Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Abstract

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone-and rod-specific PDE6 ␥-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.Phosphodiesterases of cyclic nucleotides (PDEs) 2 are essential enzymes controlling cellular levels of cAMP and cGMP. Eleven families of PDEs have been identified in mammals on the grounds of sequence homology, substrate selectivity, and regulation (1). Photoreceptor-specific PDEs in rods and cones comprise the sixth PDE family (PDE6) and serve as the effector enzymes in the vertebrate phototransduction cascade (1-5). Rod PDE6 is composed of homologous catalytic ␣-subunit (PDE6A) and ␤-subunit (PDE6B) and two copies of a small inhibitory ␥-subunit (P␥) (3). Cone PDE6 is a catalytic dimer of two identical ␣Ј-subunits (PDE6C) (3). A cone-specific inhibitory P␥-subunit is highly homologous to the rod P␥ (6). In rod photoreceptors, PDE6 is located in the specialized compartments called rod outer segments (ROS), where it associates with disc membranes. The membrane attachment of PDE6 is mediated by farnesylation of the PDE6A C terminus and geranylgeranylation of the PDE6B C terminus (7). In cones, geranylgeranylated PDE6C resides on infoldings of the cone outer segment plasma membrane (8). Following photoexcitation of rod or cone photoreceptor cells, PDE6 is activated by the GTPbound transducin ␣-subunit (G␣ t GTP) that relieves the P␥ inhibition of the enzyme. cGMP hydrolysis by active PDE6 leads to a cellular response due to a closure of cGMP-gated channels in the photoreceptor plasma membrane (2-5).Although PDE6 plays a prominent role in vertebrate vision, the structure-function relationships of PDE6 are poorly understood in comparison with other key phototransduction proteins. The lack of an expression system for PDE6 has become a major impediment for PDE6 research. Importantly, an expression system for PDE6 is required to elucidate the mechanisms of visua...

Aryl Hydrocarbon Receptor-interacting Protein-like 1 Is an Obligate Chaperone of Phosphodiesterase 6 and Is Assisted by the γ-Subunit of Its Client

“…The key characteristics of recombinant PDE6C in the PDE6C ϩ AIPL1 ϩ P␥ lysate (K m cGMP 15.6 Ϯ 1.5 M, K i (P␥) 94 Ϯ 14 pM; mean Ϯ S.E., n ϭ 3) after trypsin treatment were comparable with those reported for native trypsin-activated PDE6C (27,28). Given the effect of P␥ on the production of functional PDE6, the expression system was simplified for subsequent studies by subcloning FLAG-PDE6C and EGFP-P␥ into a single pcDNA3.1 vector with the two DNA sequences separated by an internal ribosome entry site (IRES).…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…Many PDEs (and their functional domains) aggregate at concentrations required for biophysical techniques. A greater barrier has been experienced with the PDE6 family whose members cannot be expressed as active enzymes in numerous systems; this complication has resisted resolution until recently when Muradov et al (265) reported expression of human cone PDE6 in rods of transgenic Xenopus laevis; the enzymatic characteristics of the recombinant enzyme are similar to those of native bovine PDE6C, but this system is tedious and the enzyme yield is low, which continues to restrict studies.…”

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

“…For example, phosphodiesterase 6 (PDE6), an enzyme that catalyzes hydrolysis of cGMP to 5 0 -GMP, is localized to the disk rim and incisure regions in frog photoreceptors. 30 While PDE6 is localized uniformly around the circumference of the disk, the involuted structure of the incisures accommodates more enzyme per area of proximal phototransductive plasma membrane. Thus the disk incisures may allow quick hydrolysis of cGMP in the limited cytoplasmic volumes that are proximately located to the striated features.…”