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 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.
Savage
J R
JR
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
McLaughlin
J N
JN
Skiba
N P
NP
Hamm
H E
HE
Willardson
B M
BM
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D005136:Eye Proteins / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D020349:Surface Plasmon Resonance; D015293:Transducin / Q000378:metabolism*
D002352:Carrier Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; C498320:Pdcl protein, rat; D010750:Phosphoproteins; C065631:phosducin; D012243:Rhodopsin; D015293:Transducin