BBS5 Organizes a Complex of Proteins along the Photoreceptor Axoneme that Constitutes a Functional cGMP Signaling Cascade

Abstract

Purpose: Previous studies have shown that BBS5 is present along the photoreceptor axoneme in the outer segment. Here we use affinity isolation of BBS5 to identify proteins associated with BBS5 along the photoreceptor axoneme. We show that proteins associated with BBS5 form a functional cGMP signaling cascade, suggesting that the axoneme may create a specialized “reaction chamber” for photoreceptors.

Methods: BBS5 and associated proteins were affinity isolated from extracts of rod outer segments using antibodies prepared against BBS5. Isolated proteins were identified by tandem mass spectrometry, verified by co-precipitation and co-localization. To assay for functional cGMP signaling, isolated complexes were mixed with a fluorescent analog of cGMP [2’-O-(N’-methylanthraniloyl)-cIMP] and hydrolysis was monitored fluorescently.

Results: Affinity isolation of BBS5 from rod outer segments results in the co-precipitation of a nearly complete cGMP signaling cascade, including transducin, cyclic GMP phosphodiesterase subunits, and cyclic nucleotide-gated channel subunits. Fluorescent localization studies show that these proteins co-localize with a microcomplex of the BBSome composed of BBS5, BBS8, and BBS9 along the photoreceptor axoneme. To ascertain whether this complex of proteins can perform as a functional signaling cascade, we monitored cGMP hydrolysis fluorescently in the presence 1 uM MANT-cIMP. Protein complexes isolated with anti-BBS5 supported active cGMP hydrolysis; control samples isolated with an irrelevant antibody showed no cGMP hydrolysis activity.

Conclusions: Our findings show that BBS5 isolated from photoreceptor outer segments organizes a complex of proteins that function as a cGMP signaling cascade. This study suggests that the axoneme, the evolutionarily original cilium of the photoreceptor, may do more than simply provide physical support to the outer segment or provide a pathway for delivery of material to the outer segments. Instead, the axoneme may be an active participant in photoreceptor cell biology serving as a subcellular compartment to organize cGMP signaling that may function to respond to extracellular signals beyond light.