Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor.

Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia.

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

A 500-kb sequence-ready cosmid contig and transcript map of the MEN1 region on 11q13.

We have generated a transcript map of an approximately 1.2-Mb region from human chromosome band 11q13 between the loci VEGFB and CAPN1, which flank the multiple endocrine neoplasia type 1 (MEN 1) locus. In total, we isolated 144 cosmids from this region and generated a sequence-ready cosmid contig of the approximately 500-kb region between the neurexin locus and D11S2196E. We identified 54 genes/ESTs by sample sequencing and have constructed a transcript map of this region. Genes were found to be clustered in three regions, and one of these genes was identical to the recently identified MEN1 locus. Relative to the latter, we have mapped the positions of 13 known genes, 18 genes which show homology to genes from humans or other organisms, and 22 genes/ESTs that appear novel. In addition, we have ascertained the directions of transcription of some of these genes and have determined intergenic distances between many loci. Full characterization of some of these genes, as well as the novel ESTs, will be useful in identifying candidate genes for other diseases known to map to this chromosomal region.

Characterization of the mouse Men1 gene and its expression during development.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

Mutation analysis of the MEN1 gene in multiple endocrine neoplasia type 1, familial acromegaly and familial isolated hyperparathyroidism.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disease characterized by neoplasia of the parathyroid glands, the endocrine pancreas, and the anterior pituitary gland. In addition, families with isolated endocrine neoplasia, notably familial isolated hyperparathyroidism (FIHP) and familial acromegaly, have also been reported. However, whether these families constitute MEN 1 variants or separate entities remains speculative as the genetic bases for these diseases are unclear. The gene for MEN 1 has recently been cloned and characterized. Using single strand conformation analysis (SSCA) and sequencing, we performed mutation analysis in: a) a total of 55 MEN 1 families from 7 countries, b) 13 isolated MEN 1 cases without family history of the disease, c) 8 acromegaly families, and d) 4 FIHP families. Mutations were identified in 27 MEN 1 families and 9 isolated cases. The 22 different mutations spread across most of the 9 translated exons and included frameshift (11), nonsense (6), splice (2), missense mutations (2), and in-frame deletions (1). Among the 19 Finnish MEN 1 probands, a 1466del12 mutation was identified in 6 families with identical 11q13 haplotypes and in 2 isolated cases indicating a common founder. One frameshift mutation caused by 359del4 (GTCT) was found in 1 isolated case and 4 kindreds of different origin and haplotypes; this mutation therefore represents a common "warm" spot in the MEN1 gene. By analyzing the DNA of the parents of an isolated case one mutation was confirmed to be de novo. No mutation was found in any of the acromegaly and small FIHP families, suggesting that genetic defects other than the MEN1 gene might be involved and that additional such families need to be analyzed.

Characterisation of a new human and murine member of the DnaJ family of proteins.

We report the characterisation of a human gene, designated MCG18 (multiple endocrine neoplasia type 1 candidate gene 18), that encodes a new member of the DnaJ family of proteins. Database searches indicate that MCG18 also has the locus name HSPF2. MCG18 lies 250bp centromeric of the VRF/VEGFB gene on chromosome 11q13. The MCG18 cDNA is predicted to encode a 241 amino acid product that has partial homology to Escherichia coli dnaJ in that it contains the J domain. However, MCG18 has greatest similarity to a functionally undefined protein from Caenorhabditis elegans, both of which are predicted to have a membrane-spanning region adjacent to their J domains. The cDNA encoding the murine homolog (Mcg18) was also cloned and sequenced, and the encoded protein shares 81% similarity to MCG18. The coding region of MCG18 is interrupted by 4 introns and the mRNA is expressed as a 1.4kb message in all tissues examined, including those derived from the breast, ovary, bladder, lung and keratinocytes.

Analysis of the promoter region of the human VEGF-related factor gene.

We have characterised the promoters of the human and murine VRF (vascular endothelial growth factor (VEGF) related factor) gene. A series of deletions were made of a 553-bp region 5' of the VRF initiation codon and were used in a luciferase reporter gene assay to determine the minimal promoter of the VRF gene. The region between base pairs -443 and -195 was sufficient to mediate transcription in lymphocytes and the region between -550 and -443 enhanced this promoter activity. Primer extension studies identified two regions of transcription initiation, both of which are preceded by Sp1, AP-2 and Egr-1 transcription factor binding sites. The VRF promoter is similar to VEGF in that it is associated with a CpG island, contains sites for Sp1 and AP-2, and lacks a TATA box. However, it has marked differences in that the promoter contains Egr-1 sites and lacks both hypoxia-inducible factor-1 and AP-1 sites. These data may indicate that expression of these two growth factors is regulated by different physiological stimuli.

Characterization of the murine VEGF-related factor gene.

We describe here the molecular cloning and characterization of the murine homolog of the human vascular endothelial growth factor-related factor (VRF) gene. cDNAs for two alternatively spliced forms of the murine vrf gene have been isolated, the putative translation products of which differ at their carboxyl termini due to a shift in reading frame caused by insertion, or lack thereof, of exon 6, in a similar fashion to human VRF (hVRF). The message lacking exon 6 encodes a protein (mvrf167) with 86% identity and 92% conservation of amino acid residues with hVRF. The protein coding region of the gene spans approximately 5kb of genomic DNA and is composed of 8 exons ranging in size from 36 to 398bp. The genomic structure of murine vrf is highly conserved with the human homolog in relation to position of splice junctions and the presence of contiguous exons 6 and 7. A short polymorphic AC repeat is present in the 3' untranslated region of murine vrf. A major band of approximately 1.3kb was expressed in all adult mouse tissues examined.

Cloning and characterization of a novel human gene related to vascular endothelial growth factor.

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

Characterisation of genes encoding a nucleoside monophosphate kinase and a L35 ribosomal protein from Babesia bovis.

We have sequenced a region of the Babesia bovis nuclear genome that encodes a L35 ribosomal protein homologue (bl35) and a putative nucleoside monophosphate kinase (bnmk) that is most similar to the adenylate kinase of gram-positive bacteria and the mitochondrial form of adenylate kinase in eukaryotes. BNMK appears to be unique in that it is the first eukaryotic family member to feature a putative zinc-binding domain. bnmk and bl35 are closely linked and transcribed from opposite DNA strands. Examination of the gene structures indicate that the coding regions contain small intervening sequences that obey the GT-AG rule of eukaryotic spliceosomal introns. The single intron separates the bl35 initiation codon from the remainder of the coding region and the 6-exon bnmk gene does not appear to be differentially spliced. Both genes utilise multiple polyadenylation sites and the canonical mammalian polyadenylation signal AATAAA is absent from their 3' untranslated regions. Primer extension analyses reveal that the bnmk gene utilises a cluster of transcription start points, one of which is used most frequently. The bnmk mRNA 5' end does not appear to be cis- or trans-spliced. We report here the first evidence of intronic sequences, as well as heterogeneous 5' and 3' ends for mRNA of a member of the Babesia genus.