The human S mu bp-2, a DNA-binding protein specific to the single-stranded guanine-rich sequence related to the immunoglobulin mu chain switch region

We have cloned the cDNA encoding the human homologue of S mu bp-2, which binds to single-stranded DNA with 5'-phosphorylated guanine-rich sequences related to the immunoglobulin mu chain switch (S mu) region. The deduced amino acid sequences of the mouse and human S mu bp-2 are 76.5% homologous and contain motifs conserved among helicases. We have identified a domain essential for DNA binding at residues 638-786. The binding domain is less conserved (63% homologous) than the putative catalytic domain of N-terminal half containing most of the helicase motifs (85% homologous). The human and mouse S mu bp-2 have similar, although slightly different, binding specificities. Although the mouse S mu bp-2 preferentially binds to the mouse S mu motif (GGGGT), the human S mu bp-2 binds equally well to the human (GGGCT) and mouse S mu motifs. The human S mu bp-2 gene was mapped to chromosome 11 q13.2-q13.4 by in situ hybridization.     

Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.     

A target of phosphatidylinositol 3,4,5-trisphosphate with a zinc finger motif similar to that of the ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains

We have purified a protein that binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] using beads bearing a PtdIns(3,4,5)P3 analogue. This protein, with a molecular mass of 43 kDa, was termed PtdIns(3,4,5)P3-binding protein. The partial amino acid sequences were determined and a full-length cDNA encoding the protein was isolated from bovine brain cDNA library. The clone harbored an open reading frame of 373 amino acids which contained one zinc finger motif similar to that of ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains. The entire sequence was 83% similar to centaurin alpha, another PtdIns(3,4,5)P3-binding protein. The protein bound PtdIns(3,4,5)P3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3-phosphate suggesting that the binding to PtdIns(3,4,5)P3 was specific. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each pleckstrin homology domain. Introduction of both mutations abolished the activity. These results suggest that this new binding protein binds PtdIns(3,4,5)P3 through two pleckstrin domains present in the molecule.     

Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation

An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein-protein interactions.