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Transducin ␤-Subunit Can Interact with Multiple G-Protein ␥-Subunits to Enable Light Detection by Rod Photoreceptors

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Transducin ␤-Subunit Can Interact with Multiple G-Protein ␥-Subunits to Enable Light Detection by Rod Photoreceptors New Research Sensory and Motor Systems Transducin ␤-Subunit Can Interact with Multiple G-Protein ␥-Subunits to Enable Light Detection by Rod Photoreceptors Paige M. Dexter,1 Ekaterina S. Lobanova,2 Stella Finkelstein,2 William J. Spencer,1 Nikolai P. Skiba,2 and Vadim Y. Arshavsky1,2 DOI:http://dx.doi.org/10.1523/ENEURO.0144-18.2018 1Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina 27710 and 2Albert Eye Research Institute, Duke University, Durham, North Carolina 27710 Visual Overview The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is ␥ ␥ the knockout of its rod-specific -subunit (G 1). Two stud- ies using independently generated mice documented that this knockout results in a considerable Ͼ60-fold reduction in the light sensitivity of affected rods, but provided dif- ␣ ␣ ferent interpretations of how the remaining -subunit (G t) ␤ ␥ mediates phototransduction without its cognate G 1 1- subunit partner. One study found that the light sensitivity ␣ reduction matched a corresponding reduction in G t con- tent in the light-sensing rod outer segments and proposed ␣ ␤ that G t activation is supported by remaining G 1 asso- ciating with other G␥ subunits naturally expressed in pho- toreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approx- ␣ imately sixfold, reduction of G t and proposed that the light responses of these rods do not require G␤␥ at all. To resolve this controversy and elucidate the mechanism ␥ driving visual signaling in G 1 knock-out rods, we analyzed both mouse lines side by side. We first determined ␣ ϳ that the outer segments of both mice have identical G t content, which is reduced 65-fold from the wild-type ␤ ␥ ␥ (WT) level. We further demonstrated that the remaining G 1 is present in a complex with endogenous G 2 and G 3 subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea Significance Statement Phototransduction has been a valuable system for understanding the basic principles of G-protein signaling. One question that has remained unanswered is whether the G-protein ␣-subunit can support signaling without its cognate ␤␥ partner complex. Previous studies investigating this question in photoreceptors of ␥ G 1 knock-out mice came to mutually exclusive conclusions. We now resolve this controversy by showing that phototransduction in this knockout is supported by alternative ␤␥ complexes rather than the ␣-subunit alone. Most importantly, this study highlights the functional interchangeability of different ␥-subunits in the context of an intact in vivo system. May/June 2018, 5(3) e0144-18.2018 1–9 New Research 2 of 9 ␣ ␥ ␤ ␥ that G t alone supports light responses of G 1 knock-out rods and suggest that G 1 1 is not unique in its ability to mediate vertebrate phototransduction. Key words: G-protein; phototransduction; retinal degeneration; transducin ␤ ␥ Introduction amounts of G 1, unable to fold without its G 1 partner The heterotrimeric G-protein transducin mediates visual (Lobanova et al., 2013). signal transduction in the outer segments of vertebrate In a previous attempt to understand the nature of light ␥Ϫ/Ϫ photoreceptor cells. The visual signal, or photoresponse, responses in G 1 rods, the reduction in transducin is initiated when photoexcited rhodopsin activates the subunits in their outer segments was correlated with the transducin heterotrimer by catalyzing GDPϪGTP ex- reduction in their light sensitivity (Lobanova et al., 2008). ␥Ϫ/Ϫ ϳ change on its ␣-subunit (G␣ ). G␣ subsequently dissoci- G 1 outer segments were shown to contain 50-fold t t ␣ ␤ ␤␥ ␤ less G t and G 1 than WT rods, a reduction very close to ates from the -subunit complex (consisting of G 1 and ␥ the ϳ67-fold loss in their light sensitivity also reported in G 1) to stimulate the downstream effector type 6 cGMP phosphodiesterase. The resulting reduction in cytosolic that study. Because photoreceptor light sensitivity is di- rectly proportional to the rate of transducin activation, cGMP causes cGMP-gated ion channel closure, photore- which in turn is proportional to its concentration on outer ceptor hyperpolarization, and decreased glutamate re- segment discs (Pugh and Lamb, 1993; Heck and Hof- lease from the synaptic terminal (for review, see mann, 2001; Sokolov et al., 2002; Arshavsky and Burns, Arshavsky and Burns, 2012). This signal is processed Ϫ Ϫ 2014), it was suggested that G␥ / light responses are further by downstream retinal neurons before being trans- 1 conveyed by transducin heterotrimer using an alternate mitted to the brain. G-protein ␥-subunit to enable its efficient activation by Previous studies have demonstrated that knocking out rhodopsin. However, attempts to identify this replacement individual transducin subunits produces quite different G␥ were unsuccessful due to the low transducin content ␣ phenotypes. For example, the rods of G t knock-out mice in these retinas. are completely insensitive to light (Calvert et al., 2000), More recently, an alternative G␥Ϫ/Ϫ mouse with a trans- ␥ 1 while G 1 knock-out rods retain distinct light sensitivity genic design different from that used by Lobanova et al. despite the absence of this critical component of the (2008) was characterized by Kolesnikov et al. (2011). No- transducin heterotrimer (Lobanova et al., 2008; Kolesni- tably, rod photoresponses recorded from these mice had ␣ kov et al., 2011). Next, the absence of G t does not affect characteristics very similar to those described by the first ␤ ␥ the expression levels of its cognate G 1 1 complex (Cal- study, including a 90-fold reduction in light sensitivity vert et al., 2000; Lobanova et al., 2008), whereas the from the WT level. However, the outer segment content of ␣ ␤ ␣ expression levels of both G t and G 1 are drastically G t was estimated to be only sixfold lower than normal. ␥ reduced in the G 1 knockout (Lobanova et al., 2008; Based on previously published evidence that rhodopsin ␣ ␣ ␤ ␥ Kolesnikov et al., 2011). Finally, the knockout of G t does can activate G t in vitro without G 1 1, although signifi- not affect photoreceptor viability (Calvert et al., 2000), cantly slower than normally (Fung, 1983; Kisselev et al., ␥ whereas G 1 knock-out mice undergo progressive pho- 1999; Herrmann et al., 2006), the authors argued that ␥Ϫ/Ϫ ␣ toreceptor degeneration (Lobanova et al., 2008; Kolesni- G 1 rod photoresponses are conveyed by G t acting kov et al., 2011). The latter originates from proteostatic alone. They also described a slower progression of pho- ␥Ϫ/Ϫ stress arising from the requirement to degrade vast toreceptor degeneration in G 1 retinas than observed by Lobanova et al. (2008), which was attributed to differ- ences in the genetic backgrounds of these strains. Received April 10, 2018; revised May 7, 2018; accepted May 10, 2018; First published May 17, 2018. To settle these discrepancies and elucidate the mech- Ϫ/Ϫ 1 ␥ The authors declare no competing financial interests. anism driving visual signaling in G 1 rods, we per- Ϫ Ϫ 2 ␥ / Author contributions: P.M.D., E.S.L., and V.Y.A. designed research; P.M.D., formed a side-by-side analysis of both G 1 mouse E.S.L., S.F., W.J.S., and N.P.S. performed research; P.M.D., E.S.L., S.F., ␣ ␤ strains. First, we compared the amounts of G t and G 1 in W.J.S., N.P.S., and V.Y.A. analyzed data; P.M.D. and V.Y.A. wrote the paper. retinal lysates of these strains and found that the de- 3This study was supported by the National Institutes of Health Grants EY-022959 (V.Y.A.), EY-05722 (V.Y.A.), EY-026468 (P.M.D.), and EY-025558 crease in expression levels of both subunits was identical (W.J.S.); and the Unrestricted Award from Research to Prevent Blindness, Inc. and corresponded to the decrease initially reported by to Duke University. Lobanova et al. (2008). Most importantly, this decrease Acknowledgments: We thank Dr. O.G. Kisselev for providing the StL G␥ 1 was comparable to the decrease in rod photoresponse knock-out mouse and Y. Hao for preparing the plastic-embedded retinal cross sections. sensitivity reported for both strains. We next demon- ␤ Correspondence should be addressed to Vadim Y. Arshavsky, Duke Uni- strated that the remaining G 1 is present in a complex ␥ ␥ versity Eye Center, 5012 AERI, 2351 Erwin Road, Durham, NC 27710. E-mail: with endogenous G 2 and G 3 subunits. These results [email protected]. ␤ ␥ ␤␥ indicate that G 1 1 is not the sole G complex able to E.S. Lobanova’s present address: Department of Ophthalmology, University facilitate phototransduction and that G␤ associated with of Florida, Gainesville, FL 32610. 1 DOI:http://dx.doi.org/10.1523/ENEURO.0144-18.2018 alternative, noncanonical G␥ subunits supports transdu- ␥Ϫ/Ϫ Copyright © 2018 Dexter et al. cin activation in G 1 rods at an efficiency comparable This is an open-access article distributed under the terms of the Creative ␤ ␥ to that of the canonical G 1 1 complex. Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is Given the significant difference reported in the progres- ␥Ϫ/Ϫ properly attributed. sion of photoreceptor degeneration for the two G 1 May/June 2018, 5(3) e0144-18.2018 eNeuro.org New Research 3 of 9 strains, we also performed a systematic analysis of retinal vitrogen) in PBS containing 0.1% Triton X-100. Sections degeneration in these animals.
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