Control of rhodopsin DENIS A. BAYLOR, MARIE E. BURNS activity in vision Abstract high concentration in the cytoplasm in darkness and that binds to cationic channels in the Although rhodopsin's role in activating the surface membrane, holding them open. phototransduction cascade is well known, the Hydrolysis of cGMP allows the channels to processes that deactivate rhodopsin, and thus close, interrupting an inward current of sodium, the rest of the cascade, are less well calcium and magnesium ions and producing a understood. At least three proteins appear to hyperpolarisation of the membrane. The play a role: rhodopsin kinase, arrestin and hyperpolarisation reduces the rate at which recoverin. Here we review recent physiological neurotransmitter is released from the synaptic studies of the molecular mechanisms of terminal of the rod. rhodopsin deactivation. The approach was to The purpose of this paper is to review recent monitor the light responses of individual work on the important but still poorly mouse rods in which rhodopsin was altered or understood mechanisms that terminate the arrestin was deleted by transgenic techniques. light-evoked catalytic activity of rhodopsin. Removal of rhodopsin's carboxy-terminal These mechanisms, which fix the intensity and residues which contain phosphorylation sites duration of the activation of the transduction implicated in deactivation, prolonged the flash cascade, need to satisfy strong functional response 20-fold and caused it to become constraints. Rhodopsin activity must be highly variable. In rods that did not express arrestin the flash response recovered partially, terminated rapidly so that an absorbed photon but final recovery was slowed over lOO-fold. can be registered without contamination by These results are consistent with the notion after-effects of previous absorptions, yet it must that phosphorylation initiates rhodopsin last long enough to give an easily detectable deactivation and that arrestin binding electrical response. Furthermore, activity should completes the process. The stationary night be shut off in a stereotyped fashion so that each blindness of Oguchi disease, associated with absorbed photon is encoded identically, null mutations in the genes for arrestin or allowing the brain to make an accurate rhodopsin kinase, presumably results from assessment of the number and timing of photon impaired rhodopsin deactivation, like that absorptions. Finally, shut-off should be revealed by the experiments on transgenic complete and irreversible, since escape of animals. recently deactivated rhodopsin molecules to the active state may falsely signal photon absorptions, generating masking noise that Key words Arrestin, Kinase, Night blindness, Rhodopsin, Rod, Transgenic interferes with normal vision. Given these constraints, it is perhaps not surprising that the deactivation of rhodopsin Rhodopsin's role in vision is well known; upon seems to involve fairly complex mechanisms. absorption of light it activates a G-protein Our current understanding of the process, cascade that generates an electrical response at drawn largely from in vitro biochemical the surface membrane of the retinal rod cell. DA Baylor � experiments, is summarised in Fig. 2. At least M.E. Burns This response encodes the absorption of single three proteins seem to play key roles: rhodopsin Department of photons, and upon transfer through the visual kinase, arrestin! p44 and recoverin. Neurobiology pathway it ultimately elicits visual sensations. Photo activated rhodopsin can be rapidly Stanford University School Rhodopsin's role in signal generation within the phosphorylated at multiple serine and of Medicine Stanford rod is diagrammed in Fig. l. threonine residues near its carboxy(C)­ CA94305, USA Photoisomerised rhodopsin catalyses GTP! terminus.! This phosphorylation is catalysed by Tel: +1 (650) 723-6510 GDP exchange on the G-protein transducin, a rhodopsin kinase.2 Phosphorylation of Fax: + 1 (650) 725-3958 single rhodopsin producing hundreds of copies rhodopsin reduces its ability to activate of activated transducin (T GTP) within a fraction transducin and permits the binding of the This research was supported by Grant EY05750 from the of a second. T GTP activates a cyclic GMP soluble capping protein arrestin, which further National Eye Institute, the phosphodiesterase (PDE). This enzyme quenches rhodopsin's activity?A Recoverin, a Ruth and Milton Steinbach hydrolyses cyclic GMP (cGMP), a diffusible calcium-binding protein, can inhibit rhodopsin Fund, and the McKnight internal messenger that is present at relatively phosphorylation in vitro5 by binding to Foundation Eye (1998) 12, 521-525 © 1998 Royal College of Ophthalmologists 521 phototransduction in rods of transgenic mice in