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Agonist‐sensitive binding of a photoreactive GTP analog to a G‐protein α‐subunit in membranes of HL‐60 cells
Author(s) -
Offermans S.,
Schäfer R.,
Hoffmann Barbara,
Bombien E.,
Spicher K.,
Hinsch K.-D.,
Schultz G.,
Rosenthal W.
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80054-m
Subject(s) - chemistry , pertussis toxin , g protein , gtp' , photoaffinity labeling , receptor , biophysics , agonist , protein subunit , biochemistry , gtp binding protein regulators , chemotaxis , g alpha subunit , leukotriene b4 , biology , enzyme , inflammation , immunology , gene
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. [α‐ 32 P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the G i2 α‐subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N ‐ionnyl‐methionyl‐leucyi‐phenylalanin (FMLP), absolutely required Mg 2+ ; FMLP stimulated photolabeling at all Mg 2+ concentrations employed (up to 30 mM). Addition of GDP (3–50 μM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B 4 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 Mg 2+ for basal and receptor‐stimulated activity, (ii) effective receptor‐mediated activation of G‐proteins is observed at mM concentrations of Mg 2+ , and (iii) receptor agonists apparently reduce the affinity of G‐proteins for GDP.