Transducin activates cGMP phosphodiesterase by trapping inhibitory γ subunit freed reversibly from the catalytic subunit in solution (original) (raw)

Activation of cGMp phosphodiesterase (pDe) by activated transducin α subunit (tα*) is a necessary step to generate a light response in vertebrate photoreceptors. pDe in rods is a heterotetramer composed of two catalytic subunits, PDEα and pDeβ, and two inhibitory PDEγ subunits, each binding to pDeα or pDeβ. Activation of pDe is achieved by relief of the inhibitory constraint of pDeγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to pDeγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of tα* to pDeγ still bound to pDeα or pDeβ seems to be difficult because the binding site of PDEγ to pDeα or pDeβ overlaps with the binding site to tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates pDe by trapping pDeγ released reversibly and spontaneously from the catalytic subunit. this mechanism well explains pDe activation by tα* in solution. our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment. In the vertebrate photoreceptors, an enzymatic cascade, the phototransduction cascade, is responsible for generation of a light response 1,2. Briefly, after absorption of light, light-activated visual pigment catalyzes the exchange of GDP for GTP on the α subunit of transducin (Tα) to produce a GTP-bound active form of transducin (Tα*). Tα* then activates cGMP phosphodiesterase (PDE). PDE is a heterotetrameric protein composed of two catalytic subunits of similar amino acid sequence (PDEα and PDEβ showing >70% sequence identity) and two inhibitory subunits (PDEγ), and therefore is in the form of PDEαγβγ. (We call this form of holo-PDE just PDE for simplicity.) Each catalytic subunit has an active site to hydrolyze cGMP to GMP. Tα* binds to inhibitory PDEγ, and relieves its constraint on the active site in the catalytic subunit. This activation of PDE causes hydrolysis of cGMP, leads to closure of cGMP-gated cation channels situated in the plasma membrane of the outer segment, and induces a hyperpolarization of the cell. In the activation process of PDE by Tα*, it is widely believed that Tα* directly binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the active site of a catalytic subunit 3,4. However, this mechanism seems to be difficult based on the recent structural studies on the PDEγ•PDEα complex and the PDEγ•Tα* complex: most of the amino acid residues in the C-terminal region of PDEγ, from Asp-63 to Ile-87, are in contact with Tα* 5 , and almost the same region, from Leu-60 to Ile-87 in PDEγ, is in contact with the catalytic site of PDEα or PDEβ 6. These observations suggest that PDEγ utilizes the same region to bind to Tα* and to the catalytic site of PDEα or PDEβ, and that Tα* and the catalytic subunit cannot bind to this region simultaneously.