Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits

Photoreceptor cGMP phosphodiesterases (PDE6 1 family) function as effector proteins in the vertebrate visual transduction, which is mediated by the rhodopsin-coupled G protein, transducin (1-3). Retinal rod PDE6 is composed of two catalytic PDE6␣␤ subunits each tightly associated with the smaller inhibitory ␥ subunit (P␥) (4 -6). Cone PDE consists of two identical PDE␣Ј subunits complexed with two copies of the cone-specific P␥ subunit (7-9). The catalytic subunits of rod and cone PDE, as well as the respective P␥ subunits, share a high degree of homology (9 -10). The key role of P␥ is to inhibit cGMP hydrolysis by the catalytic subunits in the dark. Upon light stimulation of photoreceptors, PDE6 is activated by GTPbound transducin-␣, which displaces P␥ from the enzyme catalytic core.Two regions of P␥ are principally involved in the interaction with the PDE6 catalytic subunits, the central polycationic region (residues 24 -45 of rod P␥) and the P␥ C terminus. The C terminus of P␥ constitutes the key inhibitory domain, whereas the polycationic region enhances the overall affinity of P␥ toward PDE6 catalytic subunits (11)(12)(13)(14). A cross-linking study localized the P␥ C-terminal binding site on PDE6␣ to residues 751-763 (residues 749 -761 of PDE6␤ or PDE6␣Ј) within the broader PDE6 catalytic domain (15). Our further analysis of the interaction between fluorescently labeled P␥ and PDE6␣␤ suggests that the C terminus of P␥ inhibits PDE6 activity by physically blocking the PDE catalytic site (16).Progress in the investigation of the structure/function of PDE6 and the mechanism of PDE6 inhibition by P␥ has been slowed by the lack of an efficient expression system for PDE6 (17, 18). Our approach to developing a system for PDE6 expression and mutagenesis included the construction of chimeras between PDE6␣Ј and cGMP-binding, cGMP-specific PDE (PDE5 family) (19). PDE5 and PDE6 share a common domain organization, i.e. two noncatalytic cGMP-binding sites located N-terminally to the conserved PDE catalytic domain (20). Furthermore, PDE5 and PDE6 display a high homology (45-48% identity) between catalytic domains, a strong substrate preference for cGMP, and similar patterns of inhibition by competitive inhibitors such as zaprinast, dipyridamole, and sildenafil (20 -23). Unlike PDE6, PDE5 is readily expressed using the baculovirus/insect cell system (24, 25). Earlier, we reported (19) the functional expression and characterization of a chimeric PDE6␣Ј/PDE5 enzyme containing the PDE6␣Ј noncatalytic cGMP-binding sites and the PDE5 catalytic domain. In this study, we generated chimeric PDE6␣Ј/PDE5 enzymes that contain the P␥ C-terminal binding site and that are potently inhibited by P␥. Ala-scanning mutational analysis of the P␥-binding site, using chimeric PDE as a template, revealed the key interaction residues and provided structural justification for the mechanism of PDE6 inhibition.

Section: Discussionmentioning

confidence: 62%

Identification of the γ Subunit-interacting Residues on Photoreceptor cGMP Phosphodiesterase, PDE6α′

Granovsky

Artemyev

2000

“…Most likely, P␤-515-539 binds P␥-24 -45, as is seen with P␣-517-541. The lower homology between rod PDE and cone PDE in this region may explain why rod P␥ inhibits rod PDE more effectively (K i , 80 pM) than cone PDE (K i , 600 pM) (Hamilton et al, 1993). The high level of homology between the P␣ and P␤ sites that bind the P␥ C terminus (Artemyev et al, 1996) and P␥-24 -45 favors a model with similar, although not necessarily identical, affinities for the P␥-P␣ and P␥-P␤ binding sites (Wensel and Stryer, 1990).…”

Section: Discussionmentioning

confidence: 99%

An Interface of Interaction between Photoreceptor cGMP Phosphodiesterase Catalytic Subunits and Inhibitory γ Subunits

