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L F Kolakowski

Publications and source records attributed to L F Kolakowski.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Co-evolution of ligand-receptor pairs in the vasopressin/oxytocin superfamily of bioactive peptides.

In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys-conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin-like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides.

Amino Acid Sequence↗
PubMed1996

Identification of cholecystokinin-B/gastrin receptor domains that confer high gastrin affinity: utilization of a novel Xenopus laevis cholecystokinin receptor.

A hallmark of the mammalian brain cholecystokinin (CCK) receptor, CCK-B/gastrin (CCK-BR), is its high affinity for two structurally related peptides, CCK and gastrin. Previous radioligand binding experiments suggested that the predominant CCK receptor from Xenopus laevis brain shares high affinity for sulfated cholecystokinin octapeptide but has > or = 1000-fold lower affinity for gastrin. To determine the molecular basis for this pharmacological divergence between mammalian and lower vertebrate receptors, we isolated a cDNA encoding the X. laevis brain CCK receptor (CCK-XLR). CCK-XLR shares approximately 50% homology at the amino acid level with both the human CCK-BR and the peripheral CCK-A receptor subtypes. The recombinant X. laevis receptor has a distinct pharmacological profile of agonist and antagonist affinities and as such offers a useful tool for structure-function studies. We used CCK-XLR to map the human CCK-BR domains that confer high affinity for gastrin. A series of chimeric CCK-BR/CCK-XLR constructs was generated and pharmacologically characterized. While maintaining wild-type affinity for sulfated cholecystokinin octapeptide, receptors with increasing amino-terminal contributions from CCK-BR demonstrated a stepwise increase in gastrin affinity. Further dissection of the amino-terminal third of the human receptor, a domain that confers a > 250-fold increase in gastrin affinity, revealed the importance of interactions among at least three subdomains. Additional structural requirements for gastrin affinity mapped to a segment spanning transmembrane domains IV and V.

Amino Acid Sequence↗
PubMed1995

The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking.

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes.

Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5' untranslated region of the cDNA, a 3' poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition.

Amino Acid Sequence↗
PubMed1995

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.

Animals↗
PubMed1995

Expression and biologic characterization of the murine chemokine KC.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was expressed in Escherichia coli by using a maltose binding protein vector and biochemically characterized as a ligand for both murine and human polymorphonuclear neutrophils (PMN). On murine PMN, KC is both a potent chemoattractant and up-regulator of Mac-1 cell surface expression. On human PMN, in contrast, KC exhibits dissociation of its chemoattractant and Mac-1 up-regulatory activities. Although KC strongly increases Mac-1 expression on human PMN, it does not induce chemotaxis in vitro. 125I-KC-Tyr binds to both mouse and human PMN with two classes of binding sites, including high affinity sites of 0.8 and 2 nM, with approximately 9,000 and 10,000 sites per cell, respectively. On mouse PMN, human macrophage inflammatory protein (MIP)-2 alpha and MIP-2 beta compete for 125I-KC-Tyr binding with high affinity, whereas the murine beta-chemokine TCA-3 does not compete. KC binds to human PMN by the IL-8 type B receptor and to murine PMN by a murine IL-8 type B receptor homologue. 125I-KC-Tyr also binds to human RBC with a single class of high affinity sites. KC mRNA is constitutively expressed in multiple murine tissues. With human IL-8 and KC cDNA as probes, a mouse neutrophil exudate library was screened: KC and MIP-2 were the dominant chemokine species found. Thus, KC appears to be intimately involved in murine inflammation and its constitutive expression may have a role in the basal trafficking of neutrophils.

Animals↗
PubMed1995

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).

