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Chemoattractant; Guanine nucleotide-binding protein; Photoaffinity labeling; (HL-60 cell)
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 Gi2 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, Gi2, 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.