Natochin

Artemyev

1996

In vertebrate photoreceptor cells, light-activated rhodopsin catalyzes GDP-GTP exchange on the rod G-protein ␣-subunit (G t␣ ), 1 which in turn activates cGMP phosphodiesterase (PDE).PDE activation leads to the rapid hydrolysis of cytoplasmic cGMP and closure of sodium channels, resulting in hyperpolarization of the rod and cone cells (for review, see Deterre (1989) andStryer (1996)). Rod photoreceptor PDE is composed of two large homologous catalytic ␣ and ␤ subunits (P␣ and P␤) of nearly identical size (M r 99,261 and 98,308, respectively) and two copies of an inhibitory ␥ subunit (P␥, M r 9700) (Ovchinnikov et al., 1986(Ovchinnikov et al., , 1987Deterre et al., 1988;Lipkin et al., 1990). The primary structures of P␣, P␤, and cone-specific P␣Ј subunits revealed that these PDEs constitute one family, that of PDE6 (Beavo et al., 1994). The photoreceptor PDEs belong to a broader group of cGMPbinding PDEs, which contain two noncatalytic cGMP binding sites located N-terminally to the conserved PDE catalytic domain (Yamazaki et al., 1980;Gillespie and Beavo, 1989;Lipkin et al., 1990;Trong et al., 1990;McAllister-Lucas et al., 1993). Unique features of photoreceptor PDEs are their high k cat /K m parameter and their ability to be inhibited by P␥ and activated by rod and cone G-protein ␣ subunits. Interactions between PDE catalytic and inhibitory subunits and the mechanism of PDE inhibition have been studied extensively. Two regions of P␥, polycationic region P␥-24 -45 and the C terminus of P␥, have been shown to participate in the interaction with P␣␤ (Lipkin et al., 1988;Brown, 1992;Takemoto et al., 1992). Both of these P␥ domains bind to P␣␤, allowing effective inhibition of PDE activity by the P␥ C terminus. Initial studies indicated that the major sites of P␣ and P␤ interaction with P␥ are different and located in the N-terminal regions (P␣, 16 -30 and 78 -90; P␤, 91-110 and 211-230) in areas with a high level of dissimilarity between catalytic subunits .More recently, using a cross-linking approach we have demonstrated that the C terminus of P␥ interacts with region P␣-751-763 located within the PDE catalytic domain (Artemyev et al., 1996). In this study, to identify a P␣ region for binding to P␥-24 -45, we have expressed several large domains of P␣ as GST fusion proteins in Escherichia coli. One of these fusion proteins contained a region unique for photoreceptor PDEs that links a second noncatalytic cGMP binding site with the catalytic domain. This protein was used to obtain a polypeptide, P␣-461-553, which blocks inhibition of PDE activity by P␥ and binds to P␥-24 -45. Characterization of the P␣-461-553 region using a set of synthetic peptides revealed that the P␣-517-541 site is primarily responsible for P␣ binding to the polycationic region of P␥.

“…PDE Activity Assay and Data Analysis-PDE activity was measured using 5 M [ 3 H]cGMP and 1 pM PDE6 or 200 M [ 3 H]cGMP and 20 pM PDE6 as described (15,25). To determine K m values for cGMP, PDE activity was measured using 10 -1000 M cGMP, and the data were fit to the equation, Y ϭ V max ⅐X/(K m ϩ X).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Muradov

Boyd

Haeri

et al. 2009

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone-and rod-specific PDE6 ␥-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.Phosphodiesterases of cyclic nucleotides (PDEs) 2 are essential enzymes controlling cellular levels of cAMP and cGMP. Eleven families of PDEs have been identified in mammals on the grounds of sequence homology, substrate selectivity, and regulation (1). Photoreceptor-specific PDEs in rods and cones comprise the sixth PDE family (PDE6) and serve as the effector enzymes in the vertebrate phototransduction cascade (1-5). Rod PDE6 is composed of homologous catalytic ␣-subunit (PDE6A) and ␤-subunit (PDE6B) and two copies of a small inhibitory ␥-subunit (P␥) (3). Cone PDE6 is a catalytic dimer of two identical ␣Ј-subunits (PDE6C) (3). A cone-specific inhibitory P␥-subunit is highly homologous to the rod P␥ (6). In rod photoreceptors, PDE6 is located in the specialized compartments called rod outer segments (ROS), where it associates with disc membranes. The membrane attachment of PDE6 is mediated by farnesylation of the PDE6A C terminus and geranylgeranylation of the PDE6B C terminus (7). In cones, geranylgeranylated PDE6C resides on infoldings of the cone outer segment plasma membrane (8). Following photoexcitation of rod or cone photoreceptor cells, PDE6 is activated by the GTPbound transducin ␣-subunit (G␣ t GTP) that relieves the P␥ inhibition of the enzyme. cGMP hydrolysis by active PDE6 leads to a cellular response due to a closure of cGMP-gated channels in the photoreceptor plasma membrane (2-5).Although PDE6 plays a prominent role in vertebrate vision, the structure-function relationships of PDE6 are poorly understood in comparison with other key phototransduction proteins. The lack of an expression system for PDE6 has become a major impediment for PDE6 research. Importantly, an expression system for PDE6 is required to elucidate the mechanisms of visua...