Asymmetric interaction between rod cyclic GMP phosphodiesterase gamma subunits and alphabeta subunits.
Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Palphabeta) and two identical inhibitory subunits (Pgamma). Palphabeta is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pgamma with Pbeta as compared with Palpha in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pgamma and Palphabeta, by differentiating Pgamma interaction with each individual Palphabeta subunit through radiolabel transfer from various positions throughout the entire Pgamma molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pgamma makes a major contribution to the interaction with Palphabeta. In addition, a striking asymmetry of interaction between the Pgamma polycationic region and the Palphabeta subunits was observed when the stoichiometry of Pgamma versus the Palphabeta dimer was below 2. Preferential photolabeling on Pbeta from Pgamma position 40 and on Palpha from position 30 increased while lowering the Pgamma/Palphabeta ratio, but diminished when the Pgamma/Palphabeta ratio was over 2. Our finding leads to the conclusion that two classes of Pgamma binding sites exist on Palphabeta, each composed of GAF domains in both Palpha and Pbeta, differing from the conventional models suggesting that each Pgamma binds only one of the Palphabeta catalytic subunits. This new model leads to insight into how the unique Palphabeta heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pgamma.
Guo
Lian-Wang
LW
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Grant
Jennifer E
JE
Hajipour
Abdol R
AR
Muradov
Hakim
H
Arbabian
Marty
M
Artemyev
Nikolai O
NO
Ruoho
Arnold E
AE
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D008027:Light; D013058:Mass Spectrometry; D008954:Models, Biological; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D012694:Serine / Q000737:chemistry; D014466:Ultraviolet Rays; D014785:Vision, Ocular
D003432:Cross-Linking Reagents; D005136:Eye Proteins; D010455:Peptides; D012694:Serine; D001710:Biotin; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003545:Cysteine