Biomedical subjects

K R Lynch

Publications and source records attributed to K R Lynch.

At least 37 records · Page 2Linked to original sources

PubMed1999

Characterization of the human cysteinyl leukotriene CysLT1 receptor.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.

Amino Acid Sequence↗

PubMed1999

Structure--activity relationships of lysophosphatidic acid: conformationally restricted backbone mimetics.

Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought to be mediated through its interaction with G-protein coupled receptor(s). In an effort to extend the structure-activity relationships of LPA, we have produced a series of LPA analogues in which the glycerol core in LPA was replaced with conformationally restricted aryl substructures. The aryl substructures encompassed aminophenol, resorcinol, dihydroxy benzophenone, and tocopherol systems. The benzophenone moiety was investigated both as a conformationally restricting substructure for LPA and as a possible photoreactive alkylating agent for the LPA receptor(s). All LPA analogues were evaluated for their potency and efficacy in mobilizing calcium ions from internal stores in MDA MB-231 cells. Ten of the 14 analogues exhibited activity in this assay at doses up to 5 microM; none of the compounds exhibited nonreceptor-mediated lytic activity at this maximal concentration. The receptor response showed surprising tolerance for manipulation in the backbone region of LPA, although none of the compounds were equipotent to LPA. This tolerance for a variety of structures has given us new leads into the realization of novel agonists and antagonists of the LPA receptor(s).

Calcium↗

PubMed1999

Discovery of three novel orphan G-protein-coupled receptors.

We have discovered three novel human genes, GPR34, GPR44, and GPR45, encoding family A G-protein-coupled receptors (GPCRs). The receptor encoded by GPR34 is most similar to the P2Y receptor subfamily, while the receptor encoded by GPR44 is most similar to chemoattractant receptors. The receptor encoded by GPR45 is the mammalian orthologue of a putative lysophosphatidic acid receptor from Xenopus laevis. Partial sequence of GPR34 was discovered during a search of the GenBank database of expressed sequence tags (ESTs). This sequence information was used both to isolate the full-length translational open reading frame from a human genomic library and to assemble a contig from additional GPR34 EST cDNAs. Northern blot and in situ hybridization analyses revealed GPR34 mRNA transcripts in several human and rat brain regions. Also, we used polymerase chain reaction (PCR) to amplify human genomic DNA using degenerate oligonucleotides designed from sequences encoding transmembrane domains 3 and 7 of opioid and somatostatin receptors. Two PCR products partially encoding novel GPCRs, named GPR44 and GPR45, were discovered and used to isolate the full-length translational open reading frames from a human genomic library. Both GPR44 and GPR45 are expressed in the central nervous system and periphery. For chromosomal localization, fluorescence in situ hybridization analysis was performed to assign GPR34 to chromosomes 4p12 and Xp11. 3, GPR44 to chromosome 11q12-q13.3, and GPR45 to chromosome 2q11. 1-q12.

Amino Acid Sequence↗

PubMed1999

Identification and cloning of three novel human G protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain.

The G protein-coupled receptor (GPCR) family share a structural motif of seven transmembrane segments with large numbers of conserved residues in those regions. Here, we report the identification and cloning of two novel human intronless GPCR genes, GPR52, GPR55 and a pseudogene PsiGPR53. GPR55 was identified from the expressed sequence tags (EST) database whereas GPR52 and pseudogene PsiGPR53 originated from the high throughput genome (HTG) database. A partial cDNA clone obtained from the IMAGE Consortium of GPR55 was used to screen a human genomic library to acquire the full length gene. GPR52 and PsiGPR53 were amplified from human genomic DNA using primers based on the HTG sequences. GPR55 and GPR52 encode receptors of 319 and 361 amino acids, respectively. GPR55 gene was mapped to chromosome 2q37, using fluorescence in situ hybridization (FISH), and its mRNA transcripts have been detected in the caudate nucleus and putamen, but not in five other brain regions. Human receptors showing the highest amino acid identity to GPR55 include P2Y5 (29%), GPR23 (30%), GPR35 (27%) and CCR4 (23%). GPR52 gene localized to chromosome 1q24 shares the highest identity with GPR21 (71%), histamine H2 (27%) and 5-HT4 (26%) human receptors. PsiGPR53 is a pseudogene mapped to chromosome 6p21 that demonstrates the highest similarity to the MRG (35%), MAS (28%) and C5a (24%) human receptor genes.

