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

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the beta gamma subunit complex (G beta gamma) and blocking G bet a gamma association with G alpha subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each co nstituting approximately half of the molecule. We investigated the function al roles of the two domains of phosducin and phosducin-like protein in bind ing retinal G(t)beta gamma. Kinetic measurements using surface plasmon reso nance showed that: 1) phosducin bound G(t)beta gamma with a 2.5-fold greate r affinity than phosducin-like protein; 2) phosphorylation of phosducin dec reased its affinity by 3-fold, principally as a result of a decrease in k(1 ); and 3) most of the free energy of binding comes from the N-terminal doma in with a lesser contribution from the C-terminal domain. In assays measuri ng the association of G(t)beta gamma with G(t)alpha and light-activated rho dopsin, both N-terminal domains inhibited binding while neither of the C-te rminal domains had any effect. In assays measuring membrane binding of G(t) beta gamma, both the N- and C-terminal domains inhibited membrane associati on, but much less effectively than the fall-length proteins. This inhibitio n could only be described by models that included a change in G(t)beta gamm a to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 +/- 0.25 kcal/moI for the transition from the G( t)alpha-binding to the Pd-binding conformation of G(t)beta gamma.