Mechanism and Regulation of GPCR Endocytosis in vivo using Rhodopsin as a Model

We explored the mechanisms and regulation of rhodopsin endocytosis in Drosophila by examining the trafficking itineraries of the major rhodopsin (Rh1) in vivo using transgenic flies expressing a modified Rh1 containing a C‐terminal mCherry tag (Rh1‐mCherry). By fluorescence microscopy, Rh1‐mCherry was detected in the visual organelles (rhabdomeres) of photoreceptors. Upon continued light stimulation, Rh1‐mCherry display light‐dependent endocytosis, and becomes enriched in the cytoplasm of photoreceptors. Internalization of rhodopsin may be mediated by the clathrin‐mediated endocytosis (CME). CME requires the presence of a minor phospholipid, phosphoinositol‐4, 5‐bisphosphate (PIP 2 ) in the membrane. Interestingly, perturbation of the PIP 2 biosynthesis fails to prevent the endocytosis of Rh1‐mCherry in rdgA mutants. The rdgA gene encodes diacylglycerol kinase critical for the recycling of PIP 2 . We also explored the contribution of two visual arrestins, Arr1 and Arr2, as adaptor proteins in promoting the endocytosis of Rh1. We introduced Rh1‐mCherry into either arr1 or arr2 mutant background and investigated how trafficking is affected. We show that internalization of Rh1‐mCherry is drastically reduced in the arr2 mutant. Arr2 is expressed 7fold more abundant than Arr1. Based on the finding, we conclude that Arr2 is critical for orchestrating the light‐dependent endocytosis of Rh1‐mCherry. Importantly, this Arr2‐mediated event does not appear to belong to the classical CME as it is insensitive to a reduction of PIP 2 in the membrane.