Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits.

Abstract

ABSTRACTcGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic a and 13 subunits (Pa and PIS) and two inhibitory 'y subunits (Py) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Py. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Py were designed for cross-linking experiments to identify regions on Pa and P18 subunits that bind to the Py C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Py were replaced with a single cysteine. This mutant, Py83Cys, was labeled with photoprobe 4-(Nmaleimido) benzophenone (MBP) at the cysteine residue. The labeled Py83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Py hydrophobic C terminus. A specific, high-yield cross-linking of up to 709% was achieved between the Py83CysMBP and PDE catalytic subunits. Pa and the N-terminally truncated P,B (lacking 147 aa residues) cross-linked to Py83CysMBP 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 Pa. The corresponding region of P18, P13-749-761, also may bind to the Py C terminus. Our data suggest that Py 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.In retinal rod cells, the visual receptor rhodopsin is activated by the absorption of a photon that leads to GTP-GDP exchange on the retinal GTP-binding protein, transducin (Gt) and to the dissociation of the a-subunit of Gt (GtaGTP) from 13yt and rhodopsin. GtaGTP then activates the effector enzyme, cGMP phosphodiesterase (PDE), by relieving the inhibitory constraint of the PDE 'y-subunits (P-y). cGMP hydrolysis by active PDE results in closure of cGMP gated channels in the plasma membrane (for reviews, see refs.

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

“…Although the last few C-terminal residues of P␥ have been reported to physically interact with the catalytic domain to block catalysis (9,(13)(14)(15)(16)(17), the shortest peptide we tested (P␥81-87) showed no ability to inhibit the catalytic activity of PDE6 under our experimental conditions (IC 50 Ͼ 2 mM; Fig. 2).…”

“…For example, C-terminally truncated P␥ mutants lacking the last 5-10 amino acids have been reported to result in no inhibition (9,22,23) up to 50% inhibition (15) of catalytic activity. There are also reports that inhibition of PDE6 catalysis can occur without an absolute requirement of the extreme C-terminal amino acids (8,(15)(16)(17).The kinetic mechanism of P␥ inhibition of PDE6 catalysis is generally believed to occur by a simple competition of P␥ and substrate for access to the active site. Although this model is supported by kinetic studies showing competition between cAMP hydrolysis and P␥ binding at the active site (12), similar * This work was supported, in whole or in part, by National Institutes of Health Grant EY-05798.…”

“…For example, C-terminally truncated P␥ mutants lacking the last 5-10 amino acids have been reported to result in no inhibition (9,22,23) up to 50% inhibition (15) of catalytic activity. There are also reports that inhibition of PDE6 catalysis can occur without an absolute requirement of the extreme C-terminal amino acids (8,(15)(16)(17).…”

“…1A) interacts with the GAF domains of the PDE6 catalytic dimer (8 -11) and stabilizes noncatalytic cGMP binding to the GAF domains (12). The C-terminal region (broadly defined as amino acids 62-87) interacts with the catalytic domain and blocks the catalytic activity (8,9,(13)(14)(15)(16)(17). Structural studies of P␥ in solution indicate that this protein is overall an intrinsically disordered protein (18) but contains ␣-helical secondary structure (␣1, ␣2, and ␣3; Fig.…”

An Interface of Interaction between Photoreceptor cGMP Phosphodiesterase Catalytic Subunits and Inhibitory γ Subunits

“…Both of these P␥ domains bind to P␣␤, allowing effective inhibition of PDE activity by the P␥ C terminus. Initial studies indicated that the major sites of P␣ and P␤ interaction with P␥ are different and located in the N-terminal regions (P␣, 16 -30 and 78 -90; P␤, 91-110 and 211-230) in areas with a high level of dissimilarity between catalytic subunits .More recently, using a cross-linking approach we have demonstrated that the C terminus of P␥ interacts with region P␣-751-763 located within the PDE catalytic domain (Artemyev et al, 1996). In this study, to identify a P␣ region for binding to P␥-24 -45, we have expressed several large domains of P␣ as GST fusion proteins in Escherichia coli.…”