease (AD). To release the amyloidogenic peptide A beta from the Alzheimer amyloid precursor protein (APP), two secretases act sequentially: first, beta-secretase cleaves close to the membrane within the ectodomain and then gamma-secretase cuts within the transmembrane domain. The sites of gamma-secretase cleavage are after residues 40 or 42 of A beta. Except in those rare cases of AD caused by a mutation, levels of secreted A beta are not elevated; thus, the secretory pathway may be unaffected, and factors other than the extracellular concentration of A beta may contribute to the aggregation properties of the peptide. A beta is also present in intracellular compartments. The two gamma-secretase cleavage products, A beta42 and A beta40, were found in different compartments: A beta42 in the endoplasmic reticulum (ER)/intermediate compartment, and A beta40 in the trans-Golgi network (TGN). The cellular compartments that harbor A beta are target sites for therapeutic intervention. Here we report that in the brain, the principal compartment in which A beta resides is a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of A beta, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs.

Rabbit aortic smooth muscle cells express inducible macrophage scavenger receptor messenger RNA that is absent from endothelial cells.

Scavenger receptors mediate uptake of modified low density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in developing atherosclerotic plaques. Smooth muscle cells, which can also be converted to foam cells in vivo, have not been shown to express the same scavenger receptor previously cloned in macrophages. We report the cloning of two cDNAs that encode type I and type II scavenger receptors isolated from rabbit smooth muscle cells. The deduced protein sequences of these isolates are highly homologous to the scavenger receptors previously isolated from macrophages. Treatment of smooth muscle cells with phorbol esters induced a marked increase in scavenger receptor mRNA and a fivefold increase in receptor degradation activity. Rabbit venous endothelial cells in primary culture and a bovine aortic endothelial cell line had no detectable scavenger receptor mRNA, despite having scavenger receptor degradation activity. The latter finding suggests that endothelial cells may possess a scavenger receptor which is structurally distinct from that found in macrophages and smooth muscle cells. The isolation of cDNAs encoding the rabbit scavenger receptor should prove useful for in vitro and in vivo studies that employ the rabbit as a model of human atherosclerosis.

Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.

To identify and clone genes that encode cell- or tissue-specific secreted and surface proteins, a polyclonal antiserum was raised against a complex mixture of surface or secreted proteins from the target cell, followed by immunodepletion of antibodies that recognize proteins from a nontarget cell or tissue. The depleted antiserum is used to screen bacteriophage cDNA expression libraries. Because of our interest in how adipocytes communicate with other cells, we have used this method to clone cDNAs encoding secreted and plasma membrane proteins that are induced during adipocyte differentiation. We describe several of these, including a novel plasma membrane-associated protein, S3-12.

Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins.

Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected.

CAP defines a second signalling pathway required for insulin-stimulated glucose transport.

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.

The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.