which the primary structure of rhodopsin was altered or one or both genes for arrestin was deleted. The molecular biology was performed in the laboratory of Professor Melvin Simon at the California Institute of Technology, using methods that have been described elsewhere.ll.12 Rh T POE Immunoblot analysis was used to compare the levels of phototransduction proteins in transgenic and control animals. The expression of truncated rhodopsin and the Na+ absence of arrestin did not appear to affect the relative expression levels of other transduction proteins, Ca++ although rearing the animals in the light caused shortening of the outer segments in some transgenics. Mg++ Phototransduction in single rods was monitored by using a suction electrode to record the reduction in inward current that resulted from flashes of 500 nm light (Fig. 3). This method allows one to observe the rod's response to a single photon. 13 Results and discussion Role of phosphorylation in deactivation of rhodopsin Previous studies demonstrated that supplying A TP to truncated, internally dialysed salamander rod outer Fig. 1. Amplifying cascade of phototransduction in the retinal rod. In segments reduced the amplitude and duration of the the dark, sodium, calcium and magnesium ions flow into the rod outer flash response, suggesting a role for phosphorylation in segment through channels held open by cyclic GMP (cGMP). Upon deactivation of the cascade.14 The C-terminus of excitation by light, rhodopsin (Rh) in the disc membrane activates the rhodopsin contains multiple serine and threonine G-protein transducin (T), which in turn activates cGMP phospho­ diesterase (POE). POE hydrolyses cGMP, causing intracellular cGMP residues that can be phosphorylated after levels to drop and channels on the surface membrane to close. The photoisomerisation occurs. Three of these sites (5334, decrease in the inward current hyperpolarises the rod, causing a 5338, 5343) have been implicated as the preferred sites reduced rate of neurotransmitter release from the synaptic terminal. for rhodopsin kinase.IS Pharmacological inhibition of rhodopsin kinase in isolated rod outer segments rhodopsin kinase under conditions of high calcium interfered with the recovery of light reponses, suggesting concentration.6.7 When the calcium level inside the rod that phosphorylation of rhodopsin by rhodopsin kinase drops during the response to light, inhibition of the reduces its catalytic activity.4 To test whether kinase by recoverin should be removed, causing phosphorylation of rhodopsin at its C-terminus is rhodopsin deactivation to proceed more rapidly, thus producing the decreased sensitivity characteristic of light adaptation. An alternative pathway for rhodopsin Rh deactivation may be provided by a truncated splice variant of arrestin, p44, which can bind to hvi nATP nADP * unphosphorylated rhodopsin and strongly inhibit its ** � Rh -! .. Rh nP activity.8 Thus, p44 may provide an important backup Rh Kinase mechanism for phosphorylation-independent shut-off of rhodopsin. Failure of proper rhodopsin deactivation is Rec·Ca presumably the cause of the functional defect in Oguchi ''''''''''"1 te r"""'" disease, a recessively inherited form of stationary night Rh-p44 blindness that is linked to homozygous deletions or �2ca RhnP-Arr Rec missense mutations in the arrestin9 or rhodopsin kinaselO (inactive) (inactive) genes. How is rhodopsin deactivated under physiological Fig. 2. Rhodopsin deactivation scheme derived from biochemical conditions? Do rhodopsin kinase and arrestin indeed experiments. After absorption of a photon (hv), rhodopsin !Rh) is function sequentially, as in the scheme of Fig. 2? What activated (Rh**) until it is phosphorylated by rhodopsin kinllse !Rh are the individual contributions of phosphorylation and kinase), which reduces its catalytic activity (Rh*). In the presence of arrestin binding to deactivation, and what happens if calcium, rhodopsin kinase is inhibited by calcium-bound recoverin (Rec-ea), which makes rhodopsin deactivation calcium-sfnsitil'f. either process fails? Which site(s) on rhodopsin are Arrestin binds to phosphorylated rhodopsin (RhI1P*),jurther qllenching phosphorylated? The purpose of this paper is to review its activity. A splice variant of arrestin (p44) can bind to Ilnd inhibit recent physiological work that has examined these the activity of unphosphorylated rhodopsin, providing an altemative questions. The approach was to observe deactivation pathway that is independent of phosphorylation. 522 10% of cases the single photon response was anomalous, with a rectangular waveform, an amplitude 2 times larger than normal, and a very prolonged time course. In 90% of the trials the single photon response was entirely normal. The relative numbers