The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

Chx10 and VSX1 are transcription factors that share a homeodomain (HD), 1 and a CVC motif, named after CHX10, VSX1, and ceh-10 (1-3). CHX10 is expressed early in development in dividing retinal progenitor cells (RPCs) and is also expressed in mature bipolar neurons and a subset of Mü ller glia (2, 4). Null mutations results in microphthalmia in humans (5) and mice (6), and antisense Chx10 RNA injected into zebrafish embryos impairs retinal development (7). Recessive mutations identified in patients with microphthalmia that modified residues in helix III of the CHX10 HD (R200P and R200Q) ablated DNA binding in vitro (5). The ocular retardation (or J ) mouse carries a mutation that generates a premature stop codon in helix

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.The mammalian retina consists of three nuclear layers. The outer nuclear layer houses the cell bodies of photoreceptors (rods and cones), whereas those of horizontal, bipolar, and amacrine interneurons and Müller glia reside in the inner nuclear layer. The innermost layer is the ganglion cell layer, which consists of a mixture of ganglion and amacrine neurons. These three cellular areas are separated by outer and inner plexiform layers that house synaptic connections. This intricate laminated structure develops through the amplification of multipotent progenitor cells, generation of more restricted post-mitotic transition cells, and maturation of these cells into terminally differentiated neurons and glia (1). In rodents, ganglion, horizontal, cone, and amacrine transition cells are born in the prenatal period, bipolar and Müller cells are born post-natally, and rods are born throughout retinal development (2). Transcription factors play critical roles in each of the stages of retinal development, but many gaps remain in our understanding of the specific target genes involved. Here, we focus on one of these factors, the homeobox gene CHX10, and its role in sculpting the characteristics of bipolar interneurons.Homeodomain (HD) 4 proteins regulate retinal development from the earliest stages of optic vesicle formation to the final stages of maturation in the adult (reviewed in Refs. 3 and 4)). Previous molecular analysis linked a naturally occurring mutation in the homeobox of Chx10 to the ocular retardation phenotype in mice (or J ) (5). These mice display a dramatic decrease in retinal progenitor cell (RPC) proliferation and lack bipolar cells, phenotypes that reflect the expression of Chx10 in both of these cell types 5 (5, 6). Molecular and genetic studies have begun to reveal some aspects of the mechanism by which Chx10 regulates retinal development...