[Small Noncoding 4.5SH and 4.5SI RNAs and Their Binding to Proteins].

The functions of small noncoding RNAs 4.5SH and 4.5SI found in murine-like rodents are unclear. These RNAs synthesized by RNA polymerase III are widely expressed in rodent organs and tissues. Using crosslinking assays, it was shown that approximately half of all 4.5SI and 4.5SH RNA molecules were bound to proteins provisionally called X and Y, respectively. An immunoprecipitation experiment showed that both these RNAs were associated with the La protein, which did not crosslink to them. The termini of 4.5SI RNA form a long duplex stem, which makes the molecule more stable than 4.5SH RNA. Modification of the 5'-end sequence destructing the stem of 4.5SI RNA altered its protein-binding properties; after the 3'-end sequence was changed to the complementary, both the stem structure and the RNA binding to protein X were restored. Presumably, this protein plays a role in increasing the half-life of 4.5SI RNA.

[A rare case of metastatic tumor of heart from bladder cancer].

The article presents a case report of metastatic heart damage which developed in association with urothelial bladder carcinoma in a 79-year old female patient. Various masses may be found in the heart. In tumors, a secondary damage to the heart is observed much more frequently than a primary damage; however, metastasis of bladder carcinoma to the heart is extremely rare. Of interest is the fact of metastatic damage to all layers of the heart, including the endocardium, pericardium, and myocardium.

[4.5SI and 4.5SH RNAs: Expression in various rodent organs and abundance and distribution in the cell].

Studying the structure, functions, and cell physiology of small RNAs remains important. The 4.5SI and 4.5SH small RNAs, which were among the first to be discovered and sequenced, share several features, i.e., they are both approximately 100 nt in size, are synthesized by RNA polymerase III, and are found only in rodents of several related families. Genes coding for these RNAs are evolutionarily related to short interspersed elements (SINEs). However, the two RNAs differ in nucleotide sequence, half-life in the cell, and the organization of their genes in the genome. Although the 4.5SI and 4.5SH RNAs have been identified more than three decades ago, several aspects of their metabolism in the cell are still poorly understood. The 4.5SI and 4.5SH RNA levels were measured in various organs of three rodent species (mouse, rat, and hamster). Both of the RNAs were found to occur at high levels, which were much the same in different organs in the case of the 4.5SI RNA and varied among organs in the case of the 4.5SH RNA. Both 4.5SI and 4.5SH RNAs demonstrated a predominantly nuclear localization with a detectable presence in the cytoplasm. The copy number per cell for the RNAs was estimated at 0.4-2.4 × 10^(6). A quantitative study for the 4.5SI and 4.5SH RNAs was performed for the first time and resolved a number of contradictions in data from other studies.

The insulin-like growth factor I receptor-induced interaction of insulin receptor substrate-4 and Crk-II.

Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y(700), Y(717), Y(743), and Y(779)). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption.

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth.

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3'-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3'-kinase or mitogen-activated protein kinase pathways.

Interplay of the proto-oncogene proteins CrkL and CrkII in insulin-like growth factor-I receptor-mediated signal transduction.

The closely related proto-oncogene proteins CrkII and CrkL consist of one SH2 and two SH3 domains and share 60% overall homology with the highest identity within their functional domains. In this study we show that CrkL and CrkII may play overlapping but different roles in insulin-like growth factor (IGF)-I receptor-mediated signal transduction. While both proteins are substrates involved in IGF-I receptor signaling, they apparently demonstrate important different properties and different biological responses. Evidence supporting this hypothesis includes (a) the oncogenic potential of CrkL versus the absence of this potential in CrkII overexpressing cell lines, (b) the inhibition of IGF-I-dependent cell cycle progression by overexpression of CrkII, and (c) the differential regulation of the phosphorylation status of selective proteins in CrkII and CrkL overexpressing cell lines. In addition we demonstrate the specific association of CrkL and CrkII with the newly characterized IRS-4 protein, again in a differential manner. Whereas CrkL strongly interacts with IRS-4 via its SH2 and N-terminal SH3 domains, CrkII interacts only via its SH2 domain, possibly explaining the unstable nature of IRS-4-CrkII association. The results obtained allow us to propose a unique mechanism of CrkL and CrkII tyrosine phosphorylation in response to IGF-I stimulation. Thus these highly homologous proteins apparently possess structural features that allow for the differential association of each protein with different effector molecules, thereby activating different signaling pathways and resulting in unique biological roles of these proteins.

Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals.

We investigated cellular proliferation, the transforming activity, and activation of known signal transduction pathways in NIH-3T3 cells stably expressing insulin-like growth factor-I receptors (IGF-IRs) with amino acid substitutions in the carboxy(C)-terminal domain. The mutant receptors contained substitutions of both tyrosines 1250 and 1251 with phenylalanine and histidine (amino acids present in the analogous positions in the insulin receptor), as well as phenylalanine 1310 replaced by tyrosine (IsY clones) to resemble the placement of tyrosine residues in the C-terminal domain of the insulin receptor. As a control for the IsY clones, a second mutant receptor was expressed with a substitution of phenylalanine 1310 with tyrosine only (DBY clones). Clones expressing IGF-IRs with the IsY substitutions had a significantly slower rate of growth compared with cells expressing an equivalent number of wild-type IGF-IRs (NWT). In contrast, the DBY clones showed relatively normal growth rates. Cells with wild-type IGF-IR demonstrated a transformed phenotype in soft agar assays. The IsY clones lost the transforming ability of the wild type IGF-IR, whereas DBY clones formed colonies. IGF-I-stimulated autophosphorylation of the IGF-IR and tyrosine phosphorylation of IRS-1 and SHC, known substrates in the IGF-IR signal transduction pathway, were studied. Mutated IGF-IRs (IsY and DBY) did not alter the IGF-I-induced tyrosine phosphorylation of these proteins. Furthermore, the mutated IGF-IRs did not alter Grb2 association with phosphorylated IRS-1 and SHC. IGF-I stimulation of Crk-II phosphorylation, a novel substrate of the IGF-IR, was similar in cells expressing mutated and wild-type IGF-IRs. IGF-I-induced activation of phosphatidylinositol (PI) 3'-kinase was equivalent in cells expressing either mutant or wild-type IGF-IRs. These data suggest that the IGF-IR mediates, at least in part, cellular proliferation and increased transforming ability through its C-terminal domain. The exact postreceptor signaling pathway(s) involved have yet to be fully elucidated.

Alternative leader sequences in insulin-like growth factor I mRNAs modulate translational efficiency and encode multiple signal peptides.

Rat insulin-like growth factor I (IGF-I) mRNAs contain multiple 5'-untranslated regions due to the use of leader exons transcribed from several transcription initiation sites and to alternative splicing within leader exon 1. Synthetic RNAs with 5'-ends corresponding to the use of exon 1 transcription initiation sites were translated in vitro into prepro-IGF-I peptides initiated at a Met-48 codon in exon 1 or a Met-22 codon in exon 3, and RNAs with a 5'-end corresponding to the major exon 2 transcription start site were translated into a prepro-IGF-I peptide initiated at a Met-32 codon in exon 2. All forms of prepro-IGF-I were processed by canine pancreatic microsomes, suggesting that all these prepeptides function as signal peptides. The translational efficiency of IGF-I RNAs was inversely proportional to the length of the 5'-untranslated region. Mutation of the first of three upstream AUG codons in exon 1, which potentially initiates a 14-amino acid open reading frame, did not affect prepro-IGF-I translation. The other two AUG codons are immediately followed by stop codons. The absence of both upstream AUG codons in a completely spliced exon 1-derived RNA enhanced the in vitro and in vivo translatability of this RNA as compared with the full-length RNA. Mutation of the downstream initiation codon in particular increased translational efficiency in vitro and in intact cells, suggesting that an inefficient reinitiation event at the Met-48 codon contributes to the poorer translation of IGF-I mRNAs in which these upstream AUGUGA motifs occur. We conclude that IGF-I mRNAs potentially encode multiple forms of preproIGF and that specific differences in their 5'-untranslated regions provide a molecular basis for translational control of IGF-I biosynthesis.

Isolation of a second nonallelic insulin-like growth factor I gene from the salmon genome.

We have characterized a second nonallelic insulin-like growth factor-I (IGF-I) gene in the chum salmon (Oncorhynchus keta) genome. This gene, IGF-I.2, differs from the previously described chum salmon IGF-I gene, IGF-I.1, in the E peptide-coding portion of exon 3; specifically, the IGF-I.2 gene lacks one codon present in the IGF-I gene and contains two potential splice donor sites at the 3' end of exon 3 rather than the single, more distal site present in the IGF-I.1 gene. The expression of these two IGF-I genes could give rise to as many as six IGF-I mRNA species, each of which would encode a unique E-peptide moiety of the IGF-I prohormone. Thus, the presence of multiple, distinct IGF genes adds an additional level of complexity to IGF-I gene expression and IGF-I biosynthesis in salmon.

