Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.     

Molecular cloning,chromosome mapping and characterization of the mouse CRTH2 gene,a putative member of the leukocyte chemoattractant receptor family

We have cloned the mouse CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) gene encoding a putative leukocyte chemoattractant receptor, of which human homologue is expressed selectively in Th2 but not in Th1 clones among T cell clones. The deduced amino-acid sequence of mouse CRTH2 bears 77% identity with its human homologue. Phylogenetic analysis suggested that both mouse and human CRTH2 are closely related to the N-formyl peptide receptor and the C5a receptor among leukocyte chemoattractant receptors. The mouse CRTH2 gene was mapped on chromosome 19c with FISH, where no other genes for leukocyte chemoattractant receptors are mapped. RT–PCR analysis revealed that mouse CRTH2 mRNA is expressed in various cell lineages, including both hematopoietic and non-hematopoietic cell lines. Expression was also observed in liver, lung, kidney, brain, heart, thymus, and spleen. These results suggest that mouse CRTH2 functions in a variety of cells, making the effects of CRTH2 pleiotropic.     

Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family

Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino acid motifs and, apart from an amino acid stretch that appears unique among mammalian sialidases, shows a high degree of homology for NEU2 and the plasma membrane-associated (NEU3) sialidases. RNA dot-blot analysis showed a low but wide expression pattern, with the highest level in liver. Transient transfection in COS7 cells allowed the detection of a sialidase activity toward the artificial substrate 4MU-NeuAc in the acidic range of pH. Immunofluorescence staining and Western blot analysis demonstrated the association of NEU4 with the inner cell membranes.     

Molecular cloning and characterization of Xenopus RGS5

We identified six genes that encode putative RGS proteins (XRGSI-VI) in developing Xenopus embryos using PCR amplification with degenerate primers corresponding to the conserved region (RGS domain) of known RGS proteins. RT-PCR analysis revealed that mRNAs of these XRGSs are differentially expressed during embryogenesis. At stage 1, only XRGSII mRNA was detected. On the other hand, expression of XRGSVI mRNA increased apparently at stage 14 and expression of three of other XRGS (III, IV, V) elevated between stage 25 and 40. To further characterize XRGS proteins expressed in Xenopus embryos, we isolated a cDNA clone for XRGSIII. Based on determined nucleotide sequence, XRGSIII was considered as a Xenopus homologue of mammalian RGS5 (XRGS5). Genetic analysis using the pheromone response halo assay showed that expression of XRGS5 inhibits yeast response to alpha-factor, suggesting that XRGS5 negatively regulates the G-protein-mediated signaling pathway in developing Xenopus embryos.     

Molecular cloning, expression and in vitro functional characterization of Myb-related proteins in Xenopus.

Two cDNAs encoding Myb-related proteins have been cloned from Xenopus laevis and they have been termed Xmyb1 and Xmyb2. The Xmyb1 cDNA clone codes for an open reading frame of 733 amino acids and exhibits a high degree of similarity over the entire predicted protein sequence with the human B-Myb protein. Xmyb2 is a partial cDNA clone encoding three copies of amino-terminal tandem repeat elements typical for the Myb DNA-binding domain. The predicted protein sequence is most closely related to the human A-Myb gene product. In vitro translation of two deletion mutants of Xmyb1, truncated in the 3'-portion of the open reading frame, results in protein products which cross-react with polyvalent as well as monoclonal antibodies directed against the human c-Myb protein. The same two XMyb1 proteins, which both contain the complete set of aminoterminal repeats, specifically bind to the c-Myb-specific DNA binding sequence as evidenced by electrophoretic mobility shift analysis in vitro. RNA expression profiles of Xmyb1 and -2 are very different from each other; Xmyb1 is present throughout oogenesis and early Xenopus embryogenesis; in adult tissue it is primarily detected in blood. In contrast, Xmyb2 is expressed at only very low levels during oogenesis, not detectable in embryonic RNA preparations, and in adult tissue it is predominantly expressed in testis, with only a very low level seen in blood.     

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.     

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.     

The Ran binding protein RanBPM interacts with Axl and Sky receptor tyrosine kinases

Axl belongs to a particular subfamily of transmembrane receptor tyrosine kinases, the biological ligand for which is the growth/survival factor Gas6. However, little is known about the molecular mechanisms for Axl activation and signal transduction. We have previously identified a novel interaction between the intracellular domain of Axl and Ran binding protein in microtubule organising centre (RanBPM). In the present study, we investigated further the nature of the RanBPM interaction with Axl. A wide distribution of RanBPM mRNA expression in human tissues and various human cancer cell lines was detected. The strength of interaction of both proteins in yeast was comparable to that with the other Axl-binding proteins phosphatidylinositol 3-kinase and Grb2. A truncated version of RanBPM with the SPRY-LisH domain region omitted failed to interact with Axl in yeast. RanBPM was also found to interact in yeast with the Axl homologue, Sky/Tyro3. The interaction between Axl intracellular domain and RanBPM was reproduced in coimmunoprecipitation experiments in both cell-free and mammalian cell systems. Furthermore, coimmunoprecipitation revealed endogenous Axl and RanBPM to interact in several mammalian cell lines in a constitutive manner. Stimulation of COS cells with Gas6 caused increased Axl tyrosine phosphorylation although appeared not to influence the RanBPM-Axl association. In conclusion, we have identified and characterised a novel interaction between RanBPM and the related receptor tyrosine kinases, Axl and Sky. This novel insight into the signalling interactions of Axl and Sky may shed further light on their suspected roles in tumourigenesis, inflammation as well as other cell proliferative diseases.     

Molecular cloning, structural characterization and functional expression of the human substance P receptor.

A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-1-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP much greater than neurokinin A greater than neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver.     

Molecular cloning and embryonic expression of the Xenopus Arnt gene

Versican, a proteoglycan recently implicated in hair follicle induction, has been shown to influence axon outgrowth in vitro and in vivo. We used immunohistochemistry to study the relationship between versican expression and innervation, during rat vibrissa follicle development and the adult hair cycle. During development, nerve fibres were commonly associated with areas of weak versican expression, and the path of axons appeared to be delineated by sharp boundaries of versican expression. Versican expression changed in the lower follicle dermis during the adult hair follicle cycle but remained strong around the follicle neck reflecting the constant innervation. Our observations show a correlation between versican expression and peripheral innervation indicating that versican may have a dual role in hair follicle biology.     

Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.     

Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family

Using a human placenta cDNA library, we cloned a novel member belonging to the scavenger receptor family. Complementary DNA of this clone encodes a type II transmembranous glycoprotein containing a collagen-like domain, which are typical structural characteristics of scavenger receptor class A. This protein also contains a C-type lectin/carbohydrate recognition domain (C-type CRD) located at the C-terminus. We designated this as Scavenger Receptor with C-type Lectin (SRCL) type I. We also isolated human SRCL type II, which lacks the C-type CRD. Northern blot analysis revealed that hSRCL type I and type II mRNAs are abundantly expressed in adult human tissues. When hSRCL type I and type II were expressed in CHO-K1 cells, they were localized in the plasma membrane forming clusters on the surface. Ligand-binding studies of CHO-K1 cells expressing hSRCL type I and type II demonstrated their specific binding capacity in Escherichia coli and Staphylococcus aureus. These results indicate that hSRCL is a novel bacteria-binding receptor containing a C-type CRD and this receptor may play an important role in host defense.     

Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.

In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.