Rhodopsin .

▲ FIGURE 13-25 Structural models of rhodopsin and its associated Gt protein. The structures of rhodopsin and the Gta and Gpy subunits were obtained by x-ray crystallography. The C-terminal segment of rhodopsin is not shown in this model. The orientation of Gta with respect to rhodopsin and the membrane is hypothetical; it is based on the charge and hydrophobicity of the protein surfaces and the known rhodopsin-binding sites on Gta. As in other trimeric G proteins, the Gta and Gy subunits contain covalently attached lipids that are thought to be inserted into the membrane. In the GDP-bound form shown here (GDP red), the a subunit (gray) and the p subunit (light blue) interact with each other, as do the p and y (purple) subunits, but the small y subunit, which contains just two a helices, does not contact the a subunit. Several segments of the a subunit are thought to interact with an activated receptor, causing a conformational change that promotes release of GDP and binding of GTP Binding of GTP in turn, induces large conformational changes in the switch regions of Gta, leading to dissociation of Gta from Gpy. The structure of a Gsa subunit in the GTP-bound form, which interacts with an effector protein, is shown in Figure 13-14b. [Adapted from H. Hamm, 2001, Proc. Natl Acad. Sci. USA 98:4819, and D. G. Lambright et al., 1996, Nature 379:311.]

Direct support for the role of cGMP in rod-cell activity has been obtained in patch-clamping studies using isolated patches of rod outer-segment plasma membrane, which contains abundant cGMP-gated cation channels. When cGMP is added to the cytosolic surface of these patches, there is a rapid increase in the number of open ion channels. The effect occurs in the absence of protein kinases or phosphatases, and cGMP acts directly on the channels to keep them open, indicating that these are nucleotide-gated channels. Like the voltage-gated K+ channels discussed in Chapter 7, the cGMP-gated channel protein contains four subunits, each of which is able to bind a cGMP molecule (see Figure 7-36a). Three or four cGMP molecules must bind per channel in order to open it; this allosteric interaction makes channel opening very sensitive to small changes in cGMP levels.

Rod Cells Adapt to Varying Levels of Ambient Light

Cone cells are insensitive to low levels of illumination, and the activity of rod cells is inhibited at high light levels. Thus when we move from daylight into a dimly lighted room, we are initially blinded. As the rod cells slowly become sensitive to the dim light, we gradually are able to see and distinguish objects. This process of visual adaptation permits a rod cell to perceive contrast over a 100,000-fold range of ambient light levels; as a result, differences in light levels, rather than the absolute amount of absorbed light, are used to form visual images.

One process contributing to visual adaptation involves phosphorylation of activated opsin (O*) by rhodopsin kinase (Figure 13-26). This rod-cell enzyme is analogous to ^-adrenergic receptor kinase (BARK) discussed previously. Each opsin molecule has three principal serine phosphorylation sites; the more sites that are phosphorylated, the less able O* is to activate Gt and thus induce closing of cGMP-gated cation channels. Indeed, rod cells from mice with mutant rhodopsins bearing zero or only one of these serine residues show a much slower than normal rate of deacti-

vation in bright light. Because the extent of opsin phosphorylation is proportional to the amount of time each opsin molecule spends in the light-activated form, it is a measure of the background (ambient) level of light. Under high-light conditions, phosphorylated opsin is abundant and activation of Gt is reduced; thus, a greater increase in light level will be necessary to generate a visual signal. When the level of ambient light is reduced, the opsins become dephosphorylated and the ability to activate Gt increases; in this case, fewer additional photons will be necessary to generate a visual signal.

At high ambient light (such as noontime outdoors), the level of opsin phosphorylation is such that the protein P-arrestin binds to the C-terminal segment of opsin. The bound P-arrestin prevents interaction of Gt with O*, totally blocking formation of the active Gta-GTP complex and causing a shutdown of all rod-cell activity. The mechanism by which rod-cell activity is controlled by rhodopsin kinase and arrestin is similar to adaptation (or desensitization) of other G protein-coupled receptors to high ligand levels.

A second mechanism of visual adaptation appears unique to rod cells. In dark-adapted cells virtually all the Gta and Gp7 subunits are in the outer segments. But exposure for 10 minutes to moderate daytime intensities of light causes over 80 percent of the Gta and Gp7 subunits to move out of the outer segments into other cellular compartments (Figure 13-27). The mechanism by which these proteins move is not yet known, but as a result of this adaptation Gt proteins are physically unable to bind activated opsin. As occurs in other signaling pathways, multiple mechanisms are thus used to inactivate signaling during visual adaptation, presumably to allow strict control of activation of the signaling pathway over broad ranges of illumination.

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