GBγ affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the γ subunit

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