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

Abstract

In view of the expanding roles of the ␤␥ subunits of the G proteins in signaling, the possibility was raised that the rich diversity of ␤␥ subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant ␣ 2A -adrenergic receptor were used to assess the contribution of the ␤␥ subunit composition. Reconstituted coupling between the receptor and heterotrimeric G i protein was assayed by high affinity, guanine nucleotide-sensitive binding of the ␣ 2 -adrenergic agonist, [3 H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various ␤␥ dimers, including those containