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Science 21 April 2006:
Vol. 312. no. 5772, pp. 443 - 446
DOI: 10.1126/science.1120378
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Reports

Differential Targeting of Gß{gamma}-Subunit Signaling with Small Molecules

Tabetha M. Bonacci1, Jennifer L. Mathews1, Chujun Yuan2, David M. Lehmann1, Sundeep Malik1, Dianqing Wu3, Jose L. Font1, Jean M. Bidlack1 and Alan V. Smrcka1,2*

1 Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
2 Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
3 Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.


Figure 1 Fig. 1. Small molecule binding to the hotspot on Gß{gamma}. (A) Structure of SIGK bound to Gß{gamma} at the hotspot. Amino acids within 6.5 Å of SIGK were targeted with the FlexX module of Sybyl virtual docking software. (B) Competition ELISA data for three of the compounds identified in the virtual screen. Compounds were tested for their ability to inhibit binding of a phage displaying the peptide SIGKAFKILGYPDYD (3, 4). M119 (NSC119910) (squares); M109 (NSC109208) (triangles); M117 (NSC117079) (circles). Data for all the binding compounds are summarized in table S1. (C) Structures of representative ß{gamma}-binding compounds. (D) Competition of M119 for interactions between G{alpha}i1 and Gß1{gamma}2. F-{alpha}i1 and M119 were simultaneously added to bGß1{gamma}2 immobilized on streptavidin beads. The amount of bead-based fluorescence was assessed by flow cytometry as described (11, 12). [View Larger Version of this Image (53K GIF file)]
 

Figure 2 Fig. 2. Differential effects of M119 and M201 on ß{gamma}-dependent regulation of downstream targets. (A) Effects of M119 and M201 (NSC201400) on Gß{gamma}-activation of PLCß. Purified PLCß2 (0.25 ng) was assayed in the presence (triangles) or absence (squares) of 100 nM purified Gß1{gamma}2. (B) Effects of M119 and M201 effects on purified PLCß3 (0.5 ng) activity in the presence (triangles) or absence (squares) of 100 nM purified Gß1{gamma}2. (C) Effects of M119 and M201 on activation of PI3K{gamma} by Gß1{gamma}2. Assays contained 10 ng of purified p101/p110 PI3K{gamma} heterodimer with or without 100 nM purified Gß1{gamma}2. Left,: (triangles) 100 nM Gß1{gamma}2 or (squares) no ß{gamma}. (D) Effects of M119 and M201 on Gß{gamma}-GRK2 interactions. M119 or M201 and 25 nM purified GRK2 were added simultaneously to 250 nM bGß1{gamma}2 immobilized on streptavidin agarose. Associated GRK2 was detected with antibody to GRK2. Data are representative experiments [mean ± SEM, except (D)] and were repeated at least three times each. The concentrations of compound required for these assays are apparently higher than predicted by the phage ELISA assays, but may reflect the different assay conditions and protein concentrations required for each assay. [View Larger Version of this Image (34K GIF file)]
 

Figure 3 Fig. 3. Effects of M119 and related compounds on Gß{gamma} signaling in dimethyl sulfoxide (DMSO)–differentiated HL-60 cells. (A) M119 and related compounds block fMLP-dependent Ca2+ release in differentiated HL-60 cells, loaded with 1 µM Fura2-AM. Cells were pretreated with DMSO or 10 µM compounds for 5 min before stimulation with 250 nM fMLP. The change in fluorescence was monitored at 340/380 nm. Data are representative of five independent experiments. Compounds are significantly different than DMSO control, P < 0.01. See fig. S4A for dose dependence. (B) Same as A except 10 µM M201 was tested. Data are representative of four independent experiments. See fig. S4B for pooled data. (C) M119 and M201 inhibition of GRK2 translocation. Differentiated HL-60 cells were treated with 10 µM compound before stimulation with 250 nM fMLP. Translocation of endogenous GRK2 was determined by Western blotting with a GRK2 antibody and quantitative chemiluminescence. Data are mean ± SEM from five experiments, *P < 0.05, **P < 0.01 analysis of variance (ANOVA). (D) M119 and M158C (NSC158110) inhibition of GFP-PHAkt translocation. Differentiated HL-60 cells stably overexpressing GFP-PHAkt were treated with 10 µM compound before stimulation with 100 nM fMLP. Translocation of GFP-PHAkt to the membrane was determined by Western blotting with antibody to GFP and quantitative chemiluminescence. Data are mean ± SEM from four experiments. ***P < 0.001 ANOVA. (E) Lack of effect of M119, M158C, and M201 on fMLP-induced ERK1 and ERK2 activation. Differentiated HL-60 cells were pretreated with 10 µM of compound prior to stimulation with 1 µM fMLP for 5 min. Levels of phosphorylated and total ERK were determined by Western blotting. This experiment was repeated three times with similar results. [View Larger Version of this Image (42K GIF file)]
 

Figure 4 Fig. 4. M119 effects on morphine-induced antinociception in (A) wild-type and (B) PLCß3–/– mice. Increasing concentrations of morphine were administered intra-cerebroventricularly to mice with (squares) or without (triangles) concomitant administration of 100 nmol M119. Antinociception was measured 20 min after injection using the 55°C tail-flick test. Data are mean ± SEM from 7 to 10 animals at each point. [View Larger Version of this Image (19K GIF file)]
 

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