Agonist-sensitive binding of a photoreactive GTP analog to a G-protein 
alpha-subunit in membranes of HL-60 cells

Offermans, S.; Schäfer, R.; Hoffmann, Barbara; Bombien, E.; Spicher, K.; Hinsch, K.-D,; Schultz, G.; Rosenthal, W.

doi:10.1016/0014-5793(90)80054-m

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Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells

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Schäfer, R.
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Offermans, S., Schäfer, R., Hoffmann, B., Bombien, E., Spicher, K., Hinsch, K.-D., et al. (1990). Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells. FEBS Letters, 260(1), 14-18. doi:10.1016/0014-5793(90)80054-m.

Cite as: https://hdl.handle.net/21.11116/0000-0007-E3E6-5

Abstract

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-³²P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the G_i2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg²⁺; FMLP stimulated photolabeling at all Mg²⁺ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B₄ and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, G_i2, requires Mg²⁺ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg²⁺, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.