Neutral endopeptidase modulation of septic shock.

Neutral endopeptidase (NEP; EC. 3.4.24.11) is a type 2 cell surface metalloprotease known by a variety of eponyms, including enkephalinase, common acute lymphoblastic leukemia antigen, and CD10. Identified substrates are largely neural or humoral oligopeptide agonists, and the enzyme functions to terminate signaling by degrading the ligand, analogously to acetylcholine/acetylcholinesterase. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin shock with a pronounced gene dosage effect. The site(s) of action appears downstream from release of tumor necrosis factor and interleukin-1 since NEP-deficient animals demonstrate increased sensitivity to these mediators as well. This unexpected finding indicates an important protective role for NEP in septic shock.

Expression cloning of an adenylate cyclase-coupled calcitonin receptor.

A calcitonin receptor complementary DNA (cDNA) was cloned by expression of a cDNA library from a porcine kidney epithelial cell line in COS cells. The 482-amino acid receptor has high affinity for salmon calcitonin (dissociation constant Kd approximately 6 nM) and is functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP). The receptor shows no sequence similarity to other reported G protein-coupled receptors but is homologous to the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor, indicating that the receptors for these hormones, which regulate calcium homeostasis, represent a new family of G protein-coupled receptors.

A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide.

The complementary DNA encoding a 585-amino acid parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor with seven potential membrane-spanning domains was cloned by COS-7 expression using an opossum kidney cell complementary DNA (cDNA) library. The expressed receptor binds PTH and PTHrP with equal affinity, and both ligands equivalently stimulate adenylate cyclase. Striking homology with the calcitonin receptor and lack of homology with other G protein-linked receptors indicate that receptors for these calcium-regulating hormones are related and represent a new family.

Melatonin receptors are for the birds: molecular analysis of two receptor subtypes differentially expressed in chick brain.

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei.

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

A time-less function for mouse timeless.

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases.

The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme. The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase. PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.

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.

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.

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

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.

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.

ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function.

This report describes the implementation of ProSearch, a computer program that can efficiently search for motifs in protein sequences. ProSearch currently uses motifs that are contained in the PROSITE database, but user-developed patterns can easily be added to any search. ProSearch can generate a report identifying the patterns present in a given protein sequence, their locations and, if desired, a short description of the identified patterns. The program is written in AWK (a small interpreted computer language), which can run on all computer platforms commonly found in laboratories. ProSearch can search a 348-amino acid protein for 690 patterns in less than 5 s on a typical workstation.

Neutral endopeptidase modulates septic shock.

Neutral endopeptidase (NEP; EC 3.4.24.11) is a type-2 cell-surface metalloproteinase known by a variety of eponyms, including enkephalinase, common acute lymphoblastic leukemia antigen (CALLA), and CD10. Identified substrates are largely neural or humoral oligopeptide agonists, and the enzyme functions to terminate signaling by degrading the ligand, analogous to the acetylcholine/acetylcholinesterase system. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin shock with a pronounced gene-dosage effect. The site(s) of action appears downstream from release of TNF and IL-1, as NEP-deficient animals demonstrate increased sensitivity to these mediators as well. This unexpected finding indicates an important protective role for NEP in septic shock.

GCRDb: a G-protein-coupled receptor database.

G-protein-coupled receptors (GCRs) are a very large protein family and are critical components in many different autocrine, paracrine and endocrine signaling systems in animals. Current estimates are that humans have several thousand GCRs encoded by only slightly fewer genes. I have developed GCRDb, a database of sequences and other data relevant to the study of the biology of the receptors. The database implementation, data collection, and query system are described. An integral component of the design of GCRDb is the classification of GCRs in families. The current composition of GCRDb is presented in a table showing the number of entries in each family and group as derived from accepted mutation parsimony analyses.

Cloning, molecular characterization and chromosome localization of the inorganic pyrophosphatase (PPA) gene from S. cerevisiae.

The gene for Saccharomyces cerevisiae inorganic pyrophosphatase, PPA, has been cloned by hybridization of "long" oligonucleotide probes with both cDNA and genomic S. cerevisiae libraries. The nucleotide sequence of 1612 bp from a genomic subclone that includes the entire coding region gives a deduced amino acid sequence that has nine differences (out of a total of 286 residues) from the previously published amino acid sequence that was determined directly. The codon usage in PPA is as expected for a "highly expressed" yeast gene. The upstream region contains a poly dA/dT sequence that might comprise a constitutive promoter. The PPA gene appears to be present in a single copy within the S. cerevisiae genome and has been localized to chromosome II.

Molecular characterization of a novel glycoprotein hormone G-protein-coupled receptor.

