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

FEBS Letters

Rosenthal, W.¹  Schultz, G.¹  Bombien, E.¹  Offermans, S.¹  Hoffmann, Barbara¹  Schäfer, R.²  Hinsch, K.-D.¹  Spicher, K.¹ 

[1] Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69173, D-1000 Berlin 33 FRG;Max-Planck-Institut für Biophysik, Kennedyallee 70, D-6000 Frankfurt 70, FRG

关键词: Chemoattractant;    Guanine nucleotide-binding protein;    Photoaffinity labeling;    (HL-60 cell);   

DOI  :  10.1016/0014-5793(90)80054-M

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-³²P]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 Mg²⁺; FMLP stimulated photolabeling at all Mg²⁺ 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 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.

【 授权许可】

Unknown