Search PubMed⌕ Search

Biomedical subjects

P M Murphy

Publications and source records attributed to P M Murphy.

At least 127 records · Page 7Linked to original sources
PubMed1994

Chemokine receptors and molecular mimicry.

Chemokines are small pro-inflammatory peptides that are best known for their leukocyte-chemoattractant activity. The cloned leukocyte chemokine receptors, interleukin 8 receptor (IL-8R) types A and B and the macrophage inflammatory protein 1 alpha (MIP-1 alpha)/RANTES receptor, are related by sequence and chemokine binding to two herpesvirus products, and to the Duffy antigen that mediates erythrocyte invasion by the malaria-causing parasite Plasmodium vivax. Here, Sunil Ahuja, Ji-Liang Gao and Philip Murphy suggest that, in addition to the activation of leukocytes, chemokines may be important in the function of erythrocytes and, through molecular mimicry, in microbial pathogenesis.

Animals↗
PubMed1994

The molecular biology of leukocyte chemoattractant receptors.

Leukocytes migrate from the blood to sites of inflammation in response to locally produced chemoattractants that activate specific cell surface receptors. The primary structures of leukocyte receptors for N-formyl peptides, C5a, platelet-activating factor, and 8 of the 18 known human chemokines (interleukin-8 and related molecules) have been deduced from cloned cDNAs. All of these are seven-transmembrane-domain rhodopsin-like G protein-coupled receptors. Biochemical and molecular genetic analysis of the chemoattractant receptors indicates that the chemoattractants may have both broadly overlapping as well as specialized roles in the regulation of acute and chronic inflammation. Interestingly, the chemokine receptors have functional homologues in human cytomegalovirus and Herpesvirus saimiri. Moreover, the Duffy antigen, which mediates invasion of erythrocytes by Plasmodium vivax, a major cause of malaria, is also a chemokine binding protein. These surprising developments suggest that in addition to leukocyte-mediated inflammation, the chemokines may also be involved in erythrocyte function and, through molecular mimicry, in microbial pathogenesis.

Amino Acid Sequence↗
PubMed1993

Species and subtype variants of the N-formyl peptide chemotactic receptor reveal multiple important functional domains.

The N-formyl peptide receptor (FPR) is a seven transmembrane-domain receptor that mediates trafficking and activation of phagocytic leukocytes in response to N-formyl oligopeptides such as fMet-Leu-Phe. cDNAs for high affinity FPRs have been cloned from both human (huFPR) and rabbit (rabFPR). To identify functional domains of FPR, we have studied two structurally related "natural mutants" that are 100-10,000-fold less sensitive than huFPR and rabFPR to fMet-Leu-Phe owing to sequence differences that are located predominantly in the proposed extracellular and transmembrane domains. The first is murine FPR (muFPR, 76% identical to huFPR) whose gene we have now cloned and expressed in Xenopus oocytes; the second is the previously reported human FPR-like 1 receptor (FPRL1R, 69% identical to huFPR) which was used to construct huFPR-FPRL1R chimeras. Comparison of the structure and function of huFPR, FPRL1R, muFPR, rabFPR, and huFPR-FPRL1R chimeras suggests that multiple non-contiguous residues must be apposed by coordinate folding of all of the extracellular and transmembrane domains in order to form the high affinity fMLF-binding site.

Amino Acid Sequence↗
PubMed1993

Sequence and organization of the human N-formyl peptide receptor-encoding gene.

The human FPR1 gene encodes the N-formyl peptide receptor, a G protein-coupled receptor (GPCR) that mediates the activation of mature myeloid cells by bacterial N-formyl oligopeptides. To investigate the molecular basis for myeloid-specific production of this receptor, we have cloned and sequenced FPR1. The gene is organized into three exons and two introns that span 6 kb. The coding block lacks introns. Yet, the transcription start point (tsp) is separated from the start codon by 4902 bp consisting of three exons and two large introns. Two mRNAs are produced by alternative splicing of exon 2 in HL-60 neutrophils and normal blood monocytes. The region 5' to the tsp contains three pyrimidine-rich segments, a feature that has been observed in other myeloid-specific genes. One complete Alu repeat is found in each intron and in the 3'-flanking region 317 bp downstream of the polyadenylation signal. Thus, FPR1 is a small myeloid-specific gene that is expressed as two alternatively spliced mRNAs encoding the same protein.