Amino Acid Sequence↗

PubMed1999

Novel GPCRs and their endogenous ligands: expanding the boundaries of physiology and pharmacology.

Nearly all molecules known to signal cells via G proteins have been assigned a cloned G-protein-coupled-receptor (GPCR) gene. This has been the result of a decade-long genetic search that has also identified some receptors for which ligands are unknown; these receptors are described as orphans (oGPCRs). More than 80 of these novel receptor systems have been identified and the emphasis has shifted to searching for novel signalling molecules. Thus, multiple neurotransmitter systems have eluded pharmacological detection by conventional means and the tremendous physiological implications and potential for these novel systems as targets for drug discovery remains unexploited. The discovery of all the GPCR genes in the genome and the identification of the unsolved receptor-transmitter systems, by determining the endogenous ligands, represents one of the most important tasks in modern pharmacology.

Animals↗

PubMed1999

Life on the edg.

Explore the source record for details and available documents.

GTP-Binding Proteins↗

PubMed1999

Panning for genes--A visual strategy for identifying novel gene orthologs and paralogs.

We have developed a rapid visual method for identifying novel members of gene families. Starting with an evolutionary tree, 20-50 protein query sequences for a gene family are selected from different branches of the tree. These query sequences are used to search the GenBank and expressed sequence tag (EST) DNA databases and their nightly updates using the tfastx3 or tfasty3 programs. The results of all 20-50 searches are collated and resorted to highlight EST or genomic sequences that share significant similarity with the query sequences. The statistical significance of each DNA/protein alignment is plotted, highlighting the portion of the query sequence that is present in the database sequence and the percent identity in the aligned region. The collated results for database sequences are linked using the WWW to the underlying scores and alignments; these links can also be used to perform additional searches to characterize the novel sequence further. With traditional "deep" scoring matrices (BLOSUM50) one can search for previously unrecognized families of large protein superfamilies. Alternatively, by using query sequences and EST libraries from the same species (e. g., human or mouse) together with "shallow" scoring matrices and filters that remove high-identity sequences, one can highlight new paralogs of previously described subfamilies. Using query sequences from the glutathione transferase superfamily, we identified two novel mammalian glutathione transferase families that were recognized previously only in plants. Using query sequences from known mammalian glutathione transferase subfamilies, we identified new candidate paralogs from the mouse class-mu, class-pi, and class-theta families.

Amino Acid Sequence↗

PubMed1998

Identification of a novel human phosphatidic acid phosphatase type 2 isoform.

Two human isoforms of membrane associated phosphatidic acid phosphatase have been described (PAP-2a and -2b), and both enzymes have been shown to have broad substrate specificity and wide tissue distribution [Kai et al., J. Biol. Chem. 272 (1997) 24572-24578]. With this report we describe a third isoform, PAP-2c, that we found by searching the database of expressed sequence tags (dbEST) with PAP-2a and PAP-2b sequences. Key structural features described previously in PAP-2a and -2b, including the glycosylation site, putative transmembrane domains, and the proposed catalytic site, are conserved in the novel phosphatase. The kinetics of the three enzymes were compared using as substrates phosphatidic acid, lysophosphatidic acid, and N-oleoyl ethanolamine phosphatidic acid. Km values for each of the substrates, respectively, were (in microM) PAP-2a: 98, 170, 116; PAP-2b: 100, 110, 56; and PAP-2c: 150, 340, 138. Expression of PAP-2c mRNA is more restricted than the two previously described isoforms.

Amino Acid Sequence↗

PubMed1998

Discovery of three novel G-protein-coupled receptor genes.

