The duration of photoresponses in vertebrate rods and cones is controlled at the level of GTP hydrolysis by a GTPase accelerating protein (GAP) whose catalytic core is provided by RGS9-1. RGS9-1 is in turn regulated by phosphorylation on serine 475, in a reaction that is dependent on Ca²⁺. In living mice, the level of phosphorylation at this site is reduced by light. Thus RGS9-1 phosphorylation provides a potential mechanism by which light-regulated changes in intracellular [Ca²⁺] may feed back on phototransduction through effects on the lifetime of activated G-protein and cGMP phosphodiesterase.

Introduction: GTP Hydrolysis and Photoresponse Kinetics

Because photoexcitation relies on activation of the heterotrimeric G protein, transducin, hydrolysis of GTP bound to this protein is one of the key determinants of recovery kinetics. Experiments with non-hydrolyzable analogues of GTP many years ago¹³ made it clear that prolonged excitation due to prolonged activation of cGMP phosphodiesterase (PDE6, here referred to simply as PDE) results when R* is formed but GTP hydrolysis is prevented. Thus GTP hydrolysis represents a point in the cascade where Ca²⁺ might potentially exert a regulatory influence. Attempts to demonstrate an effect of Ca²⁺ on PDE inactivation or GTPase kinetics have so far yielded mostly negative results.^4,5 However, recent findings have suggested that there may indeed be a link between light-controlled changes in intracellular [Ca²⁺] and molecules regulating GTPase kinetics.

RGS9-1, the GTPase Accelerating Protein (GAP) for Phototransduction

The major regulator of GTP hydrolysis in rods and cones is a member of the large RGS (Regulator of G protein Signaling)^6,7 family of GTPase accelerating proteins (GAPs),⁸ called RGS9-1.^9–11 This protein has been found only in rod and cone photoreceptor cells,¹² and its removal by gene deletion in mice leads to profoundly slowed recovery of light responses in both rods and cones.^13,14

Domain and Subunit Structure of RGS9-1

The GTPase accelerating function of RGS9-1 is localized to a catalytic core domain, known as the RGS domain or RGS box, whose basic structure is conserved among all RGS proteins. This domain is necessary and sufficient for acceleration of GTP hydrolysis by the photoreceptor G protein, transducin.^9,12,15–17

What is less certain than the basic catalytic activity of RGS9-1 and the domain responsible, is how RGS9-1 is regulated. In particular, the roles of several other domains of RGS9-1, and proteins that bind RGS9-1 (Fig. 1), are active areas of current investigations. However, it seems reasonable that because of its ability to control the timing and potentially the sensitivity as well of photoresponses, RGS9-1 activity is likely to be regulated. Likely candidates for stimuli leading to such regulation are light and intracellular calcium, based on the numerous other examples of calcium feedback signals modulating light responses described in this volume.

Figure 1

Possible role of RGS9-1 phosphorylation in Ca²⁺ feedback circuitry. When light, through activation of rhodopsin, stimulates transducin (GaGTP) and PDE, Ca²⁺ levels fall as cGMP-gated cation channels close and Ca²⁺ continues to be extruded by the exchange (more...)

Unique C-Terminus of RGS9-1

One of the interesting features of RGS9-1 is its unique C-terminal tail. Neither this 18 amino acid peptide nor any closely related sequence is found in any other RGS proteins. It is in this tail region that RGS9-1 differs from a brain-specific protein encoded by the same gene, RGS9-2. The differences between RGS9-2 and RGS9-1 arise because of differences in RNA processing in the striatum and the retina.^10,11,18 The functions of these C-terminal domains are not completely known, but there is strong evidence suggesting that the RGS9-1 C-terminal domain is important both for membrane attachment,¹⁹ and for coupling the GAP activity of RGS9-1 to interactions with the effector subunit, cGMP phosphodiesterase γ (PDEγ).^17,20

Phosphorylation of RGS9-1

Treatment of ROS membranes with radiolabeled ATP led to phosphorylation of a protein with electrophoretic mobility identical to that of RGS9-1. Unfortunately, in many gel systems RGS9-1 co-migrates with tubulin, known to be an abundant protein in rods, and a substrate for phosphorylation. Therefore it was necessary to use immunoprecipitation to verify not only that RGS9-1 is indeed phosphorylated by an endogenous protein kinase in ROS, but that it is one of the most strongly phosphorylated proteins in the membranes. Proteolytic digestion of phosphorylated RGS9-1 followed by HPLC and mass spectrometric analysis of tryptic peptides revealed a single site of phosphorylation, 475Ser. Identification of the site allowed to the synthesis of the corresponding peptide, with and without phosphate, and the preparation of phosphospecific monoclonal antibodies.²¹ These were then used to monitor RGS9-1 phosphorylation in vivo and in vitro.

Effect of Ca²⁺ on Phosphorylation

Tests of the divalent cation requirements for 475Ser phosphorylation revealed that as with other phosphorylation reactions, this one requires Mg²⁺. More interesting was he observation that Ca²⁺ also stimulates the phosphorylation. The calcium chelator EGTA greatly reduces 475Ser phosphorylation, and restoration of Ca²⁺ at levels in the physiological range of hundreds of nanomolar is sufficient to restore maximal phosphorylation. Thus RGS9-1 phosphorylation, at least in vitro, is stimulated by Ca²⁺ at the levels obtaining under darkadapted conditions, and is inhibited when Ca²⁺ levels fall to the low concentrations associated with responses to light.

Effect of Light on Phosphorylation of RGS9-1

If intracellular Ca²⁺ truly controls RGS9-1 phosphorylation under physiological conditions, then the prediction would be that light reduces levels of 475Ser phosphorylation, because of the light induced reduction in intracellular [Ca²⁺] discussed elsewhere in this volume. This prediction was tested by collecting retinas from mice exposed to light or kept in dark-adapted conditions, prior to sacrifice, detergent extraction, immunoprecipitation (with antibodies directed against RGS9-1, but not phosphorylation-specific) followed by immunoblotting with phosphopeptide-specific antibody. The result revealed that RGS9-1 phosphorylation is indeed greatly reduced by light.²¹ Thus it seems likely that light-induced decreases in intracellular [Ca²⁺] in rod outer segments do indeed regulate RGS9-1 phosphorylation in vivo.

Conclusion and Remaining Questions

The observation that RGS9-1 phosphorylation is regulated by light and intracellular calcium is tantalizing, because it suggests that changes in [Ca²⁺] feed back onto the phototransduction cascade at the level of the lifetimes of activated transducin and PDE. However, our current understanding leaves many questions unanswered. Key among these are: what are the functional consequences of 475Ser phosphorylation? Does phosphorylation increase or reduce GAP activity, or does it affect some other (possibly unknown) function of the protein? What protein kinase is responsible? What is the molecular mechanism for coupling Ca²⁺ concentrations to RGS9-1 phosphorylation? What Ca²⁺binding proteins are involved? Research aimed at answering these questions is in progress, and the answers should provide new insights into the link between Ca²⁺ and recovery kinetics in phototransduction.

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