Role of the Prenyl Group on the G Protein γ Subunit in Coupling Trimeric G Proteins to A1 Adenosine Receptors

Abstract

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein ␣ subunits and by the composition of the ␤␥ dimers. To determine the role of the ␥ subunit prenyl modification in this interaction, the CaaX motifs in the ␥ 1 and ␥ 2 subunits were altered to direct modification with different prenyl groups, recombinant ␤␥ dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the ␤␥ dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the ␣ subunit. The ␤ 1 ␥ 1 dimer (modified with farnesyl) was significantly less effective than ␤ 1 ␥ 2 (modified with geranylgeranyl) in either assay. The ␤ 1 ␥ 1 -S74L dimer (modified with geranylgeranyl) was nearly as effective as ␤ 1 ␥ 2 in either assay. The ␤ 1 ␥ 2 -L71S dimer (modified with farnesyl) was significantly less active than ␤ 1 ␥ 2 . Using 125 I-labeled ␤␥ subunits, it was determined that native and altered ␤␥ dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the ␥ subunit is an important determinant of the interaction between receptors and G protein ␥ subunits.The membranes of all cells contain multiple receptors and transmembrane signaling systems that regulate cell function (1-5). One major unsolved problem in cell signaling is understanding how a cell selects its response to a hormone or growth factor given the possibilities available. The signaling mechanism used by receptors coupled to the heterotrimeric G proteins 1 provides an excellent example of the complexities. Current evidence suggests that specificity is determined at many levels in this pathway. In addition to the selectivity provided by the receptor itself, the interaction between the intracellular loops of the receptors and the ␣␤␥ subunits in the heterotrimer is a major determinant of specificity (6, 7). Interestingly, some receptors couple selectively to certain G protein ␣ subunits. For example, the ␤-adrenergic receptor couples primarily to members of the G s ␣ family (1, 6) and rhodopsin to the G t ␣ subunit (2). Other receptors, such as the angiotensin AT 1 or muscarinic receptors couple to multiple ␣ subunits including members of the G q and G i class of ␣ subunits (6, 8), leading to activation of multiple signaling networks by a given ligand (5).Although the receptor and the ␣ subunit provide one determinant of selectivity, the ␤␥ subunit is clearly required for the receptor to couple to the ␣ subunit (9, 10). Thus, a third level of selectivity may be provided by the type of ␤␥ subunit used to form the receptor-␣␤␥ complex. In addition, since the ␤␥ dimer can activate effectors directly (11-13), the diversity of these proteins may contribute to the specificity of signaling. Two lines of experimental evidence support this conclusion. Using recombinant ␤␥ subunits purified from Sf9 insect cel...

The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

“…Although cross-linking studies have yet to detect receptor-␥ contact sites (7), several studies point to the importance of the carboxyl-terminal amino acid region and the type of prenyl group on the ␥ subunit in determining the interaction of the ␤␥ dimer with receptor (34,35). These latter studies may explain the lower reconstitutive activity of the ␤ 1 ␥ 1 dimer in the present study.…”

“…In this regard, the ␥ 1 subtype sequence is the most divergent of those determined to date, and its lipid modification is a C-15 farnesyl group rather than the C-20 geranylgeranyl group found on most other ␥ subtypes (1). With regard to the latter, a recent study comparing ␤␥ dimers with the two types of prenyl groups showed that the wild type, geranylgeranylated ␥ 2 subunit and the mutant, geranylgeranylated ␥ 1 subunit had similar abilities to interact with the A 1 adenosine receptor (34). By contrast, the wild type, farnesylated ␥ 1 subunit and the mutant, farnesylated ␥ 2 subunit were much less effective.…”

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

“…Although the influence of the C-terminal sequences and the lipid modification of the γ-subunit on GPCR coupling has been described (56)(57)(58), our data indicate the influence of small differences in the sequence. Although the N-terminal sequences distinguish γ 9 , the C-terminal one-third of all three γ-subunits is highly similar (SI Appendix, Fig.…”

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

“…The γ5-6G mutant was made to increase the distance between the geranylgeranyl modification on the Cys residue and the amino acid sequence at the C terminus previously identified as a contact site for the receptor [9]. Previous evidence from experiments with various receptors indicates that both the prenyl moiety and the C terminal amino acid sequence of the γ subunit are a requirement for receptor interaction of the G protein [13,14,30]. One possible reason for this requirement is that the receptor has contact sites for both the prenyl moiety and the C terminal residues.…”