GβγAffinity for Bovine Rhodopsin Is Determined by the Carboxyl-terminal Sequences of the γ Subunit

Abstract

Two native βγ dimers, β1γ1 and β1γ2, display very different affinities for receptors. Since these γ subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gβγ interaction with receptors. We constructed baculoviruses encoding γ1 and γ2 subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the γ chains and a set of γ1 and γ2 chimeras with the γ2 CAAX motif at the carboxyl terminus. All the γ constructs co-expressed with β 1 in Sf9 cells yielded β1γ dimers, which were purified to near homogeneity, and their affinities for receptors and Gα were quantitatively determined. Whereas alteration of the isoprenoid of γ1 from farnesyl to geranylgeranyl and of γ2 from geranylgeranyl to farnesyl had no impact on the affinities of β 1γ dimers for Gαt, the non-prenylated β1γ2 dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the β1γ2 dimer for rhodopsin and a <3-fold change for the β1γ1 dimer. In each case with identical isoprenylation, the β1γ2 dimer displayed significantly greater affinity for rhodopsin compared with the β1γ1 dimer. Furthermore, dimers containing chimeric Gγ chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Gγ subunit is essential for binding to both Gα and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Gα. Finally, the primary structure of the Gγ subunit provides a major contribution to receptor binding of Gβγ, with the carboxyl-terminal sequence conferring receptor selectivity.