Distinct patterns of compartmentalization and proteolytic stability of PDE6C mutants linked to achromatopsia

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Highlights

  • Achromatopsia-linked mutants of cone PDE6 were expressed in rods of transgenic frogs.

  • PDE6 mutants were examined for their stability and compartmentalization.

  • A spectrum of mechanisms of missense PDE6C mutations in achromatopsia is proposed.

Abstract

Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.

Introduction

Cyclic-nucleotide phosphodiesterases (PDEs) of the sixth family (PDE6) are the key effector enzymes in phototransduction in rods and cones (Fu and Yau, 2007, Arshavsky and Burns, 2012). The enzyme catalytic core is a heterodimer of PDE6A and PDE6B subunits in rod PDE6 and a homodimer of PDE6C subunits in cone PDE6. Mutations in the PDE6A and PDE6B genes are responsible for a significant fraction of recessive retinitis pigmentosa (RP), an inherited degenerative retina disease leading to blindness (McLaughlin et al., 1995, Dryja et al., 1999). Mutations in PDE6C cause autosomal recessive achromatopsia (ACHM) (Chang et al., 2009, Thiadens et al., 2009, Grau et al., 2011). ACHM results from a loss of cone function and is characterized by low visual acuity and lack of color discrimination. Many mutations in PDE6, including nonsense mutations, splice defects, and frame shifts are certain to cause loss of expression, misfolding, and/or loss of PDE6 function. However, the mechanisms of missense PDE6 mutations in rods and cones leading to retina disease are largely unknown.

Missense mutations may alter PDE6 function or interfere with transport of functional PDE6 to the outer segment (OS), a specialized ciliary compartment of photoreceptor cells. Proper transport of PDE6 in photoreceptors is critically important for the function and survival of rods and cones. Lack of functional PDE6 in the rod OS leads to elevation of cGMP levels and causes rapid retinal degeneration (RD) in animal models and humans (Farber and Lolley, 1974, Bowes et al., 1990, Pittler and Baehr, 1991, Liu et al., 2004, Ramamurthy et al., 2004). The possibility that abnormal PDE6 trafficking may underlie RP is highlighted by the PDE6B mutation L854V in the protein C-terminal CAAX motif (Veske et al., 1995). Isoprenylation modifications of PDE6 are critical to the PDE6 interaction with membranes and transport (Anant et al., 1992, Catty et al., 1992, Veske et al., 1995, Karan et al., 2008). The C-terminal CAAX boxes of mammalian PDE6A and PDE6B specify farnesylation and geranylgeranylation, respectively (Anant et al., 1992). In an attempt to characterize PDE6C mutants linked to ACHM, several missense mutations were previously introduced into a chimeric PDE5/PDE6C enzyme expressed in sf9 cells (Grau et al., 2011). These mutants indicated either a loss or reduction of the catalytic activity (Grau et al., 2011). However, the use of PDE5/PDE6C chimeras to study the effects of PDE6 mutations has severe limitations. PDE5/PDE6C chimeras contained the catalytic domain of PDE5 and thus, they are essentially PDE5-like enzymes. Furthermore, the process of PDE6 folding, assembly, and trafficking in rod and cones is complex and it involves photoreceptor-specific protein machinery (Ramamurthy et al., 2004, Karan et al., 2008). These aspects cannot be recapitulated using the chimera/insect cell system.

Our previous studies demonstrated that transgenic Xenopus laevis is a robust system to investigate PDE6 and its transport (Muradov et al., 2009, Muradov et al., 2010). Human EGFP-tagged PDE6C ectopically expressed in rods of transgenic X. laevis traffics correctly to the OS. In the OS, EGFP-PDE6C concentrates at the disc rims and co-localizes with endogenous frog rod PDE6 (Muradov et al., 2009). Recently, using transgenic X. laevis we demonstrated that the GAFa domain of PDE6 contains an OS localization signal (Cheguru et al., 2014). Here, we expressed seven known ACHM-linked missense mutants of PDE6C in rods of X. laevis to examine the mutations' effects on the protein expression, stability, and transport. The PDE6C mutants demonstrated distinct patterns of protein stability and compartmentalization depending on the domain(s) harboring the mutation and indicated a spectrum of mechanisms in achromatopsia.

Section snippets

PDE6C with ACHM mutations in the catalytic domain is properly transported to the OS

Seven missense mutations of PDE6C linked to ACHM fall into three categories based on their sequence localization (Fig. 1). Two mutations, H602L and E790K, are situated in the C-terminal catalytic domain. Three mutations are within the regulatory N-terminal GAF domains, R104W in GAFa and Y323N and P391L in GAFb. Two mutations, R29W in the N-terminus and M455V in the region linking GAFb to the catalytic domain, are located outside the conserved domains. First, we examined expression and

Discussion

The mechanisms of PDE6 mutations leading to severe retinal diseases are poorly understood, particularly since a system for expression of functional PDE6 has been lacking. This impediment was principally overcome with the demonstration that transgenic X. laevis is an excellent tool for expression and studies of PDE6 in living rods (Muradov et al., 2009, Muradov et al., 2010). Additionally, biochemical analyses of recombinant PDE6 in vitro are feasible following selective immunoprecipitation of

Generation of PDE6C mutants for expression in transgenic Xenopus rods

The pXOP-EGFP-PDE6C vector for expression of the EGFP-fused human cone PDE6 in transgenic X. laevis was used as a template for introduction of the following mutations: R29W, R104W, Y323N, P391L, M455V, H602L, and E790K. Mutations were generated using QuikChange mutagenesis (Agilent), or a two-step PCR-directed mutagenesis as described previously (Natochin et al., 1998). Briefly, in the first round PCR, mutant primers were paired with a primer carrying a unique restriction site. PCR products

Acknowledgments

We would like to thank Dr. Sheila Baker (University of Iowa) for helpful comments on the manuscript, Dr. Hakim Muradov for construction of several PDE6C mutants and Kimberly Boyd for technical assistance. This work was supported by National Institutes of Health Grant EY-10843 to N.O.A.

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