Showing papers by "ACADIA Pharmaceuticals Inc. published in 1999"

Control of cell cycle progression by c-Jun is p53 dependent

Abstract: The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Here we report that fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation.

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

Abstract: G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.