PGE, and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEGF), a secreted endothelial cellspecific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and El increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation ofVEGF expression by PGE2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism. (J. Clin. Invest. 1994.93:2490-2496

Glioma-derived vascular endothelial cell growth factor (GD-VEGF) is a 46-kDa dimeric glycoprotein mitogen with apparently greater specificity for vascular endothelial cells than the well-characterized fibroblast growth factors. The GD-VEGF cDNA sequence encodes a 190-amino acid residue subunit that is converted, by removal of an aminoterminal hydrophobic secretory leader sequence, to the mature 164-residue subunit characterized by direct amino acid sequencing. The GD-VEGF homodimeric subunit is homologous to the platelet-derived growth factor A and B chains and its oncogene homologue v-sis. We report here the complete cDNAt and amino acid sequence of rat GD-VEGF. The sequence confirms its identification as a secretory homodimeric glycoprotein and reveals an unexpected homology to platelet-derived growth factor (PDGF), a mitogen for connective tissues cells but not vascular endothelial cells from large vessels. GD-VEGF as described (5). Purity was confirmed on all samples by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Aliquots (1-2 ,.g) of the purified protein, quantitated by using an extinction coefficient based on amino acid analysis (5), were reduced and carboxymethylated with iodo[2-14C]acetic acid as described (8). The 14C-carboxymethylated GD-VEGF product was repurified on a 4.6 mm x 5 cm Vydac C4 reversed-phase HPLC column by elution at 20'C with a linear gradient of 0-67% acetonitrile in 0.1% trifluoroacetic acid over 30 min at a flow rate of 0.75 ml/min.Enzymatic Digestion and Polypeptide Purification. Reduced and carboxymethylated GD-VEGF (725 ng) was digested on the carboxyl-terminal side of most lysine and arginine residues with 30 ng of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin (Worthington) in 200 ,ul of 0.1 M ammonium bicarbonate (pH 8.3) for 6 hr at 37°C. The polypeptide digestion mixture was loaded directly on a 4.6 mm x 25 cm Vydac C18 reversed-phase column and fractionated by elution at 20°C with a 0-67% (vol/vol) linear gradient of acetonitrile in 0.1% trifluoroacetic acid over 2 hr at a flow rate of 0.75 ml/min. Polypeptide peaks were identified by monitoring A210 and individually collected.A similar digest was performed on 925 ng of carboxymethylated GD-VEGF by using 50 ng of Lys-C endoproteinase (Lys-C implies lysine specific) (Boehringer Mannheim) in 50 ,ul of 0.1 M Tris, pH 8.5/1 mM EDTA at 37°C for 8 hr. Polypeptide products, the result of cleavage on the carboxylterminal side of lysine residues, were purified as described for the tryptic digest.A final enzymatic digestion was done on 1.1 ,ug of carboxymethylated GD-VEGF by using 65 ng ofStaphylococcus aureus V8 protease (Miles). The substrate was dissolved in 5 ,ul of6 M guanidinium chloride/0.1% EDTA buffered with 0.7 M Tris to pH 7.8. The protease was added in 65 ,ul of 0.1% EDTA buffered to pH 8.0 with 0.1 M ammonium bicarbonate. The digest was incubated at 37°C for 48 hr, and the polypeptides, generated primarily by cleavage on the carboxylterminal side ofglutamic ac...

Galanin (GAL) is a widely distributed neuropeptide with diverse biological effects including modulation of hormone release, antinociception and modification of feeding behavior. Its effects are mediated through G-protein-coupled receptors (GPCR) for which only a single type has been cloned, GAL receptor 1 (GALR1). We describe the cloning of a second galanin receptor type, GALR2, from rat hypothalamus. The GALR2 amino acid sequence is 38% identical to GALR1 and is pharmacologically similar to GALR1 when expressed in COS-7 cells. GALR2 is encoded by a single gene containing at least one intron and expressed in a diverse range of tissues.

Vascular endothelial growth factor (VEGF) is a potent and selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth factor (PlGF), is also reported to be mitogenic for endothelial cells in culture. The rat GS-9L glioma cell line produces not only VEGF homodimers but also PlGF homodimers and a novel heterodimer composed of VEGF and PlGF subunits. All three dimeric forms were purified to apparent homogeneity, and their structures and mitogenic activities were compared. VEGF.PlGF heterodimers are vascular endothelial cell mitogens nearly as potent as VEGF homodimers. Therefore, some of the biological activities attributed to VEGF homodimers might be mediated by VEGF.PlGF heterodimers. In contrast, pure PlGF homodimers are mitogenic for endothelial cells only at high, possibly non-physiologic concentrations; thus the biological relevance of their mitogenic activity for these cells is not obvious. However, the existence of not only homodimers but also heterodimers clearly extends the similarity between the VEGF/PlGF and the homologous platelet-derived growth factor systems.

Abstract:Galanin is a 29-or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125l-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K 0 = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distritution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary 01-factory cortex, olfactory tubercie, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively. Key Words: Galanin receptor-G protein-coupled receptor-Cloning.

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.

No abstract