Amino Acid Sequence↗
PubMed1993

Molecular piracy of mammalian interleukin-8 receptor type B by herpesvirus saimiri.

Viruses are known to acquire and modify the genes of their hosts to attain a survival advantage in the host environment. Herpesvirus saimiri (HVS) is a T-lymphotropic virus that causes fatal lymphoproliferative diseases in several non-human primates. The gene ECRF3 of HVS was most likely acquired from a primate host. ECRF3 encodes a putative seven-transmembrane-domain receptor that is remotely related (approximately 30% amino acid identity) to the known mammalian alpha and beta chemokine receptors, namely interleukin-8 receptor (IL8R) types A and B and the MIP-1 alpha/RANTES receptor, respectively. Chemokines regulate the trafficking, activation, and, in some cases, proliferation of myeloid and lymphoid cell types. We now show that ECRF3 encodes a functional receptor for the alpha chemokines IL-8, GRO/melanoma growth stimulatory activity (MGSA), and NAP-2 but not for beta chemokines, a specificity identical to that of IL8RB. Paradoxically, IL8RA shares 77% amino acid identity with IL8RB but is not a receptor for GRO/MGSA or NAP-2. This is the first functional characterization of a viral seven-transmembrane-domain receptor. It suggests a novel role for alpha chemokines in the pathogenesis of HVS infection by transmembrane signaling via the product of ECRF3.

Amino Acid Sequence↗
PubMed1993

Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor.

The chemokine beta family is comprised of at least six distinct cytokines that regulate trafficking of phagocytes and lymphocytes in mammalian species; at least one of these, macrophage inflammatory protein 1 alpha (MIP-1 alpha), also regulates the growth of hematopoietic stem cells. We now show that MIP-1 alpha and the related beta chemokine, RANTES, induce transient alterations in intracellular Ca2+ concentration in polymorphonuclear leukocytes that can be reciprocally and specifically desensitized, suggesting a common receptor. Moreover, we have now cloned both the cDNA and the gene for this receptor, functionally expressed the receptor in Xenopus oocytes, and mapped the gene to human chromosome 3p21. Transcripts for the receptor were found in mature and immature myeloid cells as well as B cells. The receptor is a member of the G protein-coupled receptor superfamily. It has approximately 33% amino acid identity with receptors for the alpha chemokine, interleukin 8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.

Amino Acid Sequence↗
PubMed1993

Industrial EMS.

Explore the source record for details and available documents.

Career Mobility↗
PubMed1992

A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family.

Phagocytic cells of many higher species express calcium mobilizing G protein-coupled receptors for bacterial N-formyl peptides which mediate chemotaxis, degranulation, and the respiratory burst. cDNA encoding an N-formyl peptide receptor (FPR) has been reported. We now report the isolation of a closely related cDNA, 2.6 kilobase pairs in length, which we have designated as the FPRL1 receptor cDNA (FPRL1 = formyl peptide receptor like-1). FPR and the FPRL1 receptor derive from small, single-copy genes, both of which are located on human chromosome 19. The gene loci are designated FPR1 and FPRL1, respectively. Both FPR and FPRL1 cDNA cross-hybridize under high stringency conditions with a third gene, designated as FPRL2, which does not appear to be expressed in neutrophils. In contrast, transcripts for both the FPRL1 receptor and FPR are detected only in differentiated myeloid cells; the distribution of N-formyl peptide binding sites is also restricted to mature myeloid cells. FPRL1 cDNA encodes a 351-amino acid polypeptide whose sequence is 69% identical to that of FPR. G protein-coupled receptors that exhibit this degree of structural similarity typically possess a conserved ligand specificity. However, the FPRL1 receptor does not bind prototype N-formyl peptide ligands when expressed in heterologous cell types. These results suggest that FPR1 may be the only gene that is expressed by neutrophils that encodes a receptor capable of binding prototype N-formyl peptides. Moreover, discovery of the FPRL1 receptor indicates the existence of another as yet unidentified peptide that may recruit neutrophils to sites of inflammation.

