Relation between coronary artery disease, aortic stiffness, and left ventricular structure in a population sample

With the use of the degenerated nucleotides that contain the conserved sequence of G protein-coupled receptor, we have identified a 648-bp clone (HDGRC02) from human genomic DNA with significant sequence homology to human neurotransmitter receptors. HDGRC02 was then used as a probe for the screening of full length gene. From human Lambda DASH II genomic library, a 1.6 Kb clone encoded a full length gene was isolated and named putative neurotransmitter receptor (PNR). PNR has a single open reading frame which predicts a 38.3 KD protein of 338 amino acids with seven transmembrane domain topography. The amino acid sequence of PNR exhibits considerable homology to the rat 5-HR1D receptor with 35% amino acid identity and 56% amino acid similarity. PNR also shows significant sequence homology to the 5-HT1D receptor from Japanese puffer fish fugu, to the 5-HT4L receptor from mouse, to the alpha-2 adrenergic receptor and to the D2 dopamine receptor. Northern blot analysis indicates that PNR is expressed in skeletal muscle and selected areas of the brain. A chromosome mapping study located the PNR gene with human chromosome band of 6q23. The findings in the present study demonstrate that PNR is a putative neurotransmitter receptor.     

Sequence and functional analysis of cloned guinea pig and rat serotonin 5-HT1D receptors: common pharmacological features within the 5-HT1D receptor subfamily

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Molecular characterization and localization of human metabotropic glutamate receptor type 4

Oligonucleotides of consensus sequences from rat metabotropic glutamate receptor (mGluR) genes were synthesized and used to amplify human DNA by the polymerase chain reaction (PCR). Five unique human sequences homologous to these rat receptor genes were isolated including mGluR4. A human cerebellum cDNA library was screened using this amplified mGluR4 sequence as a probe and yielded clones which between them contained the complete coding sequence for human mGluR4. The coding sequence is very similar to the equivalent rat gene (90% DNA sequence identity and 97% predicted protein sequence identity). The mGluR4 cDNA was transfected in Chinese hamster ovary (CHO) cells and stable clonal cell lines were isolated. Stimulation of the expressed receptor by L-2-amino-4-phosphonobutyrate (L-AP4), L-glutamate or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) resulted in a reduction of forskolin-stimulated cyclic AMP (cAMP) with EC50 values of 0.2, 13 and 90 microM respectively. Quisqualate had little effect at concentrations up to 1 mM. In Northern blots mGluR4 mRNA appears to be brain-specific, and shows a distinct distribution (excluding the cerebellum), being expressed in the thalamus, hypothalamus and caudate nucleus. In situ hybridization studies on human brain sections confirmed this general pattern of distribution. The strongest mGluR4 mRNA signal was found in the cerebellar granule cells consistent with the reported distribution of mGluR4 in the rat brain. The major difference from the rat brain is the presence in the human brain of mGluR4 mRNA in the caudate nucleus and putamen.     

The development of low birth weight term infants and the effects of the environment in northeast Brazil

1. Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. 2. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. 3. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68+/-0.04 and 0.91+/-0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30+/-0.02 and 0.23+/-0.02. 4. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. 5. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. 6. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. 7. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly.     

Mutagenesis of the human 5-HT1B receptor: differences from the closely related 5-HT1A receptor and the role of residue F331 in signal transduction

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.     

Cloning, sequencing and functional expression of a guinea pig lung bradykinin B2 receptor

Kinin receptors are classified as B1 and B2 based upon agonist and antagonist potencies and cloning and expression studies. Using sequences from human and rat bradykinin B2 receptors, polymerase chain reaction (PCR) was utilized to isolate cDNA from guinea pig lung. The receptor obtained is predicted to have 372 amino acids and shares > 80% sequence homology with human, rat, rabbit and mouse B2 receptors. In competition binding experiments in Chinese hamster ovary (CHO-K1) cells in which the guinea pig cDNA was expressed, [3H]bradykinin was displaced by kinin receptor ligands with an order of potency consistent with a B2 subtype. In CHO cells expressing the guinea pig receptor, bradykinin caused a concentration 45Ca2+ efflux. A B1 receptor agonist, desArg9-bradykinin, also caused 45Ca2+ efflux but with a potency several orders of magnitude lower than bradykinin. Curiously, several B1 and B2 receptor antagonists induced 45Ca2+ efflux, indicating that this receptor may be coupled differently in CHO cells than in native tissues.     

Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues

Several chemokine receptors have been cloned and shown to belong to a superfamily of seven transmembrane, G protein-coupled receptors. We report here the molecular cloning of TER1, a novel human chemokine receptor-like gene. The amino acid sequence deduced from the TER1 cDNA shows 43, 40, 40, and 39% identity to CCR4, CCR5, CCR1, and CCR2B beta chemokine receptors, respectively. By the use of fluorescent in situ hybridization, we have mapped the TER1 gene to chromosome 3p21, clustered with other chemokine receptor genes. By Northern blot analysis, TER1 mRNA is found to be expressed in the thymus, spleen, and at barely detectable levels in peripheral blood lymphocytes. Moreover, TER1 message in abundant in the NK cell line NK3.3 and in the T cell line MOLT-4. The restricted TER1 expression in cells and tissues of the lymphoid lineage suggests that this receptor may play a role in regulating immune functions.     

Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover

The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a 125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of 125I-pGAL to transiently expressed GALR2 receptors was saturable (KD = 0.15 nM) and displaceable by galanin peptides and analogues in rank order: porcine galanin approximately M32 approximately M35 approximately M40 >/= galanin-(1-16) approximately M15 approximately [D-Trp2]galanin-(1-29) > C7 > galanin-(3-29). This profile resembles that of the rat GALR1 receptor with the notable exception that [D-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C.