Regulation of Photoresponses by Phosphorylation

Fig. 1. The role of phosphorylation by rhodopsin kinase in the inactivation of photoexcited rhodopsin, metarhodopsin II (MII or R*). In the dark-adapted rod, rhodopsin is not a good substrate for rhodopsin kinase (RK), so on photoactivation and conversion to R* it catalyzes rapid guanosine diphosphate–guanosine triphosphate (GDP-GTP) exchange on the α-subunit of the G protein transducin, Gαt. Activated Gαt-GTP activates cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), leading to closure of cGMP-gated cation channels and plasma membrane hyperpolarization. R* binds to and activates rhodopsin kinase, which uses adenosine triphosphate (ATP) to add multiple phosphates to the carboxyl-terminal region of R*, inducing a state that binds with high affinity to arrestin. Arrestin binding to phosphorylated rhodopsin effectively quenches the activity of R* until decay of metarhodopsin II and regeneration of rhodopsin lead to dephosphorylation by protein phosphatase 2A (PP2A). Although the decay of metarhodopsin II and regeneration eventually lead to inactivation of the phototransduction cascade in the absence of rhodopsin kinase or its target sites, this process is much too slow for normal recovery kinetics. The presence of multiple phosphorylation sites enhances the reproducibility of inactivation kinetics by decreasing the variation in the amount of time each R* remains active.

Fig. 2. Locations of domains and functional sites within the primary structure of rhodopsin kinase. Diamonds represent phosphorylation sites, with the numbers indicating sequence positions. The RGS domain is homologous to the catalytic domains of RGS proteins but is not known to demonstrate GAP (guanosine triphosphatase accelerating protein) activity. The kinase domain is homologous to other serine/threonine protein kinases. At the carboxyl terminus, the CaaX-box sequence CSVS is subject to posttranslational modification in which the last three amino acid residues are proteolytically cleaved, the cysteine is farnesylated, and the terminal carboxylate is converted to a methyl ester.

INACTIVATION OF PHOTOACTIVATED RHODOPSIN BY RHODOPSIN KINASE

Inactivation of photoexcited rhodopsin (metarhodopsin II, MII, or R*) is initiated via its enzymatic phosphorylation by rhodopsin kinase (RK, Fig. 2). The activity of RK was first described 35 years ago [16–19] when isolated rod outer segments (ROSs) were incubated with γ-32P adenosine triphosphatase (ATP) in the presence of light, causing 32P to be incorporated into MII. This light-dependent process was shown to occur in vivo [20], and later phosphorylated rhodopsin was shown to be necessary for signal attenuation in bovine rod outer segment preparations [2, 3] and subsequently in vivo [21]. After over a decade of attempts, RK was purified, stabilized, and characterized [22]. Since then, experiments from many laboratories have shed light on both the properties of the enzyme and its importance in the visual transduction process.

Rhodopsin kinase, also known as G protein-coupled receptor kinase 1 (GRK1), was the first member discovered of the GRK family of Ser/Thr kinases specific for seventransmembrane G protein-coupled receptors [23]. It is responsible for phosphorylating MII on its C-terminus (see structures in Fig. 3) with an upper limit of nine phosphorylation sites per MII [24]. This phosphorylation allows the binding of arrestin to occur, effectively quenching the signaling pathway by no longer allowing the G-protein transducin (Gt) to interact with MII [25]. Depending on reaction conditions, addition of phosphates to the C-terminus of rhodopsin can either decrease [25, 26] or fully attenuate [27] its interaction with transducin.

Rhodopsin kinase is a single-polypeptide chain enzyme with a molecular weight of 62–64kDa [28] and is posttranslationally modified: it is farnesylated at the C-terminus consensus sequence for isoprenylation, CaaX, which is followed by limited proteolysis of aaX and subsequent methyl esterification of the isoprenylated Cys [29, 30] (Fig. 2). Without these modifications, the activity of RK is approximately four-fold lower, suggesting that this hydrophobic modification is important for targeting RK to disk membranes and conferring full enzymatic activity toward MII. RK is autophosphorylated on serine residues, and the reaction is unaffected by the presence of bleached rhodopsin [11–13]. This autophosphorylation does not alter the rate of rhodopsin phosphorylation.

