06-236 Sigma-AldrichAnti-Giα1/2 Antibody

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.

Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.