The syndecans play critical roles in several signal transduction pathways. The core proteins of these heparan sulfate proteoglycans are characterized by highly conserved transmembrane and intracellular domains which are required for signaling across the membrane and for interaction with cytosolic proteins. However, regulatory mechanisms controlling these functions remain largely unknown. Here we show that, upon ligandinduced primary proteolytic cleavage within the ectodomain, the intracellular domain of syndecan 3 is released by regulated intramembrane proteolysis. The cleavage is mediated by presenilin/␥-secretase complex and negatively regulates the plasma membrane targeting of the transcriptional cofactor CASK.Heparan sulfate proteoglycans (HSPG) 1 are present on the surface of all adherent cells. Binding to a large number of ligands depends upon highly charged heparan sulfate chains, linear polysaccharide chains that can be modified by sequential enzymatic modifications. These modifications lead to enormous structural diversity (for review see Ref. 1). Although numerous functions for the heparan sulfate moiety of proteoglycans have been shown in vivo and in vitro, the roles of the core proteins remain largely elusive (2, 3). The syndecan family of HSPG, as opposed to the GPI-anchored glypicans or matrix-associated HSPG, contains a transmembrane and cytosolic domain that is highly conserved across homologues and species, suggesting an important role for this part of the protein (4). Indeed, a role in FGF signaling, neurite outgrowth, dendritic spine morphogenesis, and left-right axis formation has been demonstrated (5-8), but the regulatory mechanisms are incompletely understood. The cytosolic domain can be subdivided into a membrane-proximal C1 and a C-terminal C2 conserved region that is nearly identical in all syndecans and has a more variable V region in the middle. The most conserved residues are serines and tyrosines that at least partially undergo phosphorylation, a basic motif that binds nonreceptor tyrosine kinases and cytoskeletal proteins, and the C terminus that is required for interaction with PDZ proteins such as syntenin, CASK, synectin, or synbindin (for review see Ref. 4).The heparan sulfate-bearing syndecan ectodomain has been shown to undergo ligand-activated or stress-induced proteolytic cleavage and shedding (9 -11), but the fate of the Cterminal fragment (CTF) that remains associated with the membrane is unknown. We surmised that the remaining CTF is subject to further proteolytic degradation, ectodomain-shedding being a first step in an intracellular signaling cascade. Because regulated intramembrane proteolysis, a novel mechanism of signal transduction across the membrane (12), has been demonstrated to depend upon prior ectodomain cleavage of type I transmembrane protein substrates (13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and is mediated by presenilins (PS) as part of a larger so-called ␥-secretase complex (23), we investigated whether syndecan 3 undergoes PS-dependent cleavage. EXPERI...

SUMMARYOligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC 50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC 50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3Ј-processing reaction, and the DNA strandtransfer step. We then analyzed the kinetics of the ISC formation and 3Ј-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 Ϯ 0.01 min Ϫ1. T30177 did not inhibit 3Ј-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.For the treatment of infection with HIV-1, a number of inhibitors of both the reverse transcriptase and HIV protease have been formally approved (1). Targeting the third viral enzyme IN with effective inhibitors remains an elusive goal. The IN enzyme inserts the viral cDNA copy into the host cell chromosome; this step is essential for the replication of the virus (2, 3). Because no human counterpart of the enzyme is known, there is considerable interest in developing effective and selective inhibitors of the HIV integration process. The recent establishment of high-throughput microtiter plate assays (4, 5), on the one hand, and the elucidation of the three-dimensional structure of the catalytic domain of HIV-1 IN, on the other hand (6), will likely boost the antiviral screening of chemical libraries as well as the structure-based design of enzyme inhibitors.The only enzyme required for HIV-1 integration is IN, a protein o...

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt − hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3Ј end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5Ј end of HPRT and a Cre-encoding plasmid into the HCV + hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV + F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.