Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gβγ

Abstract

As part of a broader effort to identify postreceptor signal regulators involved in specific diseases or organ adaptation, we used an expression cloning system in Saccharomyces cerevisiae to screen cDNA libraries from rat ischemic myocardium, human heart, and a prostate leiomyosarcoma for entities that activated G protein signaling in the absence of a G protein coupled receptor. We report the characterization of activator of G protein signaling (AGS) 8 (KIAA1866), isolated from a rat heart model of repetitive transient ischemia. AGS8 mRNA was induced in response to ventricular ischemia but not by tachycardia, hypertrophy, or failure. Hypoxia induced AGS8 mRNA in isolated adult ventricular cardiomyocytes but not in rat aortic smooth muscle cells, endothelial cells, or cardiac fibroblasts, suggesting a myocyte-specific adaptation mechanism involving remodeling of G protein signaling pathways. The bioactivity of AGS8 in the yeast-based assay was independent of guanine nucleotide exchange by G␣, suggesting an impact on subunit interactions. Subsequent studies indicated that AGS8 interacts directly with G␤␥ and this occurs in a manner that apparently does not alter the regulation of the effector PLC-␤ 2 by G␤␥. Mechanistically, AGS8 appears to promote G protein signaling by a previously unrecognized mechanism that involves direct interaction with G␤␥.accessory protein ͉ signal adaptation ͉ hypoxia

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

“…AGS8 was discovered as a receptor-independent activator of G-protein signaling upregulated during transient cardiac ischemia (Sato et al, 2006b). Initial characterization of AGS8 indicated that it bound to Gβγ subunits.…”

“…In contrast, AGS2-8 were functional in the G204A Giα2 genetic background and in the presence of overexpressed RGS4. Thus, AGS2-8 differed from AGS1 and apparently activated heterotrimeric G-protein signaling by a mechanism independent of nucleotide exchange (Bernard et al, 2001;Cao et al, 2004;Cismowski et al, 1999;Sato et al, 2006b;Takesono et al, 1999) (Figure 2). AGS3-6 were active in yeast strains expressing mammalian Giα2 and Giα3 but not Gαs or Gα16.…”

“…In contrast, AGS2 and AGS7-10 were active in yeast strains expressing Giα2, Giα3, Gαs or Gα16 suggesting that their action involved an interaction with Gβγ, which was common to each of the yeast strains. AGS1-6 bind to Gα subunits, whereas AGS2 and 8 bind Gβγ (Cao et al, 2004;Sato et al, 2006b;Takesono et al, 1999). The AGS proteins may also interact at least transiently with intact Gαβγ heterotrimer.…”

“…AGS proteins that are upregulated in disease states or as part of a tissue adaptation program can likely be identified by screening a panel of cDNA libraries generated from human disease tissue in the yeast-based discovery platform. Proof of concept with this approach is provided by Sato et al (Sato et al, 2006b) and the functional screen of cDNA libraries from rat ischemic myocardium and a prostate leiomyosarcoma (Cao et al, 2004;Sato et al, 2006b). …”

“…This protein activates both heterotrimeric brain G-protein and free Gα and exhibits mechanistic properties that are distinct from receptor-mediated activation of G-protein (Ribas et al, 2002b). The second approach involved an expression cloning system utilizing the pheromone response pathway in S. cerevisiae (Cao et al, 2004;Cismowski et al, 2002;Cismowski et al, 1999;Sato et al, 2006b;Takesono et al, 1999).…”