Lnl

Inactive PDE

Active PDE

Inactive PDE

Active PDE

Rod plasma membrane

▲ FIGURE 13-24 Operational model for rhodopsin-induced closing of cation channels in rod cells. In dark-adapted rod cells, a high level of cGMP keeps nucleotide-gated nonselective cation channels open. Light absorption generates activated opsin, O* (step 11), which binds inactive GDP-bound Gt protein and mediates replacement of GDP with GTP (step 12). The free GtaGTP generated then activates cGMP phosphodiesterase (PDE) by binding to its inhibitory y subunits (step 13) and dissociating them from the catalytic a and p subunits (step 14). Relieved of their inhibition, the a and p subunits convert cGMP to GMP (step 15). The resulting decrease in cytosolic cGMP leads to dissociation of cGMP from the nucleotide-gated channels in the plasma membrane and closing of the channels (step 16). The membrane then becomes transiently hyperpolarized. [Adapted from V. Arshavsky and E. Pugh, 1998, Neuron 20:11.]

Low cytosolic cGMP( )

Closed cGMP-gated ion channel

Closed cGMP-gated ion channel

Low cytosolic cGMP( )

present in the dark acts to keep cGMP-gated cation channels open; the light-induced decrease in cGMP leads to channel closing, membrane hyperpolarization, and reduced neuro-transmitter release.

As depicted in Figure 13-24, cGMP phosphodiesterase is the effector protein for Gt. The free Gta-GTP complex that is generated after light absorption by rhodopsin binds to the two inhibitory y subunits of cGMP phosphodiesterase, releasing the active catalytic a and p subunits, which then convert cGMP to GMP. This is another example of how signal-induced removal of an inhibitor can quickly activate an enzyme, a common mechanism in signaling pathways. A single molecule of activated opsin in the disk membrane can activate 500 Gta molecules, each of which in turn activates cGMP phosphodiesterase; this is the primary stage of signal amplification in the visual system. Even though activation of cGMP phosphodiesterase leads to a decrease in a second messenger, cGMP, this activation occurs by the same general mechanism described earlier except that absorption of light by rhodopsin rather than ligand binding is the activating signal (see Figure 13-11).

Conversion of active Gta-GTP back to inactive Gta-GDP is accelerated by a GTPase-activating protein (GAP) specific for Gta-GTP. In mammals Gta normally remains in the active GTP-bound state for only a fraction of a second. Thus cGMP phosphodiesterase rapidly becomes inactivated, and the cGMP level gradually rises to its original level when the light stimulus is removed. This allows rapid responses of the eye toward moving or changing objects.

Recent x-ray crystallographic studies reveal how the sub-units of Gt protein interact with each other and with light-activated rhodopsin and provide clues about how binding of GTP leads to dissociation of Ga from Gpy (Figure 13-25). Two surfaces of Gta interact with Gp: an N-terminal region near the membrane surface and the two adjacent switch I and switch II regions, which are found in all Ga proteins. Although Gp and Gy also contact each other, Gy does not contact Gta.

Studies with adrenergic receptors discussed earlier indicate that ligand binding to a G protein-coupled receptor causes the transmembrane helices in the receptor to slide relative to one another, resulting in conformational changes in the cytosolic loops that create a binding site for the coupled trimeric G protein. The crystallographic structures in Figure 13-25 suggest that the nucleotide-binding domain of Gta, together with the lipid anchors at the C-terminus of Gy and the N-terminus of Gta, form a surface that binds to light-activated rhodopsin (O* in Figure 13-24), promoting the release of GDP from Gta and the subsequent binding of GTP. The subsequent conformational changes in Gta, particularly those within switches I and II, disrupt the molecular interactions between Gta and Gpy, leading to their dissociation. The structural studies with rhodopsin and Gt are consistent with data concerning other G protein-coupled receptors and are thought to be generally applicable to all receptors of this type.

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