Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina

Santiago López-Begines,Anna Plana-Bonamaisó,Ana Méndez

doi:10.1038/s41598-018-20893-1

Abstract

Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

Highlights

Mammalian rod and cone cells harbor two Retinal guanylate cyclase (RetGC), RetGC1 and RetGC24–7
To study whether GCAP1 requires direct binding to RetGC to be distributed to the outer segment compartment, we expressed wiltype GCAP1 and a GCAP1 mutant impaired at RetGC1 binding (K23D) as transient transgenes in the rods of GCAP1/2 double knockout mice
Based on this structural information, we here addressed whether GCAP1 direct binding to the cyclase is required for its distribution to rod outer segments in vivo

Summary

Introduction

Mammalian rod and cone cells harbor two RetGCs, RetGC1 and RetGC24–7. RetGC1 is more abundant than RetGC2 in photoreceptors, and is the isoform linked to inherited retinal dystrophies[8,9,10,11]. Fifteen different mutations in GUCA1A encoding GCAP1 have been linked to autosomal dominant cone dystrophies[18,19,20,21,22,23,24,25,26,27,28] Most of these mutations have the effect of decreasing GCAP1 Ca2+-binding affinity, resulting in constitutive activation of the cyclase independently of the light conditions, as shown by in vitro and in vivo studies[29,30,31,32,33]. Little is known about the mechanisms that regulate RetGC/GCAPs protein assembly and transport to the outer segment in the context of living cells. Whether myristoylation affects GCAPs subcellular distribution in living photoreceptors has not been directly addressed

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Results

Conclusion