Denis Banville

An amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) is mitogenic for osteoblast-like cells.

A peptide mitogen for cultured osteoblast-like cells was purified from serum-free conditioned culture medium of a human prostatic cancer cell line, PC-3. Based on amino acid sequencing and estimated molecular weight, this peptide was identified as an NH2-terminal fragment of urokinase-type plasminogen activator (uPA). Recombinant high molecular weight (HMW) uPA and the NH2-terminal growth factor domain (GFD) of uPA, but not low molecular weight (LMW) uPA (lacking the NH2-terminal region) stimulated [3H] thymidine incorporation and proliferation in osteoblast-like cells, and specific, competitive binding sites for HMW, but not LMW, uPA were demonstrable. These studies demonstrate the production of a mitogenic NH2-terminal fragment of uPA by a human prostatic cancer cell line which may be of importance in the pathogenesis of osteoblastic metastases.

Mechanism of action of somatostatin: An overview of receptor function and studies of the molecular characterization and purification of somatostatin receptor proteins☆

To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

Human α and β parvalbumins

alpha and beta parvalbumins are Ca(2+)-binding proteins of the EF-hand type. We determined the protein sequence of human brain alpha parvalbumin by mass spectrometry and cloned human beta parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. beta parvalbumin differs in 54 positions from alpha parvalbumin and lacks the C-terminal amino acid 109. From MS analyses of alpha and beta parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. alpha and beta parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase-chain-reaction techniques. Whereas alpha parvalbumin was found in a number of adult human tissues, beta parvalbumin was restricted to preterm placenta. The pattern of alpha parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, alpha parvalbumin was found in extrafusal and intrafusal skeletal-muscle fibres whereas, in man, alpha parvalbumin was restricted to the muscle spindles. Different functions for alpha and beta parvalbumins are discussed.

Retroviral long terminal repeat is the promoter of the gene encoding the tumor-associated calcium-binding protein oncomodulin in the rat

Oncomodulin is a small calcium-binding protein normally found only in extra-embryonic tissues such as the placenta, but whose presence in a variety of tumors has been documented. We have isolated the oncomodulin gene from a Buffalo rat genomic library. The rat gene is approximately 9000 bases in length and consists of five exons and four introns. The introns interrupt the coding sequence of oncomodulin in positions identical with those previously reported for the parvalbumin gene, indicating that the two genes are derived from a common ancestor. Analysis of the promoter sequence of the oncomodulin gene revealed that the gene is under the control of a solo long terminal repeat element related to intracisternal-A particles, a family of endogenous retroviral elements. This represents a unique example of a mammalian gene transcribed in normal and tumor cells, from a promoter of viral origin.

Solution Structure of the PDZ2 Domain from Cytosolic Human Phosphatase hPTP1E Complexed with a Peptide Reveals Contribution of the β2–β3 Loop to PDZ Domain–Ligand Interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.

ZRP-1, A ZYXIN-RELATED PROTEIN, INTERACTS WITH THE SECOND PDZ DOMAIN OF THE CYTOSOLIC PROTEIN TYROSINE PHOSPHATASE HPTP1E

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.

Protein-tyrosine Phosphatase SHP2 Is Positively Linked to Proteinase-activated Receptor 2-mediated Mitogenic Pathway

Proteinase-activated receptor-2 (PAR2), a new member of family of the G protein-coupled receptors, is activated by proteolytic cleavage of its extracellular amino terminus, a mechanism similar to that used by the thrombin receptor. It has been suggested that PAR2 has a potential role in the late phases of the acute inflammatory response and in tissue repair and/or skin-related disorders. Here we demonstrate that the agonist peptide (SLIGRL) stimulated c-fos-mediated mitogenic activation and tyrosine phosphorylation of cellular proteins. One of the tyrosine-phosphorylated proteins was identified as an Src homology-2 domain-containing protein-tyrosine phosphatase, SHP2. The stimulatory effect of the agonist peptide on early gene transcription was markedly blocked by pertussis toxin treatment whereas the induced tyrosine phosphorylation of SHP2 was completely abolished by the drug. More importantly, while expression of wild-type SHP2 enhanced the agonist-stimulatory mitogenic activity, overexpression of a catalytically inactive mutant of SHP2 strongly suppressed the stimulatory effect of the agonist peptide on both early gene transcription and DNA synthesis. These results suggest that SHP2 acts as a positive regulator linked to the PAR2-mediated mitogenic pathway coupled to a pertussis toxin-sensitive heterotrimeric G protein. Demonstration of SHP2 as a positive mediator in a G protein-coupled, receptor-mediated signaling adds to our understanding of the function of both SHP2 and PAR2 in the signaling pathway.

mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2

The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily. By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E. The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes. The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction. This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor. When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell-cell interactions. In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9. The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33. Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.

Parathyroid hormone-like peptide: molecular characterization and biological properties.

Parathyroid hormone-like peptide (PLP) is a newly discovered novel peptide that interacts with renal and osseous parathyroid hormone (PTH) receptors through its amino-terminal sequence, which is homologous with PTH. Human PLP is the product of a complex single-copy gene, existing in several isoforms which result from alternative exon usage. The peptide was implicated as a causal agent in the hypercalcemia associated with a variety of malignancies, but it may also modulate calcium homeostasis in some normal physiological states and mediate cellular growth or differentiation.

Identification and Characterization of S2V, a Novel Putative Siglec That Contains Two V Set Ig-like Domains and Recruits Protein-tyrosine Phosphatases SHPs

We describe the molecular cloning and characterization of S2V, a novel sialic acid binding immunoglobulin-like lectin. The cDNA of S2V encodes a type 1 transmembrane protein with four extracellular immunoglobulin-like (Ig-like) domains and a cytoplasmic tail bearing a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif. A unique feature of S2V is the presence of two V-set Ig-like domains responsible for the binding to sialic acid, whereas all other known siglecs possess only one. S2V is predominantly expressed in macrophage. In vivo S2V was tyrosine-phosphorylated when co-expressed with exogenous c-Src kinase. Upon tyrosine phosphorylation, S2V recruits both Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2, two important inhibitory regulators of immunoreceptor signal transduction. These findings suggest that S2V is involved in the negative regulation of the signaling in macrophage by functioning as an inhibitory receptor. When expressed in COS-7 cells, S2V was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that S2V may function through cell-cell interaction.