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was synthesized as a recombinant protein in Escherichia coli and binds with 0.8 nM affinity to mouse neutrophils. Human neutrophils also bind recombinant KC at a site competitive with human interleukin (IL8) and Gro-alpha/MGSA, consistent with binding at the IL8 type B receptor (IL8RB). The cDNA corresponding to human IL8RB hybridizes strongly with two restriction fragments in murine genomic DNA, representing candidate receptor genes for KC. Molecular cloning of both mouse genomic DNA and neutrophil exudate cell cDNA libraries yielded a receptor with approximately 68% sequence identity to both the human IL8 type A and B receptors. Transient expression of the murine receptor cDNA in COS cells conferred binding ability to KC and a related gene product, macrophage inflammatory protein-2 (MIP-2) with high affinity (approximately 5 nM). Human IL8 was a poor agonist for this expressed receptor (Kd = approximately 400 nM). The potent activity of human IL8 on mouse polymorphonuclear neutrophils is not consistent with binding on the cloned receptor and suggests that murine homologues of IL8 and an IL8 type A receptor remain to be identified. Our data indicate that KC is the murine homologue of human Gro-alpha, and the KC receptor is an IL8 type B receptor homologue capable of binding both KC and macrophage inflammatory protein-2 with high affinity.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Mutations in the vasopressin V2 receptor gene in two families with nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (CNDI) is a rare X-linked disorder in which the renal collecting duct is unresponsive to arginine vasopressin, and thus, the urine is consistently hypotonic to plasma. As a result, affected individuals are unable to concentrate urine and suffer from episodes of severe dehydration and hypernatremia. Recently, the association between arginine vasopressin V2 receptor gene mutations and CNDI has been demonstrated. In this report, two additional novel molecular defects of the arginine vasopressin V2 receptor gene in CNDI families are described. In one family, the affected individual demonstrated a G-->T transversion causing a nonsense mutation in codon 231. This mutation results in a glutamic acid becoming a termination codon, causing premature termination and truncation of the encoded receptor protein. This mutation causes a NciI site within the gene to be abolished and a BsaWI site to be created. In the second family, affected individuals showed a 28-basepair duplicating insertion in the very beginning of exon 2 down-stream of the splice acceptor site. It was hypothesized that an insertion mutagenesis mechanism involves the formation of a stem-loop structure within the newly synthesized DNA strand, followed by a slipped mispairing. This may be a general mechanism for the deletion or insertion of repeated sequences within the genome. Recent data show that G-protein-coupled receptors are susceptible to many different mutations that often result in the loss of function, causing a similar clinical phenotype.

Adult↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1993

The human brain cholecystokinin-B/gastrin receptor. Cloning and characterization.

The predominant brain cholecystokinin receptor (CCK-B/gastrin) has been implicated in mediating many of the central effects of cholecystokinin, including anxiety, panic attacks, satiety, and analgesia, suggesting it is an important pharmacologic target. We now report the cloning and characterization of the cDNA encoding the human brain CCK-B/gastrin receptor. The cDNA was isolated from a human brain library by low stringency screening using the canine "gastrin" receptor cDNA as a hybridization probe. Nucleotide sequence analysis revealed an open reading frame encoding a 447-amino-acid protein with seven putative hydrophobic transmembrane domains and significant homology with other known members of the gastrin/cholecystokinin receptor family. Agonist and antagonist affinities of the recombinant human brain receptor expressed in COS-7 cells are consistent with a classical "CCK-B" receptor as defined by the literature. In COS-7 cells expressing the cloned receptor, CCK-8-stimulated phosphatidylinositol hydrolysis and intracellular Ca2+ mobilization suggesting second messenger signaling through phospholipase C. CCK-B/gastrin receptor transcripts were identified in human brain, stomach, and pancreas using high stringency Northern blot analysis. Southern blot hybridization analysis of human genomic DNA indicates that a single gene encodes both the brain and the stomach CCK-B/gastrin receptors. Our data suggest that the CCK-B and gastrin receptors are identical and that the long standing distinction between them may no longer apply.

Amino Acid Sequence↗
PubMed1993

A Null mutation in the vasopressin V2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus in the Hopewell kindred.

Congenital nephrogenic diabetes insipidus (DIR) is a rare X-linked hereditary disorder in which the renal collecting duct is unresponsive to arginine vasopressin; thus, the urine is consistently hypotonic to plasma. Recently, the association between the V2 receptor gene (AVPR2) and DIR has been proven. We have determined the gene sequence of four family members, from three generations, of a large North American family with CNDI who were originally part of the study used to formulate the Hopewell hypothesis. It had been proposed that a single DIR gene defect was introduced to North America by a member of an Ulster Scot kindred arriving on the ship Hopewell in 1761. DNA sequencing of the AVPR2 has identified a single base transversion from G-->A which changes tryptophan 71 to a stop codon in affected patients. This point mutation causes a truncation of the receptor leading to an essentially null allele. These data and other recently described mutations in the AVPR2 in North American pedigrees, descended from Ulster Scot ancestors and other origins, make the assertion of a founder effect proposed in the Hopewell hypothesis invalid.

Amino Acid Sequence↗