G protein signaling in the retina and beyond: the Cogan lecture.

In order to survive and function properly, mammalian cells send and receive a vast number of signals that are used to adjust their behavior in response to changes in the environment. Among the multiple receptor systems that cells utilize for this purpose, the most prominent role is undoubtedly played by G protein-coupled receptors (GPCRs). These cell surface receptors respond to an incredibly wide repertoire of ligands, including ions, peptides, lipids, neurotransmitters and light. G protein-coupled receptors constitute the largest family in mammalian genomes, accounting for approximately 3% to 4% of all genes. Despite their versatility, the organization of all known GPCRs is rather conserved; they share a common seven transmembrane topology and the ability to activate intracellular heterotrimeric G proteins. Ligand binding causes GPCRs to undergo a conformational change, which is sensed intracellularly by G proteins, causing them to release guanosine diphosphate (GDP) in exchange for guanosine triphosphate (GTP). Nucleotide binding occurs on the Ga subunit and results in its dissociation from the Gbc subunits. In their dissociated state, both Ga-GTP and free Gbc are able to interact with and regulate the activity of downstream effectors, including proteins key to cellular homeostasis, such as ion channels, kinases, and second messenger-producing/degrading enzymes. This signaling is terminated upon the hydrolysis of GTP by the Ga subunit, causing its inactive Ga-GDP form to reassociate with the Gbc subunit. Much of what we know about the functional organization of GPCR systems is derived from the phototransduction cascade of vertebrate photoreceptors, one of the first and the beststudied G protein pathways. As a result, the lessons learned in the study of photoreceptors have had a tremendous impact on our understanding of GPCR biology and will likely continue to guide research on G protein cascades for years to come. The main sequence of the events in phototransduction is now well established and has been the subject of several excellent reviews. In prototypic rod photoreceptors, light causes a conformational change in the photosensitive GPCR rhodopsin by inducing isomerization of the receptor-bound inverse agonist 11-cis retinal into the full agonist all-trans retinal. Photoexcited rhodopsin activates G protein transducin, which in turn dissociates into Gat1-GTP and Gb1c1 subunits. Activated Gat1-GTP binds to its effector enzyme—the gamma subunit of phosphodiesterase, type 6 (PDE6c)—and relieves the inhibitory constraint that this subunit has on the catalytic PDE6ab subunits, which leads to the hydrolysis of the second messenger cGMP. The declining concentrations of cGMP allow the opening of cGMP-gated ion (CNG) channels on the plasma membrane, leading to cellular hyperpolarization and the resulting inhibition of neurotransmitter release. All components of the phototransduction cascade are delegated to a special compartment of the cell called the outer segment, which is essentially an elaboration of the primary cilia. Thus, the phototransduction cascade is highly compartmentalized, revealing the first lesson from this GPCR cascade. The second lesson is provided by studies on the mechanisms that allow photoreceptors to quickly recover from excitation, a property that is essential for achieving the high temporal resolution of our vision. This process requires the deactivation of phototransduction, which involves the termination of both rhodopsin and transducin signaling. One of the major breakthroughs in the field was the demonstration that transducin deactivation is the rate-limiting step in the termination of phototransduction reactions. Transducin, as well as all other G proteins, has a very slow GTP hydrolysis rate, with kinetics that are insufficient to explain the physiologically relevant speed of photoresponse termination. The timely deactivation of transducin requires the contribution of another element of the GPCR cascade, type 9 regulator of G protein signaling (RGS9), which functions to speed up the rate of GTP hydrolysis of this G protein. Type 9 regulator of G protein signaling belongs to a family of RGS proteins that consists of more than 30 members ubiquitously expressed in all cells and involved in the regulation of GPCR signaling. Thus, the second lesson learned from the organization of the phototransduction cascade is the key involvement of RGS proteins for achieving physiologically relevant timing. In photoreceptors, RGS9 does not act alone but requires the contribution of two proteins with which it forms a tight complex, and which are now considered to be its bona fide subunits. The first protein, an atypical member of the G protein family, type 5 beta subunit (Gb5), is required for ensuring the correct folding and stability of the complex, with additional contributions in guiding RGS9 to selectively recognize its correct substrate, the Gat1-PDE6c complex, instead of free Gat1. The second molecule, a SNARE-like transmembrane protein named RGS9 anchor protein (R9AP), delivers the complex to the outer segments of the photoreceptors, positioning it on the disc membranes; R9AP also plays an essential role in determining the proteolytic stability of the complex. Work on the organization and functional regulation of the RGS complex in photoreceptors by Vadim Arshavsky and