The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, ␣␤␥ 2 ) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, ␣␤␥ 2 ␦) can release and bind cGMP at both noncatalytic sites; the ␦ subunit is likely responsible for this difference in cGMP exchange rates. Removal of the ␦ and/or ␥ subunits yields a catalytic ␣␤ dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (K D >1 M); cGMP binding affinity at the second site (K D ϳ60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of ␥ subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the ␥ subunit with the ␣␤ dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells. The cGMP phosphodiesterase (PDE)1 present in rod and cone retinal photoreceptors is the central effector enzyme for vertebrate visual excitation. Photoactivation of the visual pigment rhodopsin leads to G-protein (transducin) activation, which then proceeds to the activation of PDE. The activated ␣ subunit of transducin (␣ t -GTP) is believed to activate the membraneassociated rod PDE holoenzyme (subunit stoichiometry, ␣␤␥ 2 ) by displacing the inhibitory ␥ subunits (P␥) from the active sites of the catalytic heterodimer (P␣␤). The enhanced hydrolytic activity of the activated PDE rapidly reduces cytoplasmic cGMP levels and then leads to dissociation of bound cGMP from the cGMP-gated ion channel, closure of the ion channel, and finally a hyperpolarization of the membrane. These steps constitute the excitation pathway for vertebrate visual transduction (for reviews see Refs. 1-3).The photoreceptor PDE is a member of a family of phosphodiesterases that all share the ability to hydrolyze cyclic nucleotides. The PDE in rods and cones (classified as PDE6 (4)) is most closely related to the cGMP-specific PDE (PDE5) based on several criteria, including overall amino acid sequence similarity, substrate preference for cGMP over cAMP, inhibition of catalysis by isozyme-selective drugs, and the presence of a consensus sequence in the N-terminal half of the protein that represents noncatalytic cGMP binding domains. In the case of PDE5, binding of cGMP to the noncatalytic cGMP-binding sites may regulate activity indirectly via protein phosphorylation of the enzyme (reviewed in Ref. 5). The cGMP-stimulated PDE (PDE2) also contains noncatalytic cGMP-binding sites that allosterically regulate catalysis at the active site (6).For the photoreceptor PDE, most...

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (␣␤); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory ␥ subunits. Rod PDE binds cGMP at two noncatalytic sites on the ␣␤ dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of ␥ to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and ␥ binding to activated PDE; dissociation of bound cGMP accompanies ␥ dissociation from PDE, whereas addition of either cGMP or ␥ to ␣␤ dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic ␣␤ dimers completely lacking bound ␥ subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether ␥ remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.Initiation of the phototransduction cascade in vertebrate rod photoreceptors by light results in the sequential activation of the visual pigment (rhodopsin), the photoreceptor G-protein (transducin), and the effector enzyme (cGMP phosphodiesterase (EC 3.1.4.35), PDE) 1 (for reviews see Refs. 1-4). The PDE present in rod and cone photoreceptors (classified as PDE6) differs in several ways from other classes of mammalian phosphodiesterases (5-7): rod photoreceptor PDE forms a catalytic dimer from two closely related ␣ and ␤ subunits (P␣␤); rod and cone PDE are directly regulated via heterotrimeric G-proteins. the catalytic constant (k cat ) for rod PDE is ϳ1000-fold greater than for any other class of PDE; the catalytic activity of photoreceptor PDE is potently inhibited by binding of an inhibitory ␥ subunit (P␥) (for reviews see Refs. 8 and 9). Transducin activation of PDE results from binding of the activated transducin ␣ t subunit (␣ t -GTP) to one or more sites on the PDE holoenzyme. One result of this interaction is the displacement of the inhibitory P␥ subunit from its binding site in the catalytic pocket of PDE. It is not clear, however, whether each PDE catalytic subunit binds P␥ with equal affinity, whether ␣ t -GTP can activate each catalytic site equally well, and under what conditions the ␣ t -GTP-P␥ complex dissociates from P␣␤. Conflicting results reported by different laboratories (10 -24) may reflect underlying...