The nature of the link between mislocalized rod opsin and AC stimulation may be multifaceted. In tests on stimulation of mislocalized opsin as a cause of cell death in isolated salamander rod cells,
19 we found that pertussis toxin reduced cell death, indicating that a G
i protein, like transducin, was involved. Moreover light-stimulation of rhodopsin in cell-free systems can enhance AC activity in a transducin-dependent manner.
79,80 Chinese hamster ovary cells expressing bovine rhodopsin showed inhibition of AC in the presence of light; however, the AC isotype in question appeared to be Type VI,
81 not Type I, the primary isotype in photoreceptors.
30,31 Since our publication, there have been mixed reports in cold-blooded vertebrate models, testing our idea that G-protein activation by mislocalized opsin is involved in rod cell death. In
Xenopus, our model was examined by creating a transgenic animal that expressed normal rod opsin as well as opsin with a double mutation at Q350ter (equivalent to Q344ter in mammals, to cause mislocalization) and K296R (to prevent 11-cis–retinal binding and therefore transducin activation).
13 Because the double mutation did not appear to reduce degeneration, the authors concluded that activation of transducin by mislocalized opsin was not a factor in rod cell death. Their conclusion seems plausible if mutated protein was the only type of opsin on the membranes of the inner segment/synaptic terminal. Immunocytochemical analysis of normal opsin localization was done at only one time point and without quantitative controls for immunolabel density. Wild-type opsin is transported to the plasmalemma of inner segments in transgenic mice with Q344ter or S334ter mutated rhodopsin.
62,82 Moreover, from other animal studies (see above), elevated amounts of normal opsin in the inner segment would be expected as outer segments degenerate. Mislocalized normal opsin could have interacted with transducin and contributed, in parallel with other causes like overexpression of opsin,
83 to cell death in the
Xenopus model. In
ovl zebra fish with a retinal degeneration caused by a mutant ciliary gene, which therefore displays opsin mislocalization, both transducin and AC activity were shown to contribute to rod cell death
84 in a pathway identical to our initial proposed pathway.
19 In murine retina, Nakao et al.
84 also demonstrated that rod cell death in rd10 mice was AC dependent. In another mouse study, a Q344ter-induced retinal degeneration was created on a rho −/− background to minimize retinal degeneration by rhodopsin overexpression.
85 In these retinas, opsin is mutated at the C-terminal and thus missorts to the inner segment but still interacts with transducin. Degeneration was exacerbated by light. Additionally, the authors demonstrated that mislocalized opsin was capable of light activation in vivo and that cell death was reduced in animals bred on a Trα −/− background, indicating dependence on transducin signaling, supporting our model. In our study,
19 and in this latter study,
85 there was the recognition that other G proteins could be involved. For us, for instance, pertussis toxin did not completely prevent rod cell death. Other opsin family members in retinal ganglion cells and invertebrate photoreceptors interact with a G
q protein to cause an increase in calcium through activation of phospholipase C.
86,87 Calcium/calmodulin in turn can stimulate photoreceptor AC type 1. Rod cells in fact contain a homolog of G
αq called G
α11.
88 Additionally, it is likely that cAMP mechanisms are not the only pathways contributing to rod cell regenerative growth. In retinal ganglion cells, for example, increased cAMP combined with controlled inflammation and decreased PTEN activity determine the length of new regenerating processes and work through separate signaling cascades.
89