Fig. 3. Models of structures of the C-terminus (residues 330–348) of rhodopsin (left) from x-ray crystallography (PDB file 1U19) of the dark state of rhodopsin [131] and (right) from nuclear magnetic resonance (NMR) (PDB file 1NZS) of the peptide chemically phosphorylated at the seven labeled serine and threonine residues and bound to arrestin [132]. These structures suggest that photoactivation, phosphorylation, or arrestin binding may involve dramatic differences in the conformation of this region.

The kinetic parameters of the purified enzyme for MII are as follows: Km= 4 M

(rhodopsin), Km = 2 M (ATP), and Vmax = 700 nmol-min−1-mg−1, corresponding to a turnover number, kcat = 0.8 s−1 [22]. However, in vivo, rhodopsin is inactivated with

a time constant of 80 ms or less [31].

Proof that RK binding to MII is required for normal inactivation of MII in rods first came from recordings of photocurrent responses of single mouse rods expressing a C-terminal truncation mutant of rhodopsin [21] and subsequently from responses of mouse rods with a null mutation of RK [32]. Mutations in RK cause defects in the kinetics of deactivation and an increase in the amplitude of the light response.

While RK can phosphorylate many serine and threonine residues on the C-terminus of rhodopsin, only a few (Ser 334, Ser338, and Ser343) have been identified biochemically as major phosphorylation sites in intact retinas [33, 34]. Based on experiments using rapid quench followed by mass spectrometry, it was reported that in mouse retinas the sites closest to the C-terminus of rhodopsin are the first to be phosphorylated; Ser343 is phosphorylated most rapidly, followed by Ser338. Ser334 is phosphorylated after a delay of more than 10 s [35]. In relating these experiments to electrophysiological results, it must be borne in mind that these experiments required supersaturating levels of light to obtain sufficient product for analysis, and therefore the kinetics may differ in the dim flash or single-photon regimes.

The requirement for multiple biochemical steps to inactivate MII has been proposed to play an important role in reducing the variability in the lifetime of catalytically active

Fig. 4. Structure of recoverin bound to an N-terminal peptide from rhodopsin kinase (RK), illustrating interactions that may help mediate Ca2+-dependent inhibition of this kinase. Structures (2I94) are based on nuclear magnetic resonance (NMR) [133] of recoverin in the presence of Ca2+, which is bound at the third and fourth EF hands, and a peptide, RK25, corresponding to the first 25 amino acid residues of rhodopsin kinase.

rhodopsin [36]. A recent electrophysiological study using rhodopsin transgenes encoding proteins with different numbers of phosphorylation sites [37] elegantly demonstrated the importance of the multiplicity of these sites in single-photon reproducibility.

The activity of RK for isolated peptide substrates derived from the C-terminal region of rhodopsin is around 1,000-fold lower than for the full-length MII [38]. Peptide competition studies and alanine-scanning mutagenesis of rhodopsin have shown the interaction site of RK with rhodopsin to include cytoplasmic loops 1 through 3 [28, 39].

Early studies of rhodopsin phosphorylation have shown that at low light levels (sufficient to activate < 1% of the total rhodopsin pool), for every mole of activated rhodopsin several hundred moles of phosphate were added to the rhodopsin pool [17, 40]. A straightforward explanation of this phenomenon, known as high-gain phosphorylation, is that the nonspecific substrate, inactive rhodopsin, is being phosphorylated in a light-dependent manner in trans. While RK has been shown to exist in two states (an inactive and an active state using proteolytically digested rhodopsin from bovine rod outer segments and synthetic peptides [41]), trans-phosphorylation does not occur in heterologously expressed chimeras of rhodopsin [42], still leaving the underlying cause of transphosphorylation uncertain.

Lack of functionally active RK in humans has been shown to cause blinding diseases: Oguchi disease, a form of congenital stationary night blindness, and retinitis pigmentosa. While Oguchi disease is characterized by profoundly slowed rod dark adaptation [43], patients with retinitis pigmentosa undergo progressive blindness [44]. The visual functions of a patient with an inactivating mutation in the RK gene have been thoroughly characterized by electrophysiological and psychophysical methods [45]. In mice, absence of RK leads to a light-dependent degeneration of the retina [32].

Rhodopsin kinase has been reported to be regulated by recoverin, a member of the neuronal calcium sensor (NCS) branch of the EF-hand superfamily [46, 47]. In vitro studies together with electrophysiological studies of recoverin knockout mice suggest that by inhibiting RK at high calcium levels (Fig. 4), recoverin prolongs the lifetime of MII [47], thereby allowing the accelerated inactivation of rhodopsin in response to lowered intracellular calcium under light conditions [48, 49].