Timing is everything: GTPase regulation in phototransduction

Abstract

As the molecular mechanisms of vertebrate phototransduction became increasingly clear in the 1980s, a persistent problem was the discrepancy between the slow GTP hydrolysis catalyzed by the phototransduction G protein, transducin, and the much more rapid physiological recovery of photoreceptor cells from light stimuli. Beginning with a report published in 1989, a series of studies revealed that transducin GTPase activity could approach the rate needed to explain physiological recovery kinetics in the presence of one or more factors present in rod outer segment membranes. One by one, these factors were identified, beginning with PDEγ, the inhibitory subunit of the cGMP phosphodiesterase activated by transducin. There followed the discovery of the crucial role played by the regulator of G protein signaling, RGS9, a member of a ubiquitous family of GTPase-accelerating proteins, or GAPs, for heterotrimeric G proteins. Soon after, the G protein β isoform Gβ5 was identified as an obligate partner subunit, followed by the discovery or R9AP, a transmembrane protein that anchors the RGS9 GAP complex to the disk membrane, and is essential for the localization, stability, and activity of this complex in vivo. The physiological importance of all of the members of this complex was made clear first by knockout mouse models, and then by the discovery of a human visual defect, bradyopsia, caused by an inherited deficiency in one of the GAP components. Further insights have been gained by high-resolution crystal structures of subcomplexes, and by extensive mechanistic studies both in vitro and in animal models.

Figure 1

Overview of phototransduction and the physiological light response. (A) Schematic representation of a rod photoreceptor cell. Phototransduction takes place in the outer segment organelle filled with several hundreds of flat disc membranes. (B) The place of transducin activation/deactivation cycle in phototransduction (note that all protein–protein interactions illustrated here take place on the surface of a photoreceptor disc). Vision begins with rhodopsin photoexcitation to produce its R* conformation, which is capable of activating transducin. Transducin activation consists of the exchange of bound GDP for GTP, followed by the separation of the trimer into Gα_t-GTP and the βγ subunits. Next, Gα_t-GTP stimulates the activity of PDE by binding to PDEγ and relieving the inhibitory action that PDEγ imposes on the PDE catalytic subunits. This results in vigorous cGMP hydrolysis in photoreceptor cytoplasm and closure of the cGMP-gated channels located in the plasma membrane enclosing the outer segment. PDE activation persists until the GTP molecule bound to Gα_t is hydrolyzed to GTP, the process facilitated by the RGS9·Gβ5·R9AP complex. The GTP hydrolysis is followed by the return of both transducin and PDE into their inactive states. (C) An example of single photon photoresponse recorded from a mouse rod cell. Reprinted with permission from Arshavsky VY, Lamb TD, Pugh EN Jr. G proteins and phototransduction. Annu Rev Physiol. 2002;64:153–187. Copyright 2002 Annual Reviews, Inc. Arshavsky VY, Burns ME. Photoreceptor signaling: supporting vision across a wide range of light intensities. J Biol Chem. 2012;287:1620–1626. Copyright 2012 American Society for Biochemistry and Molecular Biology. Burns ME, Arshavsky VY. Beyond counting photons: trials and trends in vertebrate visual transduction. Neuron. 2005;48:387–401. Copyright 2005 Elsevier.

Figure 2

Acceleration of transducin GTPase by a component of rod outer segments. Simultaneous measurements of transducin GTPase and cGMP hydrolysis by PDE were performed in suspensions of bleached photoreceptor membranes. Both reactions were initiated by an addition of GTP taken in the amount significantly smaller than that of transducin, thereby allowing only a single synchronized cycle of transducin and PDE activation/deactivation. (A) The time courses of 2 reactions followed a similar exponential trajectory. Solid line represents continuously monitored cGMP hydrolysis; solid circles represent the measurements of P_i produced in the course of the GTPase reaction. Rhodopsin concentration in this experiment was 100 μM. (B) Summary of the data obtained at multiple photoreceptor membrane concentrations. The exponential time constants of transducin GTPase (solid circles) and PDE deactivation (open circles) increased with the increase in membrane concentration, following the same trend. Reprinted with permission from Arshavsky VY, Antoch MP, Lukjanov KA, Philippov PP. Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments. FEBS Lett. 1989;250:353–356. Copyright 1989 Federation of European Biochemical Societies.

Figure 3

Structures of GAP complexes. (A) Complex of RGS9 residues 1–422 with Gβ5 (PDB file 2PBI). The N-terminal DEP domain, DEP helical extension (DHEX), GGL, and RGS domains of RGS9 are labeled. (B) The tertiary complex of the RGS domain of RGS9, a Gα_t/i1 chimeric protein in transition state for GTP hydrolysis (mimicked by bound GDP-AlF₄⁻) and the C-terminal fragment of PDEγ (residues 46–87) (PDB file 1FQJ). (C) Alignment of the RGS domains of the 2 structures in (A, B) suggests how the components may be arranged under physiological conditions. (D) Same as in (C) with space-filling representation of the RGS domain and PDEγ showing the interactions of PDEγ with both RGS9 and Gα (arrows) that allow it to overcome the inhibitory constraint imposed by Gβ5, and enhance the interactions between RGS9 and Gα to achieve highly specific acceleration of transducin GTP hydrolysis.