A Potential Role for BBS5 in Regulating Light-Driven Arrestin Translocation in Rod Photoreceptors

Abstract

Purpose: : Light-driven translocation of arrestin1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this project is to identify potential PKC substrates and identify their contribution to regulating arrestin translocation.

Methods: : Dark-adapted, adult Xenopus eye cups were incubated with 32Plabeled-ATP, followed by stimulation of PKC with 0.1 mM phorbol ester. Labeled proteins were excised after SDS-PAGE separation and identified by in gel tryptic digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Transgenic Xenopus tadpoles expressing GFP-tagged arrestin1 were used to correlate arrestin1 translocation via fluorescence confocal microscopy with target protein phosphorylation. Arrestin1 and BBS5 localization were determined under different lighting conditions via immunocytochemical labeling of frozen sections prepared from Xenopus retinas.

Results: : Eye cups from adult Xenopus were incubated with 32P-ATP and then stimulated with 0.1 mM phorbol ester. Autoradiography of samples separated on 4-16% SDS-PAGE revealed two prominent bands at 40 kD and 65 kD. These bands were excised and identified by LC-MS/MS as BBS5 (Bardet-Biedl Syndrome protein 5) and protein kinase C, respectively. Quantification of arrestin translocation under threshold conditions with increasing intensities of light showed that the initiation of arrestin1 translocation to the outer segments correlated with BBS5 phosphorylation. Immunolocalization of BBS5 revealed that BBS5 was found most abundantly at the base of the cilium and along the entire axonemal structure. This localization of BBS5 was coincident with arrestin1 along the axoneme in dark-adapted rods.

Conclusions: : BBS5 is phosphorylated in response to either light adaptation or activation of PKC by phorbol ester. The correlation of BBS5 phosphorylation with arrestin1 translocation and its localization along the axoneme of rod photoreceptors suggest that BBS5 may be an important regulator of arrestin1 mobility through the connecting cilia during light-driven translocation.

Keywords: photoreceptors • retina: distal (photoreceptors, horizontal cells, bipolar cells) • signal transduction