Determination of the G(βγ)-binding domain of phosducin. A regulatable modulator of G(βγ) signaling

Although a role for the βγ-subunits of heterotrimeric G proteins (G(βγ)) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G(βγ) have yet to be fully elucidated. The G(βγ)-binding region of β- adrenergic receptor kinase (βARK), a cytosolic enzyme recruited to the membrane receptor substrate by G(βγ), has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G(βγ)-binding retinal phosphoprotein, contains sequences homologous with the G(βγ)-binding domain of βARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G(βγ) binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G(βγ) is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G(βγ)-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G(βγ) to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.