Structural requirements for CD43 function.
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The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways. (+info)
DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain.
(42/55031)
mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA. (+info)
Two independent calcineurin-binding regions in the N-terminal domain of murine NF-ATx1 recruit calcineurin to murine NF-ATx1.
(43/55031)
Intracellular calcium regulates events controlling nuclear translocation of nuclear factor of activated T cells (NF-AT). Calcium-dependent phosphatase calcineurin (CN) plays a central role in this process. Structural and functional analyses of the N-terminal domain of murine NF-ATx1, a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. The binding of murine NF-ATx1 (mNF-ATx1) to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. We propose that activation of mNF-ATx1 can be modulated through two distinct CN target regions. Our findings provide a new opportunity for pharmacological intervention with Ca2+-dependent signaling events. (+info)
Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules.
(44/55031)
Zn-alpha2-glycoprotein (ZAG) is a soluble protein that is present in serum and other body fluids. ZAG stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. The 2.8 angstrom crystal structure of ZAG resembles a class I major histocompatibility complex (MHC) heavy chain, but ZAG does not bind the class I light chain beta2-microglobulin. The ZAG structure includes a large groove analogous to class I MHC peptide binding grooves. Instead of a peptide, the ZAG groove contains a nonpeptidic compound that may be implicated in lipid catabolism under normal or pathological conditions. (+info)
F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p.
(45/55031)
Ubiquitin-dependent proteolysis plays an important role in cell-cycle control [1] [2]. In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis [3] [4] [5] [6] [7] [8]. Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p [5] [6], while Skp1p binds to the F-box of Cdc4p [9]. In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p [10] [11] and the recently identified Sud1p/Pop2p [12], regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p. We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis. Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p. The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant. Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex. (+info)
The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins.
(46/55031)
Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution. (+info)
A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins.
(47/55031)
In recent years, a large number of coiled-coil proteins localised to the Golgi apparatus have been identified using antisera from human patients with a variety of autoimmune conditions [1]. Because of their common method of discovery and extensive regions of coiled-coil, they have been classified as a family of proteins, the golgins [1]. This family includes golgin-230/245/256, golgin-97, GM130/golgin-95, golgin-160/MEA-2/GCP170, giantin/macrogolgin and a related group of proteins - possibly splice variants - GCP372 and GCP364[2][3][4][5][6][7][8][9][10][11]. GM130 and giantin have been shown to function in the p115-mediated docking of vesicles with Golgi cisternae [12]. In this process, p115, another coiled-coil protein, is though to bind to giantin on vesicles and to GM130 on cisternae, thus acting as a tether holding the two together [12] [13]. Apart from giantin and GM130, none of the golgins has yet been assigned a function in the Golgi apparatus. In order to obtain clues as to the functions of the golgins, the targeting to the Golgi apparatus of two members of this family, golgin-230/245/256 and golgin-97, was investigated. Each of these proteins was shown to target to the Golgi apparatus through a carboxy-terminal domain containing a conserved tyrosine residue, which was critical for targeting. The domain preferentially bound to Rab6 on protein blots, and mutations that abolished Golgi targeting resulted in a loss of this interaction. Sequence analysis revealed that a family of coiled-coil proteins from mammals, worms and yeast contain this domain at their carboxyl termini. One of these proteins, yeast Imh1p, has previously been shown to have a tight genetic interaction with Rab6 [14]. On the basis of these data, it is proposed that this family of coiled-coil proteins functions in Rab6-regulated membrane-tethering events. (+info)
A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins.
(48/55031)
The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation [1]. Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain - see the paper by Munro and Nichols elsewhere in this issue) in a family of putative peripheral membrane proteins from lower and higher eucaryotes. The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus. Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p. By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), we have directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells. Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain. Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants. Thus, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus. (+info)