Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.

Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit. The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR), In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl, Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways. Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes. In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper. In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers. Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl, The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants. In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbl's signaling functions. Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbl's signaling function, modulating the activity of Cbl itself and/or affecting Cbl's associations with other signaling proteins in the cell.

During bone resorption, osteoclasts acidify the extracellular bone resorbing compartment via a vacuolar H+-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the composition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library constructed from highly purified chicken osteoclasts. Comparison of the predicted amino-acid sequence with the published sequences of isoforms of V-ATPase B-subunits from other sources revealed that the chicken osteoclast B-subunit is brain type and not kidney type. Furthermore, only clones encoding the brain type isoform of subunit B could be generated by PCR from a cDNA library prepared from human osteoclastoma osteoclast-like cells. Northern blot analysis revealed that two B-subunit mRNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mononuclear cells, brain and kidney, although the relative amounts of these two transcripts were different in each tissue. In brain, the 3.5-kb mRNA was predominantly expressed. In bone marrow cells, the levels of the 1.7-kb mRNA were higher than in other tissues and expression of this message was increased by 1,25-dihydroxyvitamin d-3, suggesting that this mRNA is specifically upregulated during osteoclast differentiation. These results indicate that the B-subunit isoforms present in the catalytic domains of the osteoclast and kidney V-H+-ATPases are different and further suggest that selective expression of isoforms of the B-subunit in these two tissues could provide a structural basis for some of the differences we have reported in the pharmacology and catalytic properties of these two enzymes.

We have identified a second isoform of the catalytic A subunit of the vacuolar H+ pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical A1 isoform. The A1-specific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the P-loop) identified in this polypeptide, as well as the pharmacologically relevant Cys(254). In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2- and A1-specific exons, as either acceptor (in A1) or donor (in A2) splice site.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5′ termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate

An RNA molecule, 340 nucleotides in length and designated H1 RNA, copurifies with RNase P activity from extracts of HeLa cells or isolated HeLa cell nuclei. When the genomic DNA of various organisms is probed with H1 cDNA in Southern hybridization assays, only mammalian DNA gives a positive signal. The gene coding for H1 RNA in human cells is present in one to three copies per cell. The nucleotide sequence of H1 RNA, which shows little homology to the known sequences of its analogs from prokaryotes and yeast, can be drawn as a two-dimensional, hydrogen-bonded structure that resembles similar structures proposed for the RNA subunit of RNase P from these other sources. Part of the hypothetical structure is virtually identical to structures that can be drawn for analogous RNAs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S. octosporus.

Sera from certain patients with systemic lupus erythematosus (SLE) and related rheumatic diseases contain antibodies that selectively deplete extracts of HeLa cells of RNase P activity. Most of the sera that recognize RNase P, an endoribonuclease with an essential RNA subunit, also contain antibodies against another small ribonucleoprotein known as the Th antigen. A species of RNA about 400 nucleotides in length is the only RNA species found in common in all immunoprecipitates prepared with anti-RNase P antibodies. The discovery of antibodies against RNase P defines a major class of antibodies produced by patients with autoimmune disease.

Ribonuclease P (RNase P) is an endoribonuclease required for the maturation of the 5′ termini of tRNAs from their precursors (for review, see Altman et al. 1986). This enzyme, which engages in a single cleavage event to generate a 5′ phosphate in its tRNA products in vivo and in vitro, has been best characterized in extracts of Escherichia coli. It has a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein; Guerrier-Takada et al. 1983). Under certain conditions in vitro, the RNA subunit alone can carry out the cleavage of tRNA precursors. This reaction, catalyzed by M1 RNA, has all the features of a true enzymatic reaction, and its mechanism differs from those of the reactions governed by other RNA enzymes (Table 1). Although RNase P generates the same end groups in its products as do group I self-splicing introns during the cleavage step performed by these introns,...