Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil

Lad, Pramod M.; Glovsky, M. Michael; Richards, John H.; Learn, David B.; Reisinger, Diane M.; Smiley, Paula A.

doi:10.1016/0161-5890(84)90048-8

Cited by 20 publications

(4 citation statements)

References 34 publications

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“…The cells were washed twice with PBS and then resuspended at 10s cells/mL in PBS supplemented with 0.1 mM EGTA. Plasma membranes were prepared according to a modification of the procedure described by Lad et al (1984). Briefly, lysis was affected by sonication for 7 s at 4 °C with a micro-tip probe sonicator at 20 W. The homogenate was layered over a 25% sucrose, 0.1 mM DTT, PBS cushion and centrifuged at 500g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Dickey¹,

Pyun²,

Navarro³

1988

Biochemistry

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We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

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Section: Methodsmentioning

confidence: 99%

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Dickey¹,

Pyun²,

Navarro³

1988

Biochemistry

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“…Distinct pools of DAG are generated in different cell compartments by activation of phospholipases C and D [Koo et al, 1983;Lad et al, 1984;Gewirtz et al, 1998]. …”

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confidence: 99%

Differentiation of HL‐60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs)

Batista

Warbington

Badwey

et al. 2004

J of Cellular Biochemistry

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Diacylglycerol Kinases (DGKs) are a family of enzymes that regulate the levels of different pools of diacylglycerol (DAG), affecting DAG-mediated signal transduction. Since DAG is known to play several important regulatory roles in granulocyte physiology, we investigated the expression pattern of DGK isoforms throughout differentiation of HL-60 cells to granulocytes. HL-60 cells were incubated with 1.25% dimethyl-sulfoxide (DMSO) to initiate differentiation and total RNA isolated at different time points. DGK expression was assessed through Northern blot, end-point PCR, and real-time PCR. The non-selective inhibitors R59022 and R59949 were used to block DGK at different time points throughout differentiation. CD11b and GPI-80, reactive oxygen species (ROS) generation, changes in the cell cycle, and apoptosis were used as markers of differentiation. Of the nine isoforms of DGK evaluated (alpha, delta, epsilon, gamma, zeta, beta, theta;, iota, eta), only five (alpha, delta, epsilon, gamma, and zeta) were expressed in HL-60 cells. DGKalpha was virtually absent in non-differentiated cells, but was markedly upregulated throughout differentiation. The other isoforms (delta, epsilon, gamma, and zeta) were expressed in undifferentiated HL-60 cells but were substantially decreased throughout differentiation. Non-selective blocking of DGK with R59022 and R59949 led to acceleration of differentiation, reducing the time necessary to observe upregulation of CD11b, GPI-80 and generation of ROS by 50%. Likewise, the cell cycle was disrupted when DGK isoforms were inhibited. These results provide evidence that DGK levels are dynamically regulated throughout differentiation and that expression of DGKs play an important regulatory function during the differentiation of neutrophils.

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“…Through the use of radioiodinated cross-linking reagents and photoaffinity labeling techniques, the human neutrophil FMLP receptor has been reported to consist of three components, the predominant glycoprotein having a molecular weight of 55,000 to 70,000 (12,17,25). Functional responses of neutrophils are the result of ligand binding to specific plasma membrane receptors and appear to be mediated through a transduction mechanism involving the activation of phospholipase C and specific guanine nucleotide-binding proteins (18), which are involved in the receptor-mediated control of neutrophil function (19). Recently, the human FMLP receptor has been proposed to be a seven-transmembrane-domain structure characteristic of the G-protein-coupled receptor superfamily (7,8).The production of synthetic peptides from the known amino acid sequence of a biologically active protein allows the characterization of the immunological and functional domains of that protein.…”

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confidence: 99%

Structural and functional characterization of the human formyl peptide receptor ligand-binding region

1994

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The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions.Polymorphonuclear leukocytes (neutrophils) provide an important host defensive mechanism against bacterial and fungal infections. Neutrophils respond to inflammation both in vitro and in vivo through a chemotactic pathway of directed locomotion towards a stimulus (22). N-formylated peptides such as N-formyl-1-methionyl-1-leucyl-1-phenylalanine (FMLP), believed to be structural analogs of bacterial metabolic products, are potent chemoattractants for neutrophils (26,33). In addition to chemotaxis, a number of neutrophil functions, including aggregation, phagocytosis, generation of cytotoxic superoxide anions, production of bioactive arachidonate metabolites, and proteolytic enzyme release, can be elicited by potent chemoattractants such as FMLP (3,6,23,28,32). Through the use of radioiodinated cross-linking reagents and photoaffinity labeling techniques, the human neutrophil FMLP receptor has been reported to consist of three components, the predominant glycoprotein having a molecular weight of 55,000 to 70,000 (12,17,25). Functional responses of neutrophils are the result of ligand binding to specific plasma membrane receptors and appear to be mediated through a transduction mechanism involving the activation of phospholipase C and specific guanine nucleotide-binding proteins (18), which are involved in the receptor-mediated control of neutrophil function (19). Recently, the human FMLP receptor has been proposed to be a seven-transmembrane-domain structure characteristic of the G-protein-coupled receptor superfamily (7,8).The production of synthetic peptides from the known amino acid sequence of a biologically active protein allows the characterization of the immunological and functional domains of that protein. This technique has been successfully utilized in the identification of functional domains of receptors such as the pho...

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Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil

Cited by 20 publications

References 34 publications

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Differentiation of HL‐60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs)

Structural and functional characterization of the human formyl peptide receptor ligand-binding region

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