A growth hormone pseudogene in the salmon genome.

Representatives of the fish family Salmonidae were reported to possess two nonallelic growth hormone (GH)-encoding genes. In addition to those, we found a third GH-like sequence in a chum salmon genomic DNA library. A number of point mutations and large deletions abolished the possibility of expressing this sequence, showing that the chum salmon genomic DNA contains a GH pseudogene besides functional GH genes.

Structure of the chum salmon insulin-like growth factor I gene.

Insulin-like growth factor I (IGF-I) plays a major role in development and metabolism. Currently, the cDNA-derived primary structure of IGF-I is known for some mammals and for chicken, frog, and salmon. Additionally, the organization of the human, rat, and chicken IGF-I genes has been established. The investigation of IGF-I gene structure in fish would extend the evolutionary picture for this hormone and facilitate our understanding of the features of the IGF-I gene that are common to all vertebrate species. The cloned chum salmon IGF-I gene appears to be much more compact than the mammalian and avian genes, being less than 20 kb in length. As in other species, however, the mature IGF-I peptide appears to consist of 70 amino acids and is encoded by exons 2 and 3. Intriguingly, exon 1-encoded 5'-untranslated region sequences are highly conserved, while the coding sequences at the 3' end of the same exon are less conserved. The amino terminus of the signal peptide is four amino acids shorter than in the mammalian and avian peptides. The end of the B domain, the C, A, and D domains, and the first part of the E peptide are encoded by exon 3, but the exon 3-encoded E peptide sequence is 27 amino acids longer than in other species. These extra 27 amino acids, encoded by both coho and chum salmon cDNAs, may be deleted by alternative splicing, as suggested from the sequence of a coho salmon IGF-I cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Phylogeny of the insulin-like growth factors (IGFs) and receptors: a molecular approach.

The IGFs (IGF-I and IGF-II) are essential for normal mammalian growth and development. Their actions are mediated primarily by their interactions with the type I IGF receptor (IGF-I receptor), a transmembrane tyrosine kinase. The ligands and the IGF-I receptor are structurally related to insulin and to the insulin receptor, respectively. Analysis of evolutionary conservation has often provided insights into essential regions of molecules such as hormones and their receptors. The genes for insulin and IGFs have been partially characterized in a number of vertebrate species extending evolutionarily from humans as far back as fish. The sequences of the exons encoding the mature insulin and IGF peptides are highly conserved among vertebrate species, and IGF-I-like molecules are found in species whose origins extend back as much as 550 million years. The insulin receptor is also highly conserved in vertebrate species, and an insulin-receptor-like molecule has been characterized in Drosophila. In contrast, IGF-I receptors have only been characterized in mammalian species and partially studied in Xenopus, in which the tyrosine kinase domain is highly conserved. Studies are presently being undertaken to analyze in more detail the regulation of the genes encoding this important family of growth factors and the structure/function relationships in the gene products themselves.

[Nucleotide sequence of chum salmon preproinsulin gene]. / Nukleotidnaia posledovatel'nosti gena preproinsulina kety.

The nucleotide sequence of chum salmon preproinsulin gene isolated from the phage library of genomic sequences is presented. The transcription initiation site of the gene was experimentally determined. The resolved upstream region contains a TATA-promoter sequence and CAAT-like-box sequence. The salmon insulin gene is split and contains two introns. The first intron, 393 b.p. long, is situated in the 5'-untranslated region. The second intron, 287 b.p. long, interrupts triplet coding for the seventh amino acid residue of the C-peptide. Thus, the overall structure of the insulin gene appears to be closely related to other sequenced insulin genes, including those of birds and mammals. Both introns in the salmon insulin gene are flanked by GC...AG pairs in contrast to GT...AG, the rule common for the majority of genes. Analysis of the cloned salmon insulin gene and cDNAs reveals a high frequency of polymorphic differences, up to 6% in the translated region. The nucleotide sequence of the 3'-untranslated region of the gene is only 70% homologous to the corresponding region in the previously cloned salmon preproinsulin cDNAs. These differences may implicate for the presence of more than one insulin gene per haploid genome in salmon.