scholarly journals Regulation of the affinity state of the N-formylated peptide receptor of neutrophils: role of guanine nucleotide-binding proteins and the cytoskeleton.

1987 ◽  
Vol 105 (6) ◽  
pp. 2959-2971 ◽  
Author(s):  
R G Painter ◽  
K Zahler-Bentz ◽  
R E Dukes
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Intact Cell ◽  
Human Neutrophils ◽  
Membrane Domain ◽  
Intact Cells ◽  
Receptor Ligand ◽  
Ligand Dissociation ◽  
Buffer Control ◽  
Gamma S

Previous studies have indicated that the receptor for N-formylated peptides present on human neutrophils can exist in several ligand-dissociation states at least one of which is sensitive to guanine nucleotides. Human neutrophil membranes rich in cell surface enzyme markers have been isolated from cells pretreated at 37 degrees C with 5 nM fluoresceinated chemotactic peptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein; Fl-peptide) or a buffer control and analyzed for receptor-ligand dissociation states using a previously published fluorescence assay for estimating ligand binding and dissociation rates (Sklar, L. A., et al. 1984. J. Biol. Chem. 259:5661-5669). Fractionation of crude microsomes derived from homogenates of unstimulated cells by ultracentrifugation on linear D2O gradients yielded two plasma membrane-rich fractions termed fast and slow microsomes. Analysis of Fl-peptide dissociation rates from receptor present in fast membrane fractions of unstimulated cells yielded data that could be best fit by assuming that the receptor exists in three distinct ligand-dissociation states. The intermediate ligand-dissociation state (state B) accounted for 47% of the total and was converted to the fastest ligand-dissociation state (state A) by incubation of membranes with GTP or GTP-gamma-S. The remainder of the receptor (17%) present in unstimulated membranes was in a state from which ligand was virtually nondissociable (state C). This form of the receptor was insensitive to GTP-gamma-S. When cells were stimulated with Fl-peptide, most of the receptor present in slow and fast membranes was of the state C type. In contrast to unstimulated cells, slow membranes derived from cells exposed to Fl-peptide contained the majority of the recoverable receptor indicating that receptor was transferred to a physically isolatable membrane domain after ligand binding to the intact cell. The ligand-induced formation of state C in both fast and slow microsome fractions was inhibited by treatment of cells with dihydrocytochalasin B. However, the drug had no effect on translocation of the receptor to slow membranes. Pertussis toxin treatment of intact cells had no effect on ligand-induced formation of state C in either fraction even though other cellular responses were inhibited. Both slow and fast membranes contained a 41-kD G protein as assayed by immunoblot analysis. The data suggest that ligand induces a segregation of receptor-ligand complexes into a membrane domain in which the receptor is functionally uncoupled from the 41-kD neutrophil G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

1989 ◽  
Vol 109 (6) ◽  
pp. 2783-2790 ◽  
Author(s):  
A J Jesaitis ◽  
J O Tolley ◽  
G M Bokoch ◽  
R A Allen
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Membrane Domain ◽  
Plasma Membrane Fraction ◽  
A Minor ◽  
Plasma Membrane Domain ◽  
Gamma S

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

scholarly journals Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

2021 ◽  
Author(s):  
Naincy R. Chandan ◽  
Saji Abraham ◽  
Shuvasree SenGupta ◽  
Carole A. Parent ◽  
Alan V. Smrcka
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Intact Cell ◽  
Human Neutrophils ◽  
Intact Cells ◽  
Functional Roles ◽  
Tandem Mass Tag ◽  
Labeling Approach ◽  
Functional Relevance

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gαi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gαi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gαi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gαi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gαi1 strongly activates PRG. Strikingly, large differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gαi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.

