Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.

A venus flytrap mechanism for activation and desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.

Desensitization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptor channels is an important process shaping the time course of synaptic excitation. Upon desensitization, the receptor channel closes and the agonist affinity increases. So far, the nature of the structural rearrangements leading to these events was unknown. On the basis of the structural homology of the ligand binding domains of AMPA receptors and of the bilobated bacterial periplasmic proteins, we now show that agonist interaction with one lobe of the GluR1 subunit of homomeric AMPA receptors controls channel activation while additional interactions with the other lobe cause channel desensitization. Accordingly, we suggest that the transition of the AMPA receptor channel to the desensitized state involves the agonist-mediated stabilization of the closed lobe conformation of its binding domain and is a process akin to that used by the venus flytrap.

CDNA cloning of chick brain alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors reveals conservation of structure, function and post-transcriptional processes with mammalian receptors.

Several types of functional ionotropic glutamate receptor have been cloned in the recent years from the mammalian central nervous system, but till now, none from other vertebrate species. Here, we report the cloning and functional analysis of four chick brain cDNAs, coding for members of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptors. These receptors are highly homologous to the mammalian GluR1-4 (A-D) receptors ( > 90%), and conserve their post-transcriptional modifications. The flip/flop exons are conserved not only at the amino acid level but also at the nucleotide level, and the intron of GluR4 involved in the RNA editing of the R/G site displays a rat-chick sequence conservation of 95%. Significant sequence differences are found only in the region containing the immunogenic epitope of neuroactive anti-GluR3 antibodies. Chick AMPA receptors are expressed in both the cerebrum and cerebellum. The ion channel activities of chick GluR1-4 were analyzed in Xenopus oocytes and found to be similar to those of mammalian AMPA receptors. Though their contribution to kainate binding activity in the cerebellum is minor, the profile of channel activity of the chick GluR1-4 suggests that they account for the kainatergic channel activity expressed by total chick cerebellar mRNAs.