The R7 regulators of G protein signaling (R7 RGS), namely RGS6, RGS7, RGS9 and RGS11, are expressed in the retina along with its binding partner Gβ5. The RGS9-1 isoform is expressed only in retinal photoreceptors and rate-limits the recovery of rod phototransduction by acting as a member of the transducin GAP complex (RGS9-1/Gβ5L/R9AP). The Gβ5L isoform is also only expressed in retinal photoreceptors and acts by stabilizing the GAP complex. The Gβ5S isoform differs from Gβ5L by the absence of exon 1 due to alternative splicing and is expressed in many other retinal cells. Gβ5L is barely detectable in RGS9-/- mice suggesting that Gβ5L has a protein degradation signal conferring instability in the absence of RGS9. To study the role of exon 1 of Gβ5L, we replaced Gβ5L with Gβ5S in rods by expressing transgenic Gβ5S under the control of the rhodopsin promoter within a Gβ5-/- mouse and determined that exon 1 of Gβ5L has two previously unidentified functions: (1) to increase the capacity of Gβ5L to bind to RGS9-1 and (2) to serve as a signal for rapid degradation of Gβ5L in RGS9-/- photoreceptors. Several groups have reported that RGS7 and RGS11 with Gβ5S reside in the dendritic tips of depolarizing bipolar cells (DBCs) and that they are involved in the mGluR6/Gαo/TRPM1 pathway, which mediates DBC light responses. The exact role of RGS7 in DBCs has not been unequivocally determined. We have contributed by making a true RGS7 null mouse line and found the RGS7-/- mice are viable and fertile, but have a small body size. Electroretinogram (ERG) b-wave implicit time in young RGS7-/- mice is prolonged at eye opening, but the phenotype disappears by 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7-/- retina, but the Gβ5S level is significantly reduced. We further generated a RGS7 and RGS11 double knockout (711dKO) mouse line and found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as well as the ERG b-wave. Ultrastructural defects similar to those of Gβ5-/- mice are present in 711dKO. Furthermore, in retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S becomes undetectable, while the photoreceptor-specific Gβ5L remains unaffected. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in the retina, the DBC-related defects found in Gβ5-/- mice appear to be caused solely by a combined loss of RGS7 and RGS11. The notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins is firmly established in this work. The availability of all four R7 RGS single knockout mouse lines enables future studies to further elucidate the roles of R7 RGS proteins in vision.

ABSTRACT RATE-LIMITING STEP OF CONE PHOTOTRANSDUCTION RECOVERY AND OGUCHI DISEASE MECHANISMS By Frank Sungping Chen Advisor: Ching-Kang Jason Chen, Ph.D. Retinal photoreceptors provide the first gateway in which light information from the environment is transformed into neuronal signals. The cone and rod photoreceptors are responsible for day and night vision, respectively. Understanding rod and cone phototransduction is to figure out how these cells differ in their temporal and spatial sensitivities to allow perception of a broad dynamic range of stimuli. Phototransduction is mediated through a Gprotein signaling cascade. Light absorption by visual pigment triggers the isomerization of 11- cis-retinal covalently attached to these pigments, which are heptahelical transmembrane Gprotein- coupled receptors. Isomerization of 11-cis-retinal to all-trans-retinal activates the receptor, which catalyzes the exchange of GDP for GTP on the α subunit of heterotrimeric Gprotein called transducin. Activated transducin relieves inhibitory constraint on cGMP-PDE, leading to rapid hydrolysis of cGMP, closure of cGMP gated cation channels, and membrane hyperpolarization. In order for photoreceptor to be responsive to light again, this robust phototransduction pathway must be deactivated in a timely fashion and this involves several reactions simultaneously. First, the activated opsin must be phosphorylated by G-protein-coupled receptor kinases (GRKs) and capped by arrestin binding. Second, activated transducin must hydrolyze bound GTP through intrinsic GTPase activity, which is accelerated by a GTPase accelerating protein (GAP) complex comprised of RGS9-1/Gβ5-L/R9AP. Mutations in human genes involved in these reactions cause various visual defects. Cone, by and large, uses the same set of genes for pigment and transducin deactivations but it has lower sensitivity and faster kinetics than rod and is responsible for high visual acuity. During phototransduction recovery in which multiple reactions take place, the slowest reaction will determine the overall rate of recovery. In rod, this so-called, rate-limiting step has been determined to be transducin deactivation. It is unknown whether cone transducin deactivation also controls the timing of conerecovery, although we and others have shown that cone possesses a higher level of GAP concentration. In this thesis, the rate-limiting step in cone phototransduction recovery has been unequivocally determined by overexpressing RGS9-1 by 2.7 fold in mouse cones, which results in accelerated cone recovery. Complementarily, we find that ectopically expressing a human cone opsin kinase GRK7 in mouse cones does not affect cone recovery. These results altogether demonstrate that the rate-limiting step of cone recovery is the GTP-hydrolysis of cone transducin, not the opsin phosphorylation by GRKs. By elucidating the rate-limiting step of photoreceptor recovery, we have revealed the importance of G-protein cycling in timing of both rod and cone photoreceptors. This may further be generalized to other physiological processes controlled by heterotrimeric G-proteins. The proper shutoff of phototransduction is essential for normal vision as recovery defects lead to visual impairment. Even though the reaction catalyzed by GRK1 is not rate-limiting, mutations of this important gene render rhodopsin phosphorylation and deactivation the slowest step in rod recovery and create a pathological condition. GRK1 mutations have been found in Oguchi disease patients, who suffer from congenital stationary night blindness. One of the mutations, V380D, is investigated in detail in this study. Transgenic expression of GRK1 V380D mutant in rods reveals a kinase with reduced expression and catalytic activity. While V380D GRK1 is found capable of inactivating rhodopsin, the reduction in kinase activity leads to a delayed dark adaptation, and is congruent with the night blindness phenotype observed in Oguchi disease patients. Finally, we have also investigated the role of post-translational isoprenylation on GRK1 function. We found that isoprenylation is required for GRK1 membrane association and outer segment targeting. Altogether our data add significantly to understanding the structure and function of GRK1, which is one of the least understood molecules involved in vertebrate phototransduction.