Transducin Activation State Controls Its Light-dependent Translocation in Rod Photoreceptors

Abstract

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-␣ subunit (Gt␣) in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant Gt␣Q200L. An illumination threshold for the Gt␣ movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic Gt␣Q200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gt␣, the Gt␣Q200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of Gt␣Q200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gt␣, and apparently, do not require Gt␣ interaction with RGS9 and PDE6.Heterotrimeric GTP-binding proteins (G proteins) propagate a variety of hormonal and sensory signals from specific cell surface receptors to intracellular effectors (1-3). The visual transduction cascade in vertebrate photoreceptors has served for many years as a paradigm for G protein signaling. In rod photoreceptor cells, illuminated rhodopsin stimulates GTP-GDP exchange on the retinal G protein, transducin (Gt), 2 resulting in dissociation of Gt␣GTP from Gt␤␥ and rhodopsin. Gt␣ in the active GTP-bound conformation stimulates the effector enzyme, cGMP phosphodiesterase (PDE6), by displacing the inhibitory ␥-subunits (P␥) from the PDE6 catalytic core (PDE6␣␤). cGMP hydrolysis by active PDE6 results in closure of cGMP gated channels in the plasma membrane (4, 5). The turn-off phase of the visual signal is determined by reactions controlling the lifetimes of photoexcited rhodopsin (R*) and activated transducin. The catalytic function of R* is blocked by the rhodopsin-kinase mediated phosphorylation and the binding of arrestin to phosphorylated R* (6 -8). The lifetime of Gt␣GTP is controlled by intrinsic GTPase activity. Hydrolysis of GTP switches the Gt␣ molecule to the inactive GDP-bound conformation and allows reinhibition of PDE␣␤ by P␥. RGS9-1, a photoreceptor-specific member of the RGS (regulators of G protein signaling) family, in the complex with G␤5L acts as a GTPase-activating protein for transducin and thus is a major regulator of the turn-off kinetics of the visual signal (9 -11). The RGS9-1/G␤5L complex is anchored to disc membranes through the interaction with R9AP (RGS-9-1-anchor protein) that enhances the complex GAP acti...

Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination

“…This suggests that, contrary to the commonly accepted notion, significant amounts of dephosphorylated Pdc are available for interaction with the ␤␥ subunits of transducin or other heterotrimeric G proteins in the dark-adapted rods. It also refutes the idea that light-evoked dephosphorylation of either serine 54 or 71 is setting up an activation threshold for rod transducin translocation, which most recently was demonstrated to be determined primarily by the capacity of the GTPase-activating complex (27,28).…”

Transducin Translocation in Rods Is Triggered by Saturation of the GTPase-Activating Complex

“…A study by Kerov et al (2005) reported that the GTPase-deficient Q200L mutation in G␣ t caused the majority of transducin subunits to localize outside rod outer segments regardless of conditions of illumination. They also showed that the translocation of G␣ t in RGS9 knock-out mice, in which the lifetime of activated transducin is increased as a result of the decreased rate of its GTPase activity, occurs at a lower light intensity than in wildtype mice.…”

“…A recent study revealed that G␣ t translocates at a lowered light intensity in mice in which its lifetime in the activated state is prolonged as a result of the knockout of RGS9 (Kerov et al, 2005). However, prolonged lifetime of activated G␣ t also prolongs activation of the entire phototransduction cascade, increases the photoresponse duration, and saturates rods at dimmer light than normally (Chen et al, 2000).…”

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

“…Mouse rod outer segments (OS) are in effect genetically manipulable, femtoliter test tubes whose protein constituents (15,16), as well as their biochemical reactions (17)(18)(19) and light-stimulated translocations (20)(21)(22), are well known and have been measured primarily with ex vivo methods. Advances in imaging technology have now made it possible to achieve subcellular-resolution imaging of the mouse retina in vivo (23)(24)(25)(26).…”