The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated.
The ct-subunit primary structure of cyclic GMP phosphodiesterase has been determined by parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The enzyme ~t-subunit contains 858 amino acid residues, its N-terminal amino group being acetylated. The partial primary structure of the enzyme fl-subunit has also been elucidated. A significant homology has been found between the ct-and fl-subunits of cGMP phosphodiesterase.
Monoclonal antibodies were prepared to the γ‐subunit of the cGMP phosphodiesterase. One of them γp‐1, suppresses the activation of phosphodiesterase through the α‐subunit of transducin. The γ‐subunit fragment 24–45 rich in Arg and Lys residues is involved in γp‐1 binding and is essential for the γ‐subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C‐terminus of the γ‐subunit resulting in phosphodiesterase activation. Thus, the C‐terminal fragment of γ‐subunit participates in phosphodiesterase inhibition.
A missense mutation, G38D, was found in the rod transducin ␣ subunit (G␣ t ) in individuals with the Nougaret form of dominant stationary night blindness. To elucidate the mechanism of Nougaret night blindness, we have examined the key functional properties of the mutant transducin. Our data show that the G38D mutation does not alter the interaction between G␣ t and G␥ t or activation of transducin by photoexcited rhodopsin (R*). The mutant G␣ t has only a modestly (ϳ2.5-fold) reduced k cat value for GTP hydrolysis. The GTPase activity of G␣ t G38D can be accelerated by photoreceptor regulator of G protein signaling, RGS9. Analysis of the G␣ t G38D interaction with cGMP phosphodiesterase revealed marked impairment of the mutant effector function. G␣ t G38D completely fails to bind the inhibitory PDE ␥ subunit and activate the enzyme. Altogether, our results demonstrate a novel molecular mechanism in dominant stationary night blindness. In contrast to known forms of the disease caused by constitutive activation of the visual cascade, the Nougaret form has its origin in attenuated visual signaling due to loss of effector function by transducin G38D mutant.The visual transduction cascade in vertebrate rod photoreceptor cells is among the best understood G protein signaling systems. In the outer segments of rod photoreceptor cells (ROS), 1 the visual G protein, transducin (G t ), couples the light receptor, rhodopsin (R), to the effector enzyme, cGMP phosphodiesterase (PDE). Photolyzed rhodopsin (R*) binds G t and induces GDP/GTP exchange on the G␣ t subunit, which upon dissociation from R* and G␥ t activates PDE. To activate the enzyme, G␣ t ⅐GTP interacts with the inhibitory PDE ␥ subunits (P␥) and prevents them from blocking catalytic sites on the PDE ␣ subunits. The following decrease in intracellular cGMP concentration leads to a closure of cGMP-gated channels in the ROS plasma membrane (1-3). An analogous visual cascade operates in cone photoreceptor cells. Unlike the cones that mediate high intensity daytime color vision, rod photoreceptors are designed for dim light vision during nighttime.
Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.
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