Amino Acid Sequence↗
PubMed1992

The guanine nucleotide-binding protein Gs activates a novel calcium transporter in Xenopus oocytes.

Calcium influx is an important aspect of receptor-mediated signal transduction, yet limited information is available regarding the pathways of calcium influx into nonexcitable cells. We show that treatment of oocytes from Xenopus laevis with cholera toxin, a potent activator of the guanine nucleotide-binding protein Gs, specifically stimulates a sustained inward whole cell flux of calcium through a novel membrane transporter. The calcium is distributed into a mobilizable pool. The flux is voltage-independent and is completely and specifically blocked by microinjection of oocytes with an antiserum directed against Gs alpha. The flux is not activated by treatment of the cells with forskolin or 8-bromo-cyclic adenosine monophosphate indicating that the effect of Gs alpha on the transporter occurs independently of adenylylcyclase activation. Transporter activity is insensitive to benzyl amiloride, does not require a sodium gradient, and is not stimulated by external calcium, indicating that it is not a sodium-calcium exchanger. The Gs-activated flux is dramatically potentiated by lanthanum ion and other trivalent cations but not by any of six divalent cations that were tested; all other known calcium channels and exchangers are, in contrast, potently blocked by lanthanum. The divalent cation cadmium inhibited transporter activity in a concentration-dependent manner. This novel calcium transporter may be important for receptor-mediated calcium influx in the oocyte and perhaps other cell types.

Animals↗
PubMed1992

Molecular evolution of the human interleukin-8 receptor gene cluster.

Interleukin-8 (IL-8) is the prototype for a family of at least eight neutrophil chemoattractants whose genes map to human chromosome 4q13-q21. Two human IL-8 receptors, IL8RA and IL8RB, are known from cDNA cloning; IL8RA is a promiscuous receptor for at least two other related ligands, GRO alpha and NAP-2. We now report cloning of the genes for IL8RA, IL8RB and a recently inactivated pseudogene of receptor A (IL8RAP). These form a cluster of only three genes in the superfamily of G protein-coupled receptors (GPCRs) and map to 2q34-q35. The coevolutionary diversity displayed by the IL-8 ligand-receptor complex--ligand promiscuity for IL-8, receptor promiscuity for IL8RA, gene duplication for both ligands and receptors and gene extinction in the case of IL8RAP--is unprecedented for the GPCR superfamily.

Amino Acid Sequence↗
PubMed1991

Cloning of complementary DNA encoding a functional human interleukin-8 receptor.

Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.

Amino Acid Sequence↗
PubMed1991

Functional expression of the human formyl peptide receptor in Xenopus oocytes requires a complementary human factor.

Human phagocytic cells express receptors for bacterial N-formyl peptides (formyl peptide receptor or FPR) which mediate chemotaxis, degranulation, and the respiratory burst. Although cDNA encoding a human phagocyte formyl peptide-binding protein has been reported recently (Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochem. Biophys. Res. Commun. 168, 1103-1109), functional coupling to signal transduction processes was not demonstrated. We describe corresponding full-length cDNA clones and prove that they encode the calcium-mobilizing human formyl peptide receptor by demonstrating functional reconstitution in the Xenopus oocyte. We further demonstrate that in contrast to all other cloned guanine nucleotide-binding regulatory protein (G-protein) coupled receptors expressed in this system, microinjection of FPR transcripts is not sufficient to confer ligand responsiveness to the oocyte: co-injection of phagocyte RNA encoding a complementary human factor that is not the alpha subunit of the heterotrimeric G-proteins Gi1, Gi2 or Gi3 is also required. Whereas a 1.4-kilobase FPR transcript is expressed exclusively in differentiated phagocytic cells, the complementary factor activity localizes to a 3.5-kilobase RNA fraction and is expressed in both differentiated and undifferentiated myeloid cells as well as in liver. The deduced human FPR protein possesses seven hydrophobic putative membrane spanning segments, three sites for N-linked glycosylation, and a short 18-amino acid predicted third cytoplasmic loop. Surprisingly, the human FPR possesses only 28% amino acid identity with the rabbit FPR reported recently by Thomas and co-workers (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20065). Moreover, the rabbit FPR does not require a complementary factor for calcium mobilization in the oocyte. Structural alignment reveals at most 20% amino acid identity of the human FPR with other G-protein coupled receptors, indicating a common ancestral gene. Functional reconstitution of the recombinant FPR will now permit precise delineation of its functional and regulatory domains. Moreover, discovery of a complementary factor for oocyte expression of the human FPR establishes a novel approach to the qualification by ligand screening of cDNA encoding other suspected G-protein coupled receptors.

