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Means for the assessment of radioligand quality and its importance in receptor-binding studies. Observations with radiolabelled formylmethionyl-leucyl-phenylalanine.

ABSTRACT: Various methods for testing the quality of radioligands were applied to two different radiolabelled forms of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The purpose of the study was both to examine the value of these methods for assessing radioligand quality and to determine the suitability of these particular radioligands for studying the chemotactic formylpeptide receptors on the rabbit neutrophil. It is useful in this context to distinguish two different aspects of radioligand quality: these are purity and equivalence to the native ligand. The two methods described for measuring receptor-reactivity (or 'bindability'), by measuring binding to an increasing excess of receptors and by a re-incubation procedure, provide a reliable measure of purity that should readily be applicable to other radioligands. Equivalence to the native ligand is more difficult to establish, and any uncertainty about the specific radioactivity of the radioligand can pose serious problems with this assessment. Commercial preparations of both tritiated and 35S-labelled fMet-Leu-Phe were found to be inadequately pure for detailed receptor studies. Repurification by t.l.c., however, consistently yielded radioligand preparations of high purity and close equivalence to the native ligand. Other radioligands may often also require a suitable repurification step before use for detailed receptor studies; this is especially important whenever a complex receptor-binding pattern is envisaged.

SUBMITTER: Kermode JC

PROVIDER: S-EPMC1149175 | biostudies-other | 1988 Jun

REPOSITORIES: biostudies-other

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Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.

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The chemotactic factor N-formylmethionyl-leucyl-phenylalanine activates microtubule-associated protein 2 (MAP) kinase and a MAP kinase kinase in polymorphonuclear leucocytes.

Project description:Incubation of human polymorphonuclear leucocytes (PMN) with either the chemotactic factor N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) activates a kinase with phosphorylating activity towards a known microtubule-associated protein-2 (MAP) kinase substrate, the epidermal growth factor receptor peptide (T669). Activation of this enzyme by FMLP was maximal at 1 min, decreasing by 10 min. Activation by PMA was slightly slower than that by FMLP, but more prolonged (maximal at 5 min, with no significant decrease by 20 min). The enzyme induced by either stimulant bound strongly to phenyl-Sepharose, had a molecular mass of 40 kDa on gel filtration and phosphorylated three MAP kinase substrates, i.e. MAP, myelin basic protein and the T669 peptide. By use of antibodies to MAP kinases and phosphotyrosine, the enzyme was identified as the 42 kDa MAP kinase (also known as extracellular-signal-regulated kinase 2, ERK2). Stimulation of PMN with FMLP or PMA was also found to induce a kinase kinase which phosphorylated human recombinant MAP kinase on threonine and tyrosine, with concomitant activation. These results suggest that MAP kinase and the kinase kinase are involved in the activation of PMN by chemotactic factors such as FMLP.

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Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and Ca2+.

Project description:The role of two G-proteins, Gp and Ge, in the stimulus-secretion pathway has been proposed on the basis of studies where GTP analogues have been introduced into permeabilized cell preparations. In this study, evidence is provided that two G-proteins are also involved when a receptor-directed agonist is used. Intact human neutrophils were made refractory to formylmethionyl-leucyl-phenylalanine (fMetLeuPhe) stimulation by metabolic inhibition and then permeabilized with streptolysin O to compare the intracellular requirements for exocytosis from specific and azurophilic granules and arachidonate release. In the presence of 1 microM-Ca2+ and 1 mM-MgATP, fMetLeuPhe or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induce secretion from both granule types as well as arachidonate release. Secretion and arachidonate release owing to fMetLeuPhe can occur in the absence of ATP, conditions under which G-protein-mediated activation of phospholipase C is suppressed. GTP[S]-induced secretion can also occur in the absence of MgATP, but GTP[S]-induced arachidonate release cannot. It is concluded that fMetLeuPhe, like GTP[S], stimulates secretion by interacting with another G-protein-mediated reaction apart from Gp. Evidence is provided that a possible target for the second G-protein-mediated reaction involved in fMetLeuPhe-induced secretion (but not GTP[S]-induced secretion) is phospholipase A2.

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ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils.

Project description:Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells 'primed' with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.

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Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Project description:Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor--G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.

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