Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.     

Sequence determinants for hnRNP I protein nuclear localization

hnRNP I, also referred to as polypyrimidine tract binding protein, is one of the proteins associated with nascent pre-mRNA in the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As for all karyophilic proteins, the nuclear import of hnRNP proteins requires specific sequence determinants that in many instances differ from the canonical import signal. In order to identify the sequences responsible for the nuclear localization, various hnRNP I portions were fused to a reporter protein (bacterial chloramphenicol acetyl transferase) and used in transient transfection assay. By this approach we identified a 60-amino-acid sequence located at the amino terminus of hnRNP I (designated NLD-I) that is both necessary and sufficient for nuclear localization. NLD-I represents a novel bipartite type of nuclear localization signal that bears no resemblance to other nuclear localization determinants so far identified in hnRNP proteins.     

Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4+8+ thymocytes upon TCR cross-linking. In contrast, expression of Tgtp is peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of approximately 50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation.     

A novel membrane protein is a mouse mammary tumor virus receptor

Mouse mammary tumor virus (MMTV) infects a number of different cell types, including mammary gland and lymphoid cells, in vivo. To identify the cellular receptor for this virus, a mouse cDNA expression library was transfected into Cos-7 monkey kidney cells, and those transfected cells able to bind virus were selected by using antibody against the virus's cell surface envelope protein, gp52. One clone isolated from a library prepared from newborn thymus RNA, called MTVR, was able to confer virus binding to both monkey and human cells; this binding was blocked by anti-MTVR antibody. Moreover, transfection of MTVR into CV1 cells rendered them susceptible to infection by a murine leukemia virus-based retrovirus vector pseudotyped with the MMTV envelope protein. An epitope-tagged MTVR cofractionated with cellular membranes. Coimmunoprecipitation of the MMTV envelope protein and a MTVR-rabbit Fc fusion protein showed that these two proteins bound to each other. The MTVR sequence clone is unique, shows no homology to known membrane proteins, and is transcribed in many tissues.     

The cloning and characterization of a new stress response protein. A mammalian member of a family of theta class glutathione s-transferase-like proteins

Using differential display, a cDNA fragment was identified as being overexpressed in a mouse lymphoma cell line that had gained resistance to cell death after exposure to a variety of agents used in cancer therapy. The full-length cDNA of 1.1 kb that was cloned contained an open reading frame coding for a previously unidentified 28-kDa mammalian protein, p28. p28 showed significant homologies to a large family of stress response proteins that contain a glutathione S-transferase (GST) domain. In correspondence with the sequence homology, p28 was found to bind glutathione; however, GST or glutathione peroxidase activity could not be demonstrated. Northern analysis of the mRNA of this protein showed abundant expression in mouse heart and liver tissues, whereas anti-p28 antibody binding identified p28 expression in mouse 3T3 cells and early passage mouse embryo fibroblasts. Subcellular protein fractionation revealed p28 localization in the cytoplasm, but with thermal stress p28 relocated to the nuclear fraction of cellular proteins. Based on sequence homology and protein activity we conclude that p28 acts as a small stress response protein, likely involved in cellular redox homeostasis, and belongs to a family of GST-like proteins related to class theta GSTs.     

Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family

We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70. The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains. Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs). Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins. We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family. Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis. Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis. From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.