Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits

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 alpha and beta subunits (P alpha and P beta) and two inhibitory gamma subunits (P gamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of P gamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of P gamma were designed for cross-linking experiments to identify regions on P alpha and P beta subunits that bind to the P gamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of P gamma were replaced with a single cysteine. This mutant, P gamma 83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled P gamma 83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the P gamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the P gamma 83CysMBP and PDE catalytic subunits. P alpha and the N-terminally truncated P beta (lacking 147 aa residues) cross-linked to P gamma 83CysMBP 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 P alpha. The corresponding region of P beta, P beta-749-761, also may bind to the P gamma C terminus. Our data suggest that P gamma 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.