Identification and characterization of a lymphocytic Rho-GTPase effector: rhotekin-2.

Rhotekin belongs to the group of proteins containing a Rho-binding domain that are target peptides (effectors) for the Rho-GTPases. We previously identified a novel cDNA with homology to human rhotekin and in this study we cloned and characterized the coding region of this novel 12-exon gene. The ORF encodes a 609 amino-acid protein comprising a Class I Rho-binding domain and pleckstrin homology (PH) domain. Cellular cDNA expression of this new protein, designated Rhotekin-2 (RTKN2), was shown in the cytosol and nucleus of CHO cells. Using bioinformatics and RTPCR we identified three major splice variants, which vary in both the Rho-binding and PH domains. Real-time PCR studies showed exclusive RTKN2 expression in pooled lymphocytes and further purification indicated sole expression in CD4(pos) T-cells and bone marrow-derived B-cells. Gene expression was increased in quiescent T-cells but negligible in activated proliferating cells. In malignant samples expression was absent in myeloid leukaemias, low in most B-cell malignancies and CD8(pos) T-cell malignancies, but very high in CD4(pos)/CD8(pos) T-lymphoblastic lymphoma. As the Rho family is critical in lymphocyte development and function, RTKN2 may play an important role in lymphopoiesis.

ORP3 splice variants and their expression in human tissues and hematopoietic cells.

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells.

Using differential display polymerase chain reaction, a gene was identified in CD34(+)-enriched populations that had with low or absent expression in CD34(-) populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34(+) cells than in CD34(-) cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34(+)38(-) phenotype and was down-regulated with the proliferation and differentiation of CD34(+) cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.