We report the molecular characterization of a novel G-protein-coupled receptor, GPR48, that resembles proteins in the glycoprotein hormone receptor family. The full-length human GPR48 cDNA is comprised of 951 amino acids. The large extracellular amino terminus of 538 residues is composed of seventeen leucine-rich repeats (LRR). The genomic structure of GPR48 has several features in common with genes in the glycoprotein hormone receptor family. Analogous to these receptors, most of the LRR are encoded on single small exons, and the last exon encodes the seven transmembrane segments. The complete gene spans more than 60 kb with 18 exons and 17 introns. Northern blot analysis demonstrated high expression of GPR48 in the adult human pancreas, with moderate levels of expression in placenta, kidney, brain, and heart. Additionally, this receptor is expressed as early as 7 days post coitus in the mouse, indicating its potential involvement in development.

The role of the cholecystokinin-B/gastrin receptor transmembrane domains in determining affinity for subtype-selective ligands.

We have examined the role of transmembrane domain amino acids in conferring subtype-selective ligand affinity to the human cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were sequentially replaced by the corresponding amino acids from the pharmacologically distinct CCK-A receptor subtype. 125I-CCK-8 competition binding experiments were performed to compare all mutant CCK-B/gastrin receptor constructs with the wild type control. Affinities for the nonselective agonist, CCK-8, as well as the subtype-selective peptide (gastrin), peptide-derived (PD135,158), and nonpeptide (L365,260) and L364,718) ligands were assessed. All of the mutants retained relatively high affinity for CCK-8, suggesting that the tertiary structure of these receptors was well maintained. Only eight of the amino acid substitutions had a significant effect on subtype selective binding. When compared with the wild type, single point mutations in the CCK-B/gastrin receptor decreased affinity for gastrin, L365,260, and PD135,158 up to 17-,23-, and 61-fold, respectively. In contrast, the affinity for L364,718 increased up to 63-fold. None of the single amino acid substitutions, however, was sufficient to fully account for the subtype selectivity of any tested compound. Rather, CCK-B/gastrin receptor affinity appears to be influenced by multiple residues acting in concert. The 8 pharmacologically important amino acids cluster in the portion of the transmembrane domains adjacent to the cell surface. The spatial orientation of these residues was analyzed with a rhodopsin-based three-dimensional model of G-protein coupled receptor structure (Baldwin, J.M. (1993) EMBO J. 12, 1693-1703). This model predicts that the 8 crucial residues project into a putative ligand pocket, similar to the one which is well established for biogenic amine receptors (Caron, M. G., and Lefkowitz, R.J. (1993) Recent Prog. Horm. Res. 48, 277-290; Strader, C.D., Sigal, I.S., and Dixon, R.A. (1989) Trends Pharmacol. Sci. 10, Dec. Suppl., 26-30).

Probing the "message:address" sites for chemoattractant binding to the C5a receptor. Mutagenesis of hydrophilic and proline residues within the transmembrane segments.

The C5a anaphylatoxin ligand-receptor interaction on polymorphonuclear granulocytes stimulates chemotaxis, degranulation, and the oxidative burst. The receptor is a member of the large G-protein-coupled family. The ligand is a cationic peptide of 72 amino acids derived from the C5 component of complement and has been shown to have a number of structural requirements for interaction with the receptor. In order to probe the potential interaction sites between ligand and receptor, we constructed a series of mutated receptor molecules, targeting cysteines, prolines, and additional amino acids of interest because of combinations of charge or hydrophobicity and putative location with respect to the membrane. Transfected mutant receptors were analyzed for cell surface expression, ligand binding, and ligand-activated phospholipase C activity. The receptors created can be placed generally in four distinct classes: those which bind and signal like the natural receptor; those which bind but fail to transduce signals; those which are expressed but neither bind nor transduce signal; and those which are not expressed at the cell surface.

Functional and biochemical analysis of the cloned Duffy antigen: identity with the red blood cell chemokine receptor.

The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen. Duffy-antigen cDNAs expressed in human embryonic kidney cells produced cell-surface proteins that reacted with two known anti-Duffy monoclonal antibodies. Direct ligand binding and displacement experiments using recombinant chemokine proteins also show that the cloned Duffy protein is the RBC chemokine receptor. Radiolabeled chemokines of both the C-C (RANTES and MCP-1) and C-X-C (IL-8 and MGSA/gro) subclasses bound reversibly to transfected cells with dissociation constants in the nanomolar range. Chemokines of either class displaced heterologous chemokines, indicating that they were competing for a single site on the transfected cells. Although the chemokines bound to the transfected cells with high affinity, there was no evidence for signal transduction, as measured by transient increases in intracellular calcium ion concentration, through the Duffy antigen/RBC chemokine receptor in transfected cells. Lastly, we have performed a computer analysis on the amino acid structure of the Duffy antigen/RBC chemokine receptor. Although the cloned Duffy antigen has been postulated to be a nine-transmembrane-spanning receptor, our analysis suggests that the molecule most likely belongs to the seven-transmembrane-spanning receptor superfamily and is therefore similar to other chemokine receptors previously identified.