We report here the molecular cloning, tissue distribution, and chromosomal localization of novel genes encoding G-protein-coupled receptors (GPCRs). A search of a mouse database of expressed sequence tags revealed an EST partially encoding a GPCR, which was used to screen a mouse genomic library to obtain the translational open reading frame (ORF). The resultant clone, GPR27, contained an intronless ORF, encoding a receptor of 379 amino acids. In an alternate strategy, human genomic DNA was subjected to polymerase chain reaction (PCR) amplification, using degenerate oligonucleotides based on GPR1. Two PCR products partially encoding GPCRs were isolated and used to screen a genomic library to obtain the translational ORF. One of the resultant clones, GPR30, contained an intronless ORF encoding a receptor of 375 amino acids. The other clone, GPR35, also contained an intronless ORF encoding a receptor of 309 amino acids. Transcripts corresponding to GPR27 and GPR30 were detected in several areas of human and rat CNS, While GPR35 expression was detected only in the rat intestine. Through fluorescence in situ hybridization analysis the gene encoding GPR30 was localized to chromosome 7p22 and GPR35 to chromosome 2q37.3.

Amino Acid Sequence↗

PubMed1998

Localization of alpha2C-adrenergic receptor immunoreactivity in catecholaminergic neurons in the rat central nervous system.

Given the importance of alpha2-adrenergic receptors in the regulation of catecholaminergic transmission, we analysed the distribution of immunoreactivity corresponding to the C-subtype of alpha2-adrenergic receptor in central catecholaminergic neurons using double-label immunohistochemistry with antibodies directed against alpha2C-adrenergic receptors and tyrosine hydroxylase. Cells exhibiting both alpha2C-adrenergic receptor and tyrosine hydroxylase immunoreactivity were found in most areas containing catecholaminergic cell groups. However, the percentage of double-labelled cells varied in a region-specific manner. In the medulla, alpha2C-adrenergic receptor immunoreactivity was characteristic of only a minority of cells exhibiting tyrosine hydroxylase immunoreactivity (40-43% in area A1/C1, 27-36% in area A2/C2, 35% in area C3) while a larger percentage of double-labelled cells was observed in the pons (65% in A5, 92% in locus coeruleus, 68% in A7). In the midbrain, alpha2C-adrenergic receptor immunoreactivity was detected in most tyrosine hydroxylase-immunoreactive cells in dopaminergic regions (63% in the retrorubral field, 77-83% in substantia nigra, 67% in ventral tegmental area). These results suggest that alpha2C-adrenergic receptors may act as autoreceptors on some central adrenergic and noradrenergic neurons. In addition, the colocalization of alpha2C-adrenergic receptor and tyrosine hydroxylase immunoreactivity in dopaminergic cell groups suggests that reported effects of alpha2-adrenergic receptor agonists in these areas may be mediated by the C-subtype.

Animals↗

PubMed1998

Characterization of a receptor subtype-selective lysophosphatidic acid mimetic.

Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A)+ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.

Adenylate Cyclase Toxin↗

PubMed1997

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes.

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3-24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22-q31.1, and GPR23 to chromosome X, region q13-q21.1.

Amino Acid Sequence↗

PubMed1997

Discovery of a novel human G protein-coupled receptor gene (GPR25) located on chromosome 1.

We amplified human genomic DNA by the polymerase chain reaction (PCR) using oligonucleotides based on the primary sequence of the genes encoding the somatostatin receptors (SSTR) and the somatostatin-like receptor gene SLC-1. One resultant DNA fragment was used to screen a genomic DNA library resulting in the isolation of a gene, GPR25, encoding an additional member of the G protein-coupled receptor family (GPCR). GPR25 is intronless throughout its open reading frame (ORF) and encodes a protein of 360 amino acids. The receptor encoded by GPR25 shares highest identity to the receptor encoded by GPR15, angiotensin II type 1A receptor, and somatostatin receptor 5. Northern analysis found no transcripts expressed in liver or any of the 12 brain regions analyzed. Fluorescence in situ hybridization analysis localized GPR25 to chromosome 1q32.1.

Amino Acid Sequence↗

PubMed1997

Structure/activity relationships in lysophosphatidic acid: the 2-hydroxyl moiety.

Although lipid phosphoric acid mediators such as lysophosphatidic acid (LPA) are now recognized widely as intercellular signaling molecules, the medicinal chemistry of these mediators is poorly developed. With the goal of achieving a better understanding of the structure activity relationships in LPA, we have synthesized and tested a series of LPA analogs that lack the 2-hydroxyl moiety. Our series consisted of compounds with 2, 3, or 4 carbon diol or amino alcohol backbones and oleoyl or palmitoleoyl acyl groups. These molecules cannot be acylated further to form phosphatidic acids, nor do they have chiral centers. The rank order potency of these compounds in mobilization of calcium in MDA MB-231 cells suggested a maximum optimal chain length of 24-25 atoms. However, high potency for the inhibition of adenylyl cyclase in these cells was achieved only by one compound that also contained a dissociable proton five bond lengths from the phosphorus atom. That compound, N-oleoyl-2-hydroxyethyl-1-phosphate, was nearly equipotent to 1-oleoyl LPA in both assays. The striking mimicry of LPA by the ethanolamine-based compound and the presence of fatty acid amides in tissue prompts us to propose that phosphorylated N-acyl ethanolamides occur naturally.

