Transducin (Gt) is a protein complex naturally expressed in vertebrate retina rods and cones and it is very important in vertebrate phototransduction. It is a type of heterotrimeric G-protein with different α subunits in rod and cone photoreceptors. Light leads to conformational changes in the G protein–coupled receptor rhodopsin, which in turn leads to the activation of transducin. Transducin activates phosphodiesterase, which results in the breakdown of cyclic guanosine monophosphate (cGMP). The intensity of the flash response is directly proportional to the number of transducin activated.
Function in phototransduction Transducin is activated by metarhodopsin II, a conformational change in rhodopsin caused by the absorption of a photon by the rhodopsin moiety retinal. The light causes isomerization of retinal from 11-cis to all-trans. Isomerization causes a change in the opsin to become metarhodopsin II. When metarhodopsin activates transducin, the guanosine diphosphate (GDP) bound to the α subunit (Tα) is exchanged for guanosine triphosphate (GTP) from the cytoplasm. The α subunit dissociates from the βγ subunits (Tβγ). Activated transducin α-subunit activates cGMP phosphodiesterase. cGMP phosphodiesterase breaks down cGMP, an intracellular second messenger which opens cGMP-gated cation channels. Phosphodiesterase hydrolyzes cGMP to 5’-GMP. Decrease in cGMP concentration leads to decreased opening of cation channels and subsequently hyperpolarization of the membrane potential. Transducin is deactivated when the α-subunit-bound GTP is hydrolyzed to GDP. This process is accelerated by a complex containing an RGS (Regulator of G-protein Signaling)-protein and the gamma-subunit of the effector, cyclic GMP phosphodiesterase.
Mechanism of activation The Tα subunit of transducin contains three functional domains: one for rhodopsin/Tβγ interaction, one for GTP binding, and the last for activation of cGMP phosphodiesterase. There are different isoforms of Tα, seen in rod and cone cells. However, the isoforms exhibit functional interchangeability in the phototransduction cascade and shouldn't solely account for differences in light sensitivity. Although the focus for phototransduction is on Tα, Tβγ is crucial for rhodopsin to bind to transducin. The rhodopsin/Tβγ binding domain contains the amino and carboxyl terminal of the Tα. The amino terminal is the site of interaction for rhodopsin while the carboxyl terminal is that for Tβγ binding. The amino terminal might be anchored or in close proximity to the carboxyl terminal for activation of the transducin molecule by rhodopsin. Interaction with photolyzed rhodopsin opens up the GTP-binding site to allow for rapid exchange of GDP for GTP. The binding site is in the closed conformation in the absence of photolyzed rhodopsin. Normally in the closed conformation, an α-helix located near the binding site is in a position which hinders the GTP/GDP exchange. A conformational change of the Tα by photolyzed rhodopsin causes the tilting of the helix, opening the GTP-binding site. Once GTP has been exchanged for GDP, the GTP-Tα complex undergoes two major changes: dissociation from photolyzed rhodopsin and the Tβγ subunit and exposure of the phosphodiesterase (PDE) binding site for interaction with latent PDE. The conformational changes initiated in the transducin by binding of GTP are transmitted to the PDE binding site and cause it to be exposed for binding to PDE. The GTP-induced conformational changes could also disrupt the rhodopsin/Tβγ binding site and lead to dissociation from the GTP-Tα complex.
The Tβγ complex An underlying assumption for G-proteins is that α, β, and γ subunits are present in the same concentration. However, there is evidence that there are more Tβ and Tγ than Tα in rod outer segments (ROS). The excess Tβ and Tγ have been concluded to be floating freely around in the ROS, though it cannot be associated with the Tα at any given time. One possible explanation for the excess Tβγ is increased availability for Tα to rebind. Since Tβγ is crucial for the binding of transducin, reacquisition of the heterotrimeric conformation could lead to more rapid binding to another GTP molecule and thus faster phototransduction. Though Tβγ has been mentioned to be crucial for Tα binding to rhodopsin, there is also evidence that Tβγ may have a crucial, possibly direct role in nucleotide exchange than previously thought. Rhodopsin was found to specifically cause a conformational switch in the carboxyl terminal of the Tγ subunit. This change ultimately regulates the allosteric nucleotide exchange on the Tα. This domain could serve as a major area for interactions with rhodopsin and for rhodopsin to regulate nucleotide exchange on the Tα. Activation of the G protein transducin by rhodopsin was thought to proceed by the lever mechanism. Rhodopsin-binding causes helix formation at the carboxyl terminal on the Tγ and brings the Tγ carboxyl and Tα. Carboxyl terminals closer together to facilitate nucleotide exchange. Tα can accelerate the rate of activation of light-off induced Protein Kinase A due to binding to rhodopsin. As well as, transducin achieves full functional activation upon binding to activated rhodopsin. Mutations in this domain abolish rhodopsin-transducin interaction. This conformational switch in the Tγ may be preserved in the G protein γ subunit family.
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