Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-Å crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches. C yclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of 3Ј, 5Ј cyclic nucleotides to the inactive 5Ј monophosphates. Five of the 11 PDE families contain regulatory segments consisting of one or two so-called GAF-domain modules (1), which is one of the largest families of small moleculebinding regulatory domains. Among PDEs, cGMP is the only ligand known to bind this domain. The structure of a single GAF domain from a putative protein from yeast (YKG9) has been solved recently (2). However, yeast do not make cGMP, nor does this protein bind cGMP when tested directly (2). cGMP binding to one of two GAF domains (3) in the photoreceptor PDE6 family provides one mechanism for regulating visual signal transduction. cGMP also binds to one or more of the GAF domains of PDE5 (4), the target of the drug, Viagra. The binding and subsequent phosphorylation of an adjacent domain activates the catalytic domain of the enzyme (5). In PDE2A, the catalytic activity is allosterically stimulated by cGMP binding to its GAF domain (6), an event important for several pathways that PDE2A has been shown to regulate (7-12). For example, atrial natriuretic peptide stimulation of cGMP and subsequent activation of PDE2A in the adrenal cortex decreases the secretion of aldosterone and, thereby, mediates much of the effect of this hormone on blood pressure (13). Each PDE2A monomer contains an N-terminal (Ϸ200 residues) domain of unknown function, tandem GAF domains (GAF A and GAF B), and a C-terminal catalytic domain. What seems to be a functionally very similar tandem set of GAF domains is also present in Anabaena adenylyl cyclase. This GAF domain has a preference for cAMP where it functions to confer cAMP activation of cyclase activity (14). Here, we report the 2.9-Å crystal structure of the regulatory segment of murine PDE2A, which reveals the structure of two GAF domains with entirely different functions, dimerization, and binding of cGMP. Amazingly, this binding motif and mechanism has apparently been preserved for over 2 billion years in evolution. Methods and MaterialsCrystallization and Data Collection. Crystals were grown at ...

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type–specific RNA splicing was discovered and analyzed across tissues within an individual.

Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphateribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were ϳ4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B؉C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.

Joubert syndrome is a group of rare disorders that stem from defects in a sensory organelle, the primary cilia. Affected patients often present with disorders involving multiple organ systems, including the brain, eyes, and kidneys. Common symptoms include breathing abnormalities, mental developmental delays, loss of voluntary muscle coordination, and abnormal eye movements, with a diagnostic “molar tooth” sign observed by magnetic resonance imaging (MRI) of the midbrain. We reviewed the ocular phenotypes that can be found in patients with Joubert syndrome. Ocular motor apraxia is the most frequent (80% of patients), followed by strabismus (74%) and nystagmus (72%). A minority of patients also present with ptosis (43%), chorioretinal coloboma (30%), and optic nerve atrophy (22%). Although mutations in 34 genes have been found to be associated with Joubert syndrome, retinal degeneration has been reported in only 38% of patients. Mutations in AHI1 and CEP290, genes critical to primary cilia function, have been linked to retinal degeneration. In conclusion, Joubert syndrome is a rare pleiotropic group of disorders with variable ocular presentations.