Humans↗

PubMed1996

Characterization of a human gene related to genes encoding somatostatin receptors.

We report the identification of a gene, named SLC-1(1), encoding a novel G protein-coupled receptor (GPCR). A customized search procedure of a database of expressed sequence tags (dbEST) retrieved a human cDNA sequence that partially encoded a GPCR. A genomic DNA fragment identical to the cDNA was obtained and used to screen a library to isolate the full-length coding region of the gene. This gene was intronless in its open reading frame, and encoded a receptor of 402 amino acids, and shared -40% amino acid identity in the transmembrane (TM) regions to the five known human somatostatin receptors. Northern blot analysis revealed that SLC-1 is expressed in human brain regions, including the forebrain and hypothalamus. Expression in the rat was highest in brain, followed by heart, kidney, and ovary. Expression of SLC-1 in COS-7 cells failed to show specific binding to radiolabelled Tyr1-somatostatin-14, naloxone, bremazocine, 1,3-di(2-tolyl)-guanidine (DTG), or haloperidol. A repeat polymorphism of the form (CA)n was discovered in the 5'-untranslated region (UTR) of the gene and SLC-1 was mapped to chromosome 22, q13.3.

Amino Acid Sequence↗

PubMed1996

A novel gene codes for a putative G protein-coupled receptor with an abundant expression in brain.

Following the cloning of the dopamine receptors we continued a search of the human genome for related genes. We searched an EST data base and discovered cDNA fragments encoding novel G protein-coupled receptor genes. The available GenBank sequence of one of these EST fragments showed that it encoded a receptor with closest similarity to the D2 dopamine and adrenergic receptors. This cDNA was used to isolate the gene (GPR19), and the encoded receptor also demonstrated similarity with the neuropeptide Y receptor. The gene was mapped to chromosome 12, in region p13.2-12.3. Northern blot analysis revealed expression of GPR19 in peripheral regions, and brain regions significantly overlapping with the D2 receptor gene expression. A sequence of the rat orthologue of GPR19 was obtained and in situ hybridization analysis demonstrated a very abundant expression in rat brain.

Amino Acid Sequence↗

PubMed1996

Distribution of alpha 2A-adrenergic receptor-like immunoreactivity in the rat central nervous system.

In this study, we analyzed immunohistochemically the distribution of the A subtype of alpha 2-adrenergic receptor (alpha 2A-AR) in the rat central nervous system using light level immunohistochemistry. By using affinity-purified antisera, we found perikaryal labeling was diffuse and/or punctate; immunoreactive puncta were heterogeneous in size and number in a region-specific manner. Dense deposits of immunoreaction product were found associated with neuropil also, particularly in the lateral parabrachial nucleus, locus coeruleus, lateral septum, diagonal band, stratum lacunosum-moleculare of CA1, and various nuclei of the amygdala and extended amygdala. Prominently immunoreactive olfactory structures include the anterior olfactory nucleus and the granular layer of the olfactory bulb. The cortex was generally light to moderately labeled with greater immunoreactivity in the cingulate and insular cortices. alpha 2A-AR-like immunoreactivity was intense in the basal forebrain and continuous from the nucleus accumbens through the substantia innominata and fundus of the striatum. Most immunoreactivity in the diencephalon was restricted to the hypothalamus with light to moderate labeling in the thalamus. Generally light immunoreactivity was observed in midbrain structures. In the pons and medulla, both perikaryal and neuropil labeling were observed. Together with the accompanying paper describing the neural distribution of alpha 2C-AR-like immunoreactivity, our results provide an extensive immunohistochemical cartography of alpha 2-ARs in the adult rat central nervous system.

Animals↗

PubMed1996

Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Animals↗