Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

Abstract

Partial amino acid sequence has been determined for the cone, a' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the at' subunit as the product of a gene that is distinct from those encoding the a or .3 subunits of the membraneassociated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the a and a' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of -340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMPbinding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.whereas the cone PDE (5) has two a' subunits (93.5 kDa) and three small subunits (11, 13, and 15 kDa).The cGS-PDE is a homodimer of two identical (105 kDa) subunits and can hydrolyze both cAMP and cGMP with nearly equal efficiency (7,8). cGMP is a positive allosteric regulator of this enzyme, and micromolar concentrations produce a 10-fold stimulation of cAMP hydrolysis (7-10). A cGMP-specific, allosteric site has been demonstrated in several types of cyclic nucleotide binding experiments (7,(9)(10)(11).In this report, we describe the partial amino acid sequence of the cone a' subunit and nucleotide sequences from a partial cDNA clone encoding it. § The protein sequence data confirms the identity of the full-length cDNA clone described in the companion paper by Li et al. (14) and shows that the a, a', and p subunits of photoreceptor PDEs are distinctive gene products. The a and a' subunits and the cGS-PDE have extensive sequence similarity within single segments that are not part of their conserved, catalytic domains (15, 16). Each segment displays internal homology and appears to comprise a regulatory, cGMP-binding domain.Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are found in many eukaryotic species; in some cases, distinct isozymes exist within a single cell type (1, 2). Within several PDE families, additional diversity is created by apparent alternative mRNA splicing (3, 4). Tissue and celltype-specific expression of the isozymes has been demonstrated for several of the isozymes and is suspected in others (1-6). As part of an effort to analyze structure-function relationships among the PDE isozymes, we are determining the sequence of the bovine cone photoreceptor PDE (5) and the cGMP-stimulated (cGS) PDE (7,8). Bot...

Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse.

“…Since a truncated form of the p-subunit would be produced in the rd retina, the 88-kDa immunoreactive component described must be the a-subunit. The presence of a 230-residue domain that is conserved in cyclic nucleotide PDEs in the normal a-and p-subunits (18,23) raised the possibility that the two subunits may be independently active enzymes that can be activated by transducin-a/GTP and inhibited by the PDE t-subunit. The presence of the a-subunit and most likely the y-subunit in the rd retina but the absence of PDE activity strongly suggest that the a-and y-subunits alone are unable to effectively regulate cGMP levels in photoreceptors.…”

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

“…In mammals, GAF domains are mostly found in cyclic nucleotide phosphodiesterases (PDEs), which are crucial cellular enzymes controlling cGMP and cAMP second-messenger levels. Five of the 11 mammalian PDE families contain two GAF domains (PDE2, -5, -6, -10, and -11), all in tandem and N-terminal to the catalytic domain (3)(4)(5). So far, two separate functions, namely cyclic nucleotide binding and dimerization, have been described for different PDE GAF domains.…”

Cross-regulation of Phosphodiesterase 1 and Phosphodiesterase 2 Activities Controls Dopamine-mediated Striatal α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking

“…However, this cGMP regulation of PDE2 depends on a number of factors: the cellular levels of cGMP and cAMP, the affinity of cGMP for the allosteric site on PDE2, the cellular concentration of PDE2, and the catalytic activity of PDE2. Furthermore, reports that recombinant GAF domains can bind cGMP with high affinity, the activation constant for PDE2 is much higher (ϳ0.2-0.5 M) (43,44). Based on all of these variables, it is challenging to intuitively predict the effect of cGMP on PDE2 activity and GluA1 trafficking.…”