Molecular cloning and tissue expression of a novel orphan G protein-coupled receptor from rat lung

G protein-coupled receptors transduce the signal of a wide variety of hormones, neurotransmitters, cytokines, and other molecules across the cell membrane to elicit the corresponding response inside the target cells. We describe in this paper the molecular cloning and tissue distribution of a novel rat G protein-coupled receptor, GPR41, with highest homology to the human orphan G protein-coupled receptor DRY12. A lower degree of homology was seen with the receptors for bradykinin, angiotensin, and IL8. The expression of GPR41 appears to be the highest in brain and lung tissues, with lesser expression in heart, skeletal muscle, and kidney, as assayed by northern blotting. No GPR41 message was seen in spleen, liver, or testes. GPR41 failed to bind any of the ligands tested.     

Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.     

Molecular cloning of a novel melanocortin receptor

Using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, we isolated three full-length human genes that encode a novel subgroup of this receptor family. Recently, two of these receptors were identified as specific for alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone. We report the molecular cloning and pharmacologic characterization of a third member of this subgroup. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacology characterizes it as an MSH receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. These findings may yield insight into the physiology of peptides derived from pro-opiomelanocortin post-translational processing.     

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.     

Molecular cloning of a new unc-33-like cDNA from rat brain and its relation to paraneoplastic neurological syndromes

Anti-CV2-autoantibodies from patients with paraneoplastic neurological syndromes were used to purify protein(s) related to this disease. A novel cDNA, c-22, was obtained by PCR with primers based on amino-acid sequence of peptides obtained from this protein and rat brain cDNA as template. The deduced amino-acid sequence of c-22 shows homology to the Unc-33 gene from C. elegans in which mutations lead to defects in neuritic outgrowth and axonal guidance and cause uncoordinated movements of the nematode. Several consensus sites for putative protein kinase C phosphorylation were found, suggesting that the c-22 gene product may be a phosphoprotein. Northern hybridizations show that the apparently unique 3.8-kb mRNA of c-22 is present in rat brain tissue and its expression is developmentally regulated: the levels of C-22 mRNA, detectable in brain at embryonic day 17 (E17), increase up to post-natal day 7 (P7) and decline rapidly to an almost undetectable level in adult.     

Asymmetric distribution of functional sodium-calcium exchanger in primary osteoblasts

To understand calcium translocation in osteoblasts, we have determined the location of sodium-calcium (Na-Ca) exchanger (NCX) in relation to actin and alpha-tubulin in primary cultures of avian osteoblasts. Osteoblasts derived from the periosteal surface of tibias from growing chickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodies raised against the canine cardiac Na-Ca antibodies revealed a 70 kDa exchanger protein. Cross-reactivity of the anti-NCX antibody was confirmed by enriching for NCX in protein samples derived from plasma membrane vesicles by affinity chromatography using the exchanger inhibitory peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, revealing an intense single band at 70 kDa. Examination of live cells loaded with Calcium Green-1 AM ester by confocal microscopy demonstrated sodium-dependent calcium uptake, confirming the presence of functional NCX in intact cells. Immunolocalization studies of osteoblasts stained with anti-NCX antibodies revealed asymmetric localization of the exchanger in cultured osteoblasts, residing almost entirely within two 0.5-microm optical sections along the substrate adherent side of the cells. Since NCX is known to be a low-affinity, high-capacity calcium translocating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.     

Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.     

Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain

BACKGROUND: Skeletal muscle abnormalities contribute considerably to the clinical expression of heart failure. Deconditioning, underperfusion and an increased number of type IIb glycolytical fibres lead to early lactate production and muscle fatigue at low exercise levels. Aerobic muscle metabolism may also be impaired, as suggested by biopsy studies. Thus far, no data are available from non-invasive studies to indicate the extent of aerobic muscle dysfunction during low-grade exercise which does not induce acidosis. METHODS AND RESULTS: Mitochondrial function of skeletal muscle during fibre type I activation was studied in 22 patients with chronic heart failure [NYHA class III, left ventricular ejection fraction 28 +/- 2%, (patients)] on ACE inhibitors, diuretics and digoxin, and in 20 normal subjects, using 31P NMR spectroscopy of a single right forearm flexor muscle during three mild intermittent exercise levels (0-40% of maximum voluntary contraction) and recovery time. At rest, the inorganic phosphate/phosphocreatine ratio was different [0.13 +/- 0.005 (patients) vs 0.09 +/- 0.002 (normal subjects), P = 0.0001]. However, intracellular pH was comparable. Local acidosis (tissue pH < 6.9) was avoided to prevent fibre type IIb activation. Calculated resting phosphate potential levels were comparable, but the slope and intercept of the linear relationship of phosphate potential and workload were significantly lower in patients than in normal subjects (11.7 +/- 0.7 vs 15.8 +/- 0.6 and 139 +/- 7 vs 196 +/- 7, patients vs normal subjects, indicating early exhaustion of intracellular energy at lower exercise levels. Also, maximum calculated workload at which tissue ADP stabilized was lower in patients than in normal subjects (88 +/- 7% vs 120 +/- 4% of maximum voluntary workload, patients vs normal subjects, P < 0.05). Time to recovery to pre-test phosphocreatine levels was prolonged by 46% in patients compared to normal subjects (P < 0.05). CONCLUSIONS: In heart failure, oxidative fibre mitochondrial function in skeletal muscle is impaired, as reflected by the reduced phosphate potential and oxidative phosphorylation rate, early exhaustion and slowed recovery of intracellular energy reserve at workloads, which do not affect intracellular pH.     

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide-resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E.coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA. Furthermore, intracellular accumulation of [14C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Molecular cloning and characterization of rat lin-10

In Caenorhabditis elegans, the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of the lin-3, let-23, and let-60 products. In addition to these gene products, the lin-2, lin-7, and lin-10 products are also implicated in this pathway. Lin-2 encodes a MAGUK and lin-7 encodes a small protein with one PDZ domain. The lin-10 product has no homology to known proteins. Here, we have cloned a rat homologue of the lin-10 product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.     

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The 3-years-old patient presents bilateral exophthalmia and papillar stasis at both eyes. The cranial examination shows the frontal boss at the level of the anterior fontanelle and the hypoplasia of the inferior facial floor. The cranial radiography shows digital impressions on the entire projection surface of the cap. The child also presents mental retardation. The affection integrates in the category of craniofacial dysostosis described by Crouzon, maladies which are transmitted autosomal with great penetrance.     

Molecular cloning and expression of the functional rat C5a receptor

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.     

Molecular cloning of a novel ADP-ribosylation factor (ARF) expressed in planarians

In part I of this study, a method for the objective evaluation of profile nasal esthetics was detailed, and normative values for males and females were presented. In part II, identical methodology was applied to the preoperative lateral cephalograms of 13 female patients with vertical maxillary excess to determine how the nasal profile esthetics of these patients varies from the norm. This data was compared to the normative data established for females in part I. Results determined that patients with vertical maxillary excess have increased nasal length caused by a more superiorly positioned soft tissue nasion, decreased thickness of soft tissue at rhinion, increased nasal form angle, and increased absolute nasal tip angle related to an increased inclination of the cranial base, increased forward projection of the anterior nasal spine (tip support), and decreased incidence of supratip break. These findings suggest a characteristic appearance similar to the type II nasal characteristic (aquiline form) established in part I.     

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.