Modulation of glutathione level during butyrate-induced differentiation in human colon derived HT-29 cells

Glutathione plays an important role in various cellular functions including cell growth and differentiation. In the present study, cell differentiation was induced by butyrate in human colon cell line HT-29 and cellular thiol status was assessed. It was observed that butyrate-induced differentiation was associated with decrease in cellular GSH level and this was prominent at early stages of differentiation. Buthionine sulfoximine (BSO), a specific cellular GSH depleting agent, did not induce differentiation in cells but potentiated the differentiation induced by butyrate. Both BSO and butyrate individually and together inhibited cell growth. These studies suggest that cellular GSH level is modulated in butyrate-induced differentiation and decrease of GSH at the initial stage might facilitate cellular differentiation.     

Ggamma13 interacts with PDZ domain-containing proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.     

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.