Functional roles of the two domains of phosducin and phosducin-like protein

Abstract

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the βγ subunit complex (Gβγ) and blocking Gβγ association with Gα subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal G(t)βγ. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G(t)βγ with a 2.5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k1; and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of G(t)βγ with G(t)α and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of G(t)βγ, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in G(t)βγ to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 ± 0.25 kcal/mol for the transition from the G(t)α-binding to the Pd-binding conformation of G(t)βγ.