# Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation

## TL;DR

Different light stimuli cause rhodopsin to activate in ways that lead to distinct trafficking and recycling processes in fly photoreceptors.

## Contribution

The study reveals two distinct endocytic and recycling mechanisms of rhodopsin triggered by different light stimulations.

## Key findings

- Blue light stimulation leads to clathrin-mediated endocytosis of rhodopsin and arrestin.
- Green light stimulation causes a non-CME pathway with faster recycling of rhodopsin.
- Differential phosphorylation patterns of rhodopsin affect protein interactions and recycling outcomes.

## Abstract

Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.