Development of Drug Screening Methods Using the Activities of GTP-binding Proteins

点击翻译按钮获取中文摘要

GTP-binding proteins consisting of alpha-, beta- and gamma-subunits (G proteins) carry signals from membrane receptors to effectors such as enzymes or ion channels. Recent studies have indicated that several peptides, such as mastoparan, directly interact with G proteins resulting in the activation of the signal-coupling proteins in manners similar to receptors stimulated by agonists.In the present studies, possible interactions of various venom peptides (and related compounds) with G proteins and/or membrane receptors were studied in reconstituted phospholipid vesicles. Mast cell-degranulating (MCD) peptide stimulated the steady-state rate of GTP hydrolysis catalyzed by the reconstituted G proteins. Synthetic D-MCD peptide, the optical isomer of MCD peptide, was also effective in the activation of G proteins as L-MCD peptide. The stimulations by L- and D-peptides were both abolished in G proteins that had been ADP-ribosylated by pertussis toxin. In comparison of the amino acid sequences of various peptides tested, the extent of the activation of G proteins appeared to be correlated with the number of basic amino acid residues in the alpha-helix of peptides. These results suggest that cationic clusters at one side of the alpha-helical surface are more important in the direct activation of G proteins than a specific, alpha-helical structure. Moreover, mastoparan induced conversion of a high- and a low-affinity states of membrane receptors for agonists to a new middle-affinity state, which was resulted from coupling of the receptors with G proteins. Thus, the various activities G proteins appeared to be utilizable for elucidation of some types of drug screening.