Amino Acid Sequence↗
PubMed1991

Viability and distribution of bacteria after passage through a circle anaesthetic system.

We have assessed in vitro the viability of eight species of micro-organism suspended as aerosols and passed through a soda-lime absorber rebreathing system. As had been predicted, the soda-lime exerted a potent cidal effect on non-sporing organisms, all of which were rendered non-viable. One percent of the spore bearing organism, Bacillus subtilis, was still viable after 30 min contact. Although Bacillus subtilis is an organism of low pathogenicity, spores may be more resistant to the alkaline medium of the soda-lime. The micro-organisms were observed to lodge in all components of the breathing system, with the greatest concentration being recovered from the corrugated tubing. We recommend that disposable components of the circle breathing system should be changed, and the non-disposable parts sterilized or disinfected, daily. Soda-lime cannisters should be sterilized or disinfected when changing the soda-lime.

Anesthesia, Closed-Circuit↗
PubMed1990

Characterization of human phagocytic cell receptors for C5A and platelet activating factor expressed in Xenopus oocytes.

Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.

Animals↗
PubMed1990

Characterization of phagocyte P2 nucleotide receptors expressed in Xenopus oocytes.

Stimulation of phagocytic cells with micromolar concentrations of extracellular ATP primes the production of toxic oxygen metabolites in response to chemoattractants and independently activates a secretory response in vitro. It is hypothesized that extracellular ATP derived from platelet storage granules and damaged endothelium at sites of localized tissue damage or infection may potentiate the pro-inflammatory effects of phagocytic cells in vivo. ATP-dependent functional responses in the phagocyte appear to be due to stimulation of putative P2 purinoreceptors that are coupled to the activation of a phospholipase C via a pertussis toxin-sensitive G-protein. The existence in nature of at least four subtypes of P2 purinoreceptors has been proposed based on the rank order of potency of nucleotide analogs of ATP studied in a variety of cell types. However, no studies involving the structural identification and characterization of the putative receptors have been reported. We have used the Xenopus oocyte expression system to demonstrate acquired adenosine 5'-(thio) triphosphate (ATP gamma S) responsiveness in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 by measuring the accelerated efflux of intracellular calcium. Two peaks of ATP gamma S responsiveness (Peak I and Peak II) were detected in sucrose gradient fractionated RNA that corresponded to transcript sizes of 4 and 6 kilobases and that were distinct from a third peak previously shown to be enriched in formyl peptide chemoattractant receptor activity. Peak I and Peak II RNA endowed receptor activity in the oocyte that was pharmacologically indistinguishable: ADP and AMP were inactive whereas UTP and ITP exhibited activity that was similar in potency to that of ATP gamma S. Both Peak I and Peak II ATP gamma S-dependent activity was inhibited by pertussis toxin. These data strongly support the concept of phagocytic cell receptors for extracellular nucleotide triphosphates whose ligand specificity is distinct from all other previously described P2 purinoreceptor subtypes, with the exception of the P2 receptor described in Ehrlich ascites tumor cells, by virtue of the ineffectiveness of ADP as a stimulus. These receptors are most likely composed of a single polypeptide chain that can be expressed in the Xenopus oocyte in a functional form regulated by a pertussis toxin-sensitive G-protein.

Adenosine Triphosphate↗