<p>Rod photoreceptors are specialized neurons responsible for capturing photons and translating visual information into electrical signals. Visual signal transduction in rods is confined to the unique outer segment organelle, a modified primary cilium consisting of a stack of hundreds of flattened disc membranes enveloped by a single plasma membrane. By concentrating important signaling molecules on disc membranes, the outer segment provides an ideal biochemical environment for the production of vision with high sensitivity and temporal resolution.</p><p>This dissertation focuses primarily on a molecular dissection of two multifunctional outer segment proteins, R9AP and rhodopsin, and also reassesses the localization of Glut1, a third protein formerly believed to reside in the outer segment. All three experimental lines relied on in vivo expression of novel protein constructs in vertebrate rods using several gene delivery strategies: conventional transgenics, retinal electroporation, and retinal infection with recombinant adeno-associated virus.</p><p>The tail-anchored protein R9AP, in conjunction with RGS9-1 and G-beta5, comprises the transducin GTPase activating complex, which catalyzes the rate-limiting step in rod photoresponse recovery. In addition to maximizing the enzymatic activity of the complex, R9AP is responsible for both the post-translational stability and outer segment targeting of RGS9-1-G-beta5. We investigated the mechanism behind R9AP's poorly understood function in protecting RGS9-1-G-beta5 from proteolysis and found that it is performed simply by recruiting the complex to cellular membranes and can be entirely dissociated from R9AP's outer segment targeting function. Furthermore, we demonstrated that replacement of R9AP's transmembrane domain with a lipid anchor preserves the ability of the GTPase activating complex to function in outer segments.</p><p>Rhodopsin, the visual pigment of rods, has a second important, yet poorly defined, function as a rod outer segment building block: outer segments disc membranes fail to form in the absence of rhodopsin. Our goal was to identify the molecular features of rhodopsin mechanistically involved in outer segment morphogenesis by designing artificial membrane proteins that could fully substitute for rhodopsin in performing this function. We observed that rhodopsin's C-terminal VXPX outer segment targeting motif is unnecessary for outer segment disc formation since it could be replaced with a targeting motif from an unrelated protein, peripherin. Furthermore, we obtained surprising evidence that rhodopsin's role in this process is limited to providing an abundance of transmembrane protein material to disc membranes.</p><p>Finally, while attempting to find a targeting motif to substitute for the VXPX motif of rhodopsin, we made an unexpected observation that the facilitative glucose transporter Glut1, long thought to reside in the outer segment, is actively excluded from this organelle. This revises our understanding of the energy flow in rods by showing that the outer segment is entirely dependent on the inner segment for its energy supply.</p> / Dissertation

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.