scholarly journals Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

1990 ◽  
Vol 1 (9) ◽  
pp. 615-620 ◽  
Author(s):  
G F Verheijden ◽  
I Verlaan ◽  
J Schlessinger ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
G Protein ◽  
Egf Receptor ◽  
Phosphoinositide Hydrolysis ◽  
3T3 Cells ◽  
Guanosine Triphosphate ◽  
Intact Cells ◽  
Epidermal Growth ◽  
Gamma S

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor–Ligand Binding and β-Arrestin Assay

2013 ◽  
Vol 85 (4) ◽  
pp. 2276-2281 ◽  
Author(s):  
Kari Kopra ◽  
Markus Kainulainen ◽  
Piia Mikkonen ◽  
Anita Rozwandowicz-Jansen ◽  
Pekka Hänninen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Receptor Ligand ◽  
Homogeneous Method ◽  
G Protein Coupled

scholarly journals Neutrophil enhancement of Pseudomonas aeruginosa biofilm development: human F-actin and DNA as targets for therapy

2009 ◽  
Vol 58 (4) ◽  
pp. 492-502 ◽  
Author(s):  
Quinn M. Parks ◽  
Robert L. Young ◽  
Katie R. Poch ◽  
Kenneth C. Malcolm ◽  
Michael L. Vasil ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aspartic Acid ◽  
Biofilm Formation ◽  
Intact Cell ◽  
Relative Increase ◽  
Bacterial Biofilm ◽  
Human Neutrophils ◽  
Similar Response ◽  
Intact Cells ◽  
Biofilm Development

In the cystic fibrosis (CF) airway, chronic infection by Pseudomonas aeruginosa results from biofilm formation in a neutrophil-rich environment. We tested the capacity of human neutrophils to modify early biofilm formation of P. aeruginosa strain PAO1, and an isogenic CF strain isolated early and years later in infection. In a static reactor, P. aeruginosa biofilm density of all strains was enhanced at 24 h in the presence of neutrophils, with the greatest relative increase associated with the lowest inoculum of P. aeruginosa tested. Previously, neutrophil-induced biofilm enhancement was shown to largely result from the incorporation of F-actin and DNA polymers into the bacterial biofilm. This finding was advanced by the comparison of biofilm enhancement from intact unstimulated neutrophils and from lysed or apoptotic neutrophils. Apoptotic neutrophils, with an intact cell membrane, were unable to contribute to biofilm enhancement, while lysed neutrophils evoked a similar response to that of intact cells. Using F-actin and DNA as targets, the capacity of negatively charged poly(amino acids) to disrupt, or prevent, early biofilm formation was tested. Anionic poly(aspartic acid) effectively prevented or disrupted biofilm formation. Combination of poly(aspartic acid) with DNase resulted in a synergistic increase in biofilm disruption. These results demonstrate that the presence of dying neutrophils can facilitate the initial stages of biofilm development by low inocula of P. aeruginosa. Neutrophil F-actin represents a potential new therapeutic target for disruption of pathogenic biofilms.

scholarly journals Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

1992 ◽  
Vol 3 (11) ◽  
pp. 1229-1234 ◽  
Author(s):  
M Pupillo ◽  
R Insall ◽  
G S Pitt ◽  
P N Devreotes
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Fold Increase ◽  
Wild Type ◽  
Null Cell ◽  
Intact Cells ◽  
G Protein Alpha Subunit ◽  
Camp Receptor ◽  
System 1 ◽  
Gamma S

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.

scholarly journals Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.

1989 ◽  
Vol 109 (6) ◽  
pp. 2791-2799 ◽  
Author(s):  
E Särndahl ◽  
M Lindroth ◽  
T Bengtsson ◽  
M Fällman ◽  
J Gustavsson ◽  
...  
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Human Neutrophils ◽  
Receptor Complex ◽  
Chemotactic Peptide ◽  
Filamentous Actin ◽  
High Concentration ◽  
Intact Cells ◽  
Peptide Receptor ◽  
Receptor Complexes

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet-Leu-Phe-associated G-protein.

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