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

Abstract

Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory P␥-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and P␥ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants.Cyclic nucleotide phosphodiesterases of the sixth family (PDE6) 2 are the key effectors in the visual transduction cascade in rod and cone photoreceptors. In the dark, activity of the PDE6 catalytic dimers is restrained by two tightly bound inhibitory ␥-subunits (P␥). This allows cGMP to maintain depolarizing "dark" current through a cGMP-gated channel in the photoreceptor plasma membrane. Photoexcitation leads to G-protein-mediated activation of PDE6 followed by a drop in cytoplasmic cGMP, channel closure, and propagation of an electrical signal to downstream retinal neurons (1, 2). In addition to being essential to photoreceptor physiology, PDE6 is critical to the health and survival of rods and cones. Malfunctions caused by mutations in genes that encode either PDE6 or its putative chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), lead to severe blinding retinal diseases.Mutations in the PDE6A, PDE6B, and PDE6G genes, which encode the catalytic PDE6AB subunits and P␥ of rod PDE6, respectively, are responsible for a significant proportion of cases of recessive retinitis pigmentosa (3-5) and can also lead to autosomal dominant congenital stationary night blindness (6). Mutations in their cone counterparts, PDE6C and PDE6H, cause autosomal recessive achromatopsia (7-10).PDE6 deficiency also appears to underlie one of the most severe forms of Leber congenital amaurosis (LCA type 4), a condition caused by mutations in the AIPL1 gene (11,12). LCA is an early onset inherited retinopathy and one of the main causes of blindness in children (13). The link between PDE6 and AIPL1 was discovered in studies of AIPL1 knock-out and knockdown mouse models, which revealed rapid severe retinal degeneration and a marked reduction in PDE6 protein levels and activity prior to the loss of photoreceptor cells (14, 15). Thus, the animal models recapitulated the hallmarks of LCA and suggested that AIPL1 is a potential chaperone of PDE6. This notion is consistent with the fact that AIPL1 contains an FK506-binding pro...

Unique structural features of the AIPL1–FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness

“…In lysates of HEK293T cells cotransfected with PDE6C and Pγ, cGMP hydrolysis did not exceed the very low levels in lysates from untransfected cells (7.6 ± 2.7 pmol cGMP hydrolyzed per milligram protein per minute). Consistent with previous observations (22), coexpression of PDE6C and Pγ with WT AIPL1 led to robust cGMP hydrolysis in cell lysates (14.7 ± 1. in transfected HEK293T cells were similar to those of the wildtype AIPL1 (SI Appendix, Fig. S10).…”

“…Recently, we confirmed that three AIPL1 mutations linked to LCA (V71F, W72S, and C89R) are pathogenic, by demonstrating that they fail to chaperone PDE6 in HEK293T cells (22). W72S and C89R formed perinuclear aggregates, an outcome consistent with protein instability.…”

“…To this end, we used cultured HEK293T cells transfected with PDE6C and Pγ in the absence and presence of the WT and N65K/M66K/E70P forms of AIPL1 (22). In lysates of HEK293T cells cotransfected with PDE6C and Pγ, cGMP hydrolysis did not exceed the very low levels in lysates from untransfected cells (7.6 ± 2.7 pmol cGMP hydrolyzed per milligram protein per minute).…”

“…The mechanism whereby AIPL1 exerts its biological effects remained obscure until an AIPL1 knockout mouse model revealed that the rapid retinal degeneration that characterizes this disease is caused by marked reduction in protein levels and activity of the phototransduction effector enzyme phosphodiesterase 6 (PDE6) (20,21). The resulting hypothesis that AIPL1 is a chaperone for PDE6 was recently validated by the demonstration that AIPL1 is required for heterologous expression of functional PDE6 (22). This chaperone activity involves interaction of the FKBP domain with the farnesyl, and geranylgeranyl modifications of the catalytic subunits of PDE6 (23,24).…”

“…This chaperone activity involves interaction of the FKBP domain with the farnesyl, and geranylgeranyl modifications of the catalytic subunits of PDE6 (23,24). Interestingly, AIP fails to appreciably bind the isoprenyl moieties and to chaperone PDE6 (22), indicating that the AIPL1-FKBP domain is uniquely specialized.…”