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Guhan Iyer, Sahil Gulati, Krzysztof Palczewski, Vladimir Kefalov; Modulation of mouse rod signaling by llama-derived transducin nanobody. Invest. Ophthalmol. Vis. Sci. 2019;60(9):568.
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© ARVO (1962-2015); The Authors (2016-present)
G protein-coupled receptor (GPCR) signaling is the most widely used receptor mechanism observed in many organisms. The dissociated Gα-GTP and Gβγ dimer each regulate a variety of signaling pathways that plays crucial role in human physiology. The llama-derived nanobody Nb5 has been previously reported to bind Gβγ and inhibit its signalling. However, the effects of Nb5 on Gα signaling in living organisms are still unclear. To address this question, we generated transgenic mice expressing Nb5 in rod photoreceptors (Nb5+) and studied its effects on phototransduction, a process mediated by the α-subunit of transducin (Gαt).
The effect of Nb5 on the activation of Gαt by rhodopsin was measured in vitro by fluorescence measurements. The in vivo effect of Nb5 on rod phototransduction was studied by ERG recordings that collect rod-driven responses to light. All mice were in C57Bl/6 background and tested between 6-8 weeks of age.
Pre-treatment of heterotrimeric Gt with a 2-fold molar excess of Nb5 resulted in a significant decrease in the Gαt activation rate, suggesting that trapping of Gβ1γ1 by Nb5 reduces the effective heterotrimeric population of Gt. In vivo ERG recordings from Nb5+ mice showed that the maximal scotopic a- and b-wave responses are normal, indicating that the expression of Nb5 did not cause rod degeneration. However, rods from Nb5+ mice required over 3-fold brighter test flashes to elicit a threshold response compared to controls. Thus, expression of Nb5 resulted in a reduction of rod sensitivity, indicating partial inhibition of Gαt signaling in rod photoreceptors. However, rod signaling was only partially inhibited, as Nb5+ mice were more sensitive than Gnat1 knockout animals that lack rod response. Interestingly, the dim flash b-wave response of Nb5+ mice was also shortened by about 25% compared to controls.
Nb5 serves as a dynamic scavenger of the Gβγ dimer that reduces the effective heterotrimeric population of Gt. The resulting reduction in the effective heterotrimeric population of Gt causes suppressed Gαt-mediated signaling in rods. Nb5 provides precise suppression of transducin as the quantity of the expression determines the extent of the suppression. The amplitude of the photoreceptor response is directly influenced by the number of transducin molecules activated, therefore fine modulation of rod signaling can be achieved through the desired expression of Nb5.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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