Analysis of PDE6 function using chimeric PDE5/6 catalytic domains.
cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.
Muradov
Hakim
H
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Boyd
Kimberly K
KK
Artemyev
Nikolai O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004926:Escherichia coli / Q000378:metabolism; D055537:Light Signal Transduction / Q000502:physiology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6