Abstract
Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the betagamma subunit complex (Gbetagamma) and blocking Gbetagamma association with Galpha 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)betagamma. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G(t)betagamma 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 k(1); 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)betagamma with G(t)alpha 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)betagamma, 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)betagamma 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)alpha-binding to the Pd-binding conformation of G(t)betagamma.
Highlights
Of Pd and PhLP is responsible for inhibition of Gt␣1⁄7Gt␥ interactions, we measured the ability of Pd, PhLP, and their individual domains to block the binding of 125I-Gt␣ and Gt␥ to Rho* in UROS membranes (Fig. 2)
To further test the functional independence of the two domains, inhibition of Gt␥ binding to UROS membranes was measured in the presence of both domains
This domain appears to inhibit membrane binding by stabilizing a conformation that does not have the farnesyl group available for insertion into the membrane
Summary
Preparation of Domain Constructs—The pET 15b vector (Novagen, Madison, WI) containing the recombinant rat Pd cDNA with a His tag as well as the pET 11a vectors containing the amino-terminal (Pd-N, residues 12–107) and carboxyl-terminal (Pd-C, residues 108 –246) domains of rat Pd [33] were a gift from Dr R. To measure the binding kinetics, purified Gt␥ in Eluent buffer (10 mM HEPES, 150 mM NaCl, 0.05 mM EDTA, 0.005% surfactant P20, pH 7.4) at five known concentrations (ϳ5-fold above and below the Kd) was injected at 20 l/min over the test flow cell surface containing the immobilized Pd and over a control flow cell surface without immobilized protein. A set of curves was generated for each Pd species from the background-subtracted curves at each of the five Gt␥ concentrations tested This set of five curves was simultaneously fit to a simple one-to-one bimolecular binding model with base-line drift by a global analysis using the BIAevaluation software. Binding of 125I-Gt␥ (0.36 M) to unilluminated UROS membranes (10 M Rho) was measured at the indicated Pd, PhLP, and their N- and C-terminal domain concentrations as described previously [17]. The quadratic terms for the equation describing this model were as follows
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