The C-terminus of the oncoprotein TGAT is necessary for plasma membrane association and efficient RhoA-mediated signaling.

BACKGROUND: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. METHODS: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. RESULTS: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. CONCLUSION: Together, these results show that membrane association of TGAT is critical for its activity.

Acute abdomen as the first presentation of a malignant lymphoma of the small intestine.

Malignant small intestinal lymphoma is a quite rare disease that often has an atypical presentation with symptoms of abdominal pain, nausea and weight loss. However, as illustrated in the following cases, acute abdomen due to an intestinal perforation can be the first sign of malignant lymphoma.

Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3)).

BACKGROUND AND PURPOSE: Recently, some ligands targeting the sphingosine-1-phosphate receptor subtype 3 (S1P(3)) have become available. The characterization of these compounds was mainly based on one functional read-out system, although S1P(3) receptors are known to activate different signal transduction pathways. Therefore, this study pharmacologically characterizes these compounds using different assays. EXPERIMENTAL APPROACH: Using CHO-FlpIn cells expressing the human S1P(3) receptor the potencies and maximal effects of S1P, FTY720-P, VPC23019, VPC23153 and VPC24191 were determined in three different assays [inhibition of cAMP accumulation, elevation of intracellular calcium concentrations ([Ca(2+)](i)) and S1P(3) receptor internalization]. KEY RESULTS: All compounds tested inhibited forskolin-induced cAMP accumulation, increased [Ca(2+)](i) and induced S1P(3) receptor internalization but with different potencies and maximal effects. S1P was the most potent compound in all assays followed by FTY720-P. The VPC compounds were generally less potent than S1P and FTY720-P. Regarding the maximal effects, all compounds except VPC23153, behaved as full agonists in the cAMP accumulation assay. In the calcium assay, FTY720-P, VPC23019 and VPC24191 displayed partial and VPC23153 weak partial agonist activity, relative to S1P. Interestingly, treatment with the G(i) inactivator Pertussis toxin, did not affect S1P-induced [Ca(2+)](i) elevations but inhibited those in response to the other compounds, by about 50%. CONCLUSIONS AND IMPLICATIONS: This study demonstrated differential response patterns at the S1P(3) receptor for a range of ligands. These differences could indicate the presence of functional selectivity at this receptor as FTY720-P and the VPC compounds seemed to signal predominantly via G(i)- whereas S1P activated G(i) and G(q)-coupled pathways.