The Functional and Pathophysiological Consequences of Transducin γ-Subunit Knockout

Abstract

The initial steps of vertebrate vision take place in the retina, where light-sensitive rod and cone photoreceptor cells translate light into an electrical signal through a biochemical process called phototransduction. Transducin, a heterotrimeric G protein, is central to this process in rod photoreceptors. In rods, phototransduction begins when transducin is activated by the light-stimulated G protein-coupled receptor rhodopsin, setting off a cascade of cellular events that ultimately generates the visual signal, or photoresponse. Many aspects of transducin’s function were uncovered through studies of knockout mice lacking its individual subunits. Of particular interest is the knockout mouse lacking the transducin γ-subunit, Gγ1 (the Gγ1-/- mouse), which exhibits unique characteristics that have thus far remained incompletely understood. First, Gγ1-/- rods retain the ability to detect light, despite lacking the canonical transducin Gβ1γ1 complex. Second, these cells experience chronic proteostatic stress, consisting of an insufficient capacity for protein degradation by the ubiquitin-proteasome system (UPS), which leads to the progressive dysfunction and eventual death of Gγ1-/- rods.This dissertation focuses on uncovering the molecular mechanisms underlying the unique functional and pathophysiological consequences of Gγ1 knockout in rod photoreceptors. In Chapter 3, we investigate the mechanism driving light-signaling in Gγ1-/- rods. In Chapter 4, we evaluate whether the chronic proteostatic stress observed in degenerating rods could result from insufficient activity of a specific component of the UPS: the substrate-processing complex formed by the AAA+ ATPase P97 (aka VCP) and associated cofactors.We determined that the level of photoresponse sensitivity of Gγ1-/- rods was comparable to the expression levels of Gαt and Gβ1, the remaining components of the transducin heterotrimer, in the outer segments of these cells. We found that two additional G protein γ-subunits (Gγ2 and Gγ3) are present in the outer segments of both WT and Gγ1-/- rods. Finally, we demonstrated that Gβ1, which normally forms an inseparable heterodimer with Gγ1, also forms complexes with Gγ2 and Gγ3 in both WT and Gγ1-/- rods. Thus, we conclude that the canonical transducin Gβ1γ1 complex is not the sole Gβγ complex able to facilitate phototransduction and that transducin complexes utilizing alternative γ-subunits support transducin activation in Gγ1-/- rods.Our examination of proteostatic stress in degenerating rods focused on two mouse models of retinal degeneration: the Gγ1-/- mouse and the knockin mouse expressing a single copy of the rhodopsin P23H mutation (the P23H mouse). Rods of both strains exhibit proteostatic stress, consisting of an insufficient capacity for protein degradation by the UPS, linked to the requirement to degrade misfolded photoreceptor proteins. We investigated whether insufficient UPS function in these cells results from an insufficient cellular capacity for substrate processing by P97 complexes, a critical step in the proteasomal degradation of a large subset of UPS targets. Gγ1-/- and P23H retinas displayed strikingly different patterns of accumulation of two complementary in vivo proteasomal activity reporters whose degradation is either P97-dependent or P97-independent. Based on these patterns, we conclude that the proteostatic stress observed in Gγ1-/- and P23H rods likely originates from distinct pathophysiological mechanisms in which protein degradation by the UPS may or may not be limited by the cellular capacity for substrate processing by P97 complexes. We show that UPS function in Gγ1-/- rods is likely limited by insufficient P97-dependent substrate processing, whereas proteasomal degradation itself limits UPS function in P23H rods. Finally, we found that, despite being aphenotypic in several other tissues, P97 overexpression is toxic to rod photoreceptors and increases proteostatic stress in Gγ1-/- rods. Together, these studies broaden our understanding of photoreceptor cell biology. In addition to illuminating the mechanisms underlying light-signaling in rods, the work described in this dissertation highlights phototransduction in Gγ1-/- rods as a compelling example of the functional interchangeability of G protein γ-subunits. To our knowledge, this represents the first direct demonstration of multiple Gβγ complexes performing the same function in a living animal. Further, this work highlights the complexity of pathophysiological mechanisms related to degrading misfolded proteins in mutant photoreceptors, which must be accounted for in the development of effective strategies to ameliorate these blinding conditions.