cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca2+ mobilization

AbstractThe serine protease α-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin

BAD: a good therapeutic target?

The major goal in cancer treatment is the eradication of tumor cells. Under stress conditions, normal cells undergo apoptosis; this property is fortunately conserved in some tumor cells, leading to their death as a result of chemotherapeutic and/or radiation-induced stress. Many malignant cells, however, have developed ways to subvert apoptosis, a characteristic that constitutes a major clinical problem. Gilmore et al. recently described the ability of ZD1839, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR), to induce apoptosis of mammary cells that are dependent upon growth factors for survival. Furthermore, they showed that the major effector of the EGFR-targeted therapy is BAD, a widely expressed BCL-2 family member. These results are promising in light of the role of the EGFR in breast cancer development

Insulin induces suppressor of cytokine signaling-3 tyrosine phosphorylation through janus-activated kinase

Suppressor of cytokine signaling (SOCS) proteins were originally described as cytokine-induced molecules involved in negative feedback loops. We have shown that SOCS-3 is also a component of the insulin signaling network (1), Indeed, insulin leads to SOCS-3 expression in 3T3-L1 adipocytes. Once produced, SOCS-3 binds to phosphorylated tyrosine 960 of the insulin receptor and inhibits insulin signaling. Now we show that in 3T3-L1 adipocytes and in transfected COS-7 cells insulin leads to SOCS-3 tyrosine phosphorylation. This phosphorylation takes place on Tyr(204) and is dependent upon a functional SOCS-3 SH2 domain. Purified insulin receptor directly phosphorylates SOCS-3, However, in intact cells, a mutant of the insulin receptor, IRY960F, unable to bind SOCS-3, was as efficient as the wild type insulin receptor to phosphorylate SOCS-3, Importantly, IRY960F is as potent as the wild type insulin receptor to activate janus-activated kinase (Jak) 1 and Jak2, Furthermore, expression of a dominant negative form of Jak2 inhibits insulin-induced SOCS-3 tyrosine phosphorylation, ks transfected Jaks have been shown to cause SOCS-3 phosphorylation, we propose that insulin induces SOCS-3 phosphorylation through Jak activation. Our data indicate that SOCS-3 belongs to a class of tyrosine-phosphorylated insulin signaling molecules, the phosphorylation of which is not dependent upon a direct coupling with the insulin receptor but relies on the Jacks