Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells.

Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Assembly of multimeric von Willebrand factor directs sorting of P-selectin.

We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.

Small GTP-binding protein RalA associates with Weibel-Palade bodies in endothelial cells.

In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules. so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Small GTP-binding proteins in human endothelial cells.

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP-binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP-binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope-tagged Rab30 cDNA in monkey kidney COS-1 cells. Immunoelectron-microscopy of transfected COS-1 cells indicated that Rab30 is associated with Golgi stacks.

Conformational analysis of m4(2)C-m4(2)C-m6(2)A: a chemically modified 3'-acceptor end of tRNA, studied by NMR and CD methods.

A study on the conformation of the title compound, C-C-A, and on its constituent dinucleotides is presented. 1H-NMR spectra at 360 and 500 MHz were completely assigned by decoupling experiments. Computer simulation of the spectra yielded precise proton-proton and proton-phosphorus coupling constant values. The coupling constants are analyzed in terms of torsion angles and of N- and S-type sugar pucker. 31P-NMR spectra gave some information about P-O backbone torsion angles alpha and zeta. CD spectroscopy was used to obtain insight in the base-base interaction. The C(1) and C(2) unit in C-C-A show normal preference for N-type conformation of the sugar ring, whereas the A(3) residue appears rather biased towards the S-conformation. The zeta and alpha backbone torsion angles in the C-C phosphodiester linkage in C-C-A appear to assume normal g-, g- conformation, the zeta, alpha combination in the C-A linkage is proposed to have a g+, t conformation. In the C-C fragment in C-C-A a regular stack is indicated; it is suggested that the C-A part adopts an unusual antiparallel base stack.

Influence of the 2'-hydroxyl group and of 6-N-methylation on the conformation of adenine dinucleoside monophosphates in solution. A nuclear magnetic resonance and circular dichroism study.

Proton NMR studies at 360 MHz are reported on the adenine dinucleoside monophosphates N6-dimethyladenyly(3'-5')-N6-dimethyladenosine (m(6)(2)Apm(6)(2)A), ApA, rApdA, dAprA and on the methyl phosphate esters of the monomers m(6)(2)Ap, pm(6)(2)A, Ap and pA. Complete 1H-NMR spectral assignments are given. The dimers were also investigated by means of circular dichroism to obtain accurate thermodynamic parameters of the stacking equilibrium. With the aid of the thermodynamic data NMR coupling constants are extrapolated to values appropriate to the stacked conformers. A modernized version of pseudorotation analysis is used to delineate the conformational behaviour of the ribose and 2'-deoxyribose rings. It is shown that the unmethylated dimers can be arranged in two groups (dApdA/dAprA vs ApA/rApdA) according to their melting temperatures. ApA and the fully N6-methylated dimer m(6)(2)Apm(6)(2)A prefer to adopt the classical right-handed N-N stacked conformation. Both dimers with a 2'-deoxyribose ring at the 5'-OH end (dApdA and dAprA) behave similarly and occur in solution as a 75:25 mixture of S-S and S-N stacked states. The fully stacked hybrid dimer rApdA displays an unexpectedly large amount of S conformers (greater than 40%) in both sugar rings. This finding is rationalized by the postulation of a right-handed helical S-S stacked state on the basis of NMR and circular dichroic data.