Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Differential expression of alpha-tubulin mRNA in rat cerebellum as revealed by in situ hybridization

Nucleotide sequence analysis of two rat alpha-tubulin cDNA clones showed a marked divergence in their 3'-untranslated regions. However, each of the alpha-tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with alpha-tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of alpha-tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.     

Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation

Hyperglycemia leads to vascular disease specific to diabetes mellitus. This pathology, which results from abnormal proliferation of smooth muscle cells in arterial walls, may lead to cataract, renal failure, and atherosclerosis. The hexosamine biosynthetic pathway is exquisitely responsive to glucose concentration and plays an important role in glucose-induced insulin resistance. UDP-GlcNAc: polypeptide O-N-acetylglucosaminyltransferase (O-GlcNAc transferase; OGTase) catalyzes the O-linked attachment of single GlcNAc moieties to serine and threonine residues on many cytosolic or nuclear proteins. Polyclonal antibody against OGTase was used to examine the expression of OGTase in rat aorta and aortic smooth muscle (RASM) cells. OGTase enzymatic activity and expression at the mRNA and protein levels were determined in RASM cells cultured at normal (5 mM) and at high (20 mM) glucose concentrations. OGTase mRNA and protein are expressed in both endothelial cells and smooth muscle cells in the aorta of normal rats. In both cell types, the nucleus is intensely stained, while the cytoplasm stains diffusely. Immunoelectron microscopy shows that OGTase is localized to euchromatin and around the myofilaments of smooth muscle cells. In RASM cells grown in 5 mM glucose, OGTase is also located mainly in the nucleus. Hyperglycemic RASM cells also display a relative increase in OGTase's p78 subunit and an overall increase protein and activity for OGTase. Biochemical analyses show that hyperglycemia qualitatively and quantitatively alters the glycosylation or expression of many O-GlcNAc-modified proteins in the nucleus. These results suggest that the abnormal O-GlcNAc modification of intracellular proteins may be involved in glucose toxicity to vascular tissues.     

Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor.

A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.     

Molecular cloning and nucleotide sequence of a bovine alpha-lactalbumin cDNA

A cDNA clone for the bovine milk protein, alpha-lactalbumin (alpha LA), has been identified using a rat cDNA probe. The bovine cDNA clone is 703 nucleotides (nt) long, contains 8 nt of 5'-untranslated sequence and 269 nt of 3'-untranslated sequence. When compared with previously reported sequences, the bovine alpha LA mRNA sequence has 74% similarity with rat alpha LA mRNA, 79% similarity with human mRNA and 74% similarity with guinea pig mRNA.     

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.     

Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins

A lambda gt 11 library prepared from human umbilical vein endothelial cell RNA was screened for cDNAs encoding thrombospondin. Reagents included a monospecific antibody to human thrombospondin and a mixture of four synthetic oligodeoxyribonucleotides derived from an amino acid sequence near the NH2 terminus of mature human thrombospondin. Two series of cDNA clones coding for sequences at the 5' and 3' ends of thrombospondin mRNA, respectively, were isolated. The nucleotide sequence of a 1.3-kilobase (kb) 5' clone (lambda TS-33) coded for 99 bases of 5' untranslated RNA, a signal peptide of 18 amino acids, and the first 379 amino acids of thrombospondin. Northern blot analysis with lambda TS-33 detected a single mRNA species of approximately 6.0 kb in rat aortic smooth muscle cell RNA. Thrombospondin mRNA levels increased rapidly, but transiently, in quiescent smooth muscle cells treated with platelet-derived growth factor. The kinetics of this response were very similar to those of the thrombospondin protein to this growth factor. There was significant homology in amino acid sequence between thrombospondin and a conserved region in the circumsporozoite protein of two malarial sporozoites. This region of thrombospondin may therefore represent a potential recognition site for a cell surface thrombospondin receptor.     

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.     

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [¹²⁵l]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 μg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 μg/kg/day, sc), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03). J. Cell. Physiol. 170:106–114, 1997. © 1997 Wiley-Liss, Inc.     

Atrial natriuretic factor inhibits mitogen-induced growth in aortic smooth muscle cells

Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular homeostasis exhibiting diuretic, natriuretic, and vasorelaxant activities. ANF shows antimitogenic effects in different cell types acting through R(2) receptor. Excessive proliferation of smooth muscle cells is a common phenomenon in diseases such as atherosclerosis, but the role of growth factors in the mechanism which modulate this process has yet to be clarified. The potential antimitogenic role of ANF on the cell growth induced by growth factors appears very intriguing. Aim of the present study was to investigate the possible involvement of ANF on rat aortic smooth muscle (RASM) cells proliferation induced by known mitogens and the mechanism involved. Our data show that ANF, at physiological concentration range, inhibits RASM cell proliferation induced by known mitogens such as PDGF and insulin, and the effect seems to be elicited through the modulation of phosphatidic acid (PA) production and MAP kinases involvement.     

Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.     

Mammalian nonsarcomeric myosin regulatory light chains are encoded by two differentially regulated and linked genes [published erratum appears in J Cell Biol 1990 Nov;111(5 Pt 1):2207]

The myosin 20,000-D regulatory light chain (RLC) has a central role in smooth muscle contraction. Previous work has suggested either the presence of two RLC isoforms, one specific for nonmuscle and one specific for smooth muscle, or the absence of a true smooth muscle-specific isoform, in which instance smooth muscle cells would use nonmuscle isoforms. To address this issue directly, we have isolated rat RLC cDNAs and corresponding genomic sequences of two smooth muscle RLC based on homology to the amino acid sequence of the chicken gizzard RLC. These cDNAs are highly homologous in their amino acid coding regions and contain unique 3'-untranslated regions. RNA analyses of rat tissue using these unique 3'-untranslated regions revealed that their expression is differentially regulated. However, one cDNA (RLC-B), predominantly a nonmuscle isoform, based on abundant expression in nonmuscle tissues including brain, spleen, and lung, is easily detected in smooth muscle tissues. The other cDNA (RLC-A; see Taubman, M., J. W. Grant, and B. Nadal-Ginard. 1987. J. Cell Biol. 104:1505-1513) was detected in a variety of nonmuscle, smooth muscle, and sarcomeric tissues. RNA analyses comparing expression of both RLC genes with the actin gene family and smooth muscle specific alpha-tropomyosin demonstrated that neither RLC gene was strictly smooth muscle specific. RNA analyses of cell lines demonstrated that both of the RLC genes are expressed in a variety of cell types. The complete genomic structure of RLC-A and close linkage to RLC-B is described.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)