cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its χ and δ subunits

Abstract

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, αβχ2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, αβχ2δ) can release and bind cGMP at both noncatalytic sites; the δ subunit is likely responsible for this difference in cGMP exchange rates. Removal of the δ and/or χ subunits yields a catalytic αβ dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (K(D) > 1 μM); cGMP binding affinity at the second site (K(D) ~60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of χ subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the χ subunit with the αβ dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.