The Regulation of Visual Transduction and Olfaction

transduction mechanisms, the ensuing step takes place without amplification. The effector, cGMP phosphodiesterase, is a heterotetramer having two independent catalytic subunits, and , and two inhibitory -subunits.

These impede the access of the substrate, cGMP, to the catalytic sites.18 The nucleotide exchange catalysed by Rh* occurs on two transducin molecules that are bound to the inhibitory -subunits of the phosphodiesterase (Figure 6.9). When GDP is replaced by GTP, the t.GTP relieves the inhibition. As pointed out in Chapter 4, this is a cooperative process that involves both the conserved Ras-like (rd) domain of the t that interacts with the -subunits of the phosphodiesterase, and the unique helical domain (hd: see Figure 4.16, page 103) that interacts with its catalytic units. The t remains associated with the phosphodiesterase, so preventing it from interacting with further effectors and by its proximity ensuring rapid deactivation of the complex following GTP hydrolysis.19,20

Switching off the mechanism

We have described how a transient light signal rapidly initiates the phototransduction pathway and we have seen how the transduction of a single photon results in a hyperpolarization that lasts for about a second. However, this does not accord with our perception of light flashes that can be much briefer than this; the flicker-fusion frequency of 24 Hz corresponds to an image every 40 ms. This implies that there is a mechanism that shuts down visual transduction as soon as the stimulus is removed. Thus it is important

Fig 6.9  Activation of cyclic GMP phosphodiesterase by the -subunit of transducin is a cooperative process. In the resting state t.GDP (shown as a disc) interacts through its helical domain (hd) with the catalytic

- and -subunits of the phosphodiesterase (grey). This reduces their affinity for their inhibitory -subunits. Interaction of the Ras-like domain (rd) of t with the -subunits then acts as the switch, enabling access of the substrate cGMP to the active sites on the - and - subunits. The return to the resting state is determined by the hydrolysis of GTP on the -subunit of transducin. This is assisted by the immediate proximity of the phosphodiesterase, acting as a component of the GTPase activating protein complex (GAP).

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for photoreceptor cells to be able to terminate signals emanating from the activated pigment and also to be able to terminate any signals that are in progress further down the cascade. These terminations therefore involve inhibition of the activities of the photopigment (the receptor), transducin (the transducer), and cGMP phosphodiesterase (the catalytic unit or effector). cGMP is replenished through activation of guanylyl cyclase.

The conversion of metarhodopsin-I to metarhodopsin-II involves the loss of the proton from the Schiff base attaching the ligand to the protein. After

metarhodopsin-II has activated transducin and over the next minute or so, the linkage is hydrolysed to yield all-trans-retinal and the colourless apoprotein opsin. The pigment is said to be bleached. Later on, rhodopsin will be reformed by the binding of 11-cis-retinal regenerated from all-trans-retinal in the adjacent cells of the pigment epithelium.21,22

Retinal, an inverse agonist?

A potential problem here is that opsin, although insensitive to visible light, is capable in the presence of all-trans-retinal of catalysing guanine nucleotide exchange on transducin, albeit to a small extent.23–25 Furthermore, as noted in Chapter 3, the very high levels of rhodopsin in photoreceptor cells must pose the risk that spontaneous activation might give rise to some form of spurious ‘dark vision’ (see page 68). Of course, the catalytic potency of opsin is very small, only 10 6 that of photo-excited rhodopsin.25 However, with 108 molecules of rhodopsin per cell, even a very low degree of spontaneous activation must be sufficient to cause the sensation of light even in total darkness. It is now thought that this is prevented by the ligand, 11-cis-retinal, acting as an inverse agonist that stabilizes the inactive conformation of the photoreceptor molecule23 (Figure 6.10). Indeed, in the cell-free situation, the

Fig 6.10  Four-state model of rhodopsin.

Of the four states of the rhodopsin molecule that are shown, both Rh* and Rh can activate transducin. Rh is a non-covalent complex of opsin and all-trans-retinal and it is a weak activator of the G protein. Rhodopsin itself does not activate Gt because of the presence of 11-cis-retinal, acting as an inverse agonist.

After Surya et al.23

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apoprotein opsin can catalyse guanine nucleotide exchange on transducin and this is inhibited by 11-cis-retinal. Conversely, all-trans-retinal behaves as a somewhat inefficient activator24 in a manner reflecting the process of activation of other receptors by their specific ligands.

Activation of transducin by the apoprotein is avoided when it is phosphorylated. Rhodopsin kinase is activated by the transducin -subunits in a manner similar to the action of the receptor kinases (see page 98). As a result, the phosphorylated C-terminal domain of rhodopsin acts as a binding site for arrestin, the most abundant protein in the cytosol of the outer segments. This effectively blocks any further interaction with transducin and prevents any signals emanating from the light-insensitive opsin (Figure 6.11).

The rate of decline of the photoresponse is primarily determined by the deactivation of transducin which constitutes the rate limiting step in the sequence of reactions leading from activated rhodopsin to the cGMP phosphodiesterase.26 The persistence of the active form of the -subunits must depend entirely on the rate of hydrolysis of the bound GTP. The hydrolysis rate by isolated t subunits is far too slow to account for the physiological rate of recovery, but this is accelerated about 100-fold when it is reconstituted in vitro together with the phosphodiesterase, approaching the rate that can be measured in disrupted retinal rod outer segments.27 In this sense, the phosphodiesterase appears to act not only as the downstream effector of transducin, but also as a GTPase-activating protein

However, this is not the full story. RGS9, a member of the RGS family of GAP proteins, is present uniquely in rod outer segments.28 For knock-out mice that lack RGS9, recovery is not only slow, but is insensitive to the presence of the phosphodiesterase.29 From this it appears that GAP activity also resides in the RGS–G 5 complex, which lies in wait until the t has established communication with its effector phosphodiesterase, before it pounces. In single rods from RGS9 knock-out mice, the time constant of recovery from

a light flash, normally 0.2 s, is increased to 9 s.29 This system has the advantage that it allows t to remain active until it scores an effective hit on its

Fig 6.11  Phosphorylation of activated rhodopsin and the binding of arrestin shuts off the signal.

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