Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its γ-subunits

Abstract

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic α and β subunits (Pα and Pβ) and two inhibitory γ subunits (Pγ) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pγ. The last C-terminal residues -Helle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pγ were designed for cross-linking experiments to identify regions on Pa and Pβ subunits that bind to the Pγ C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pγ were replaced with a single cysteine. This mutant, Pγ83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pγ83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pγ hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pγ83CysMBP and PDE catalytic subunits. Pα and the N-terminally truncated Pβ (lacking 147 aa residues) cross-linked to Pγ83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Pα. The corresponding region of Pβ, Pβ-749-761, also may bind to the Pγ C terminus. Our data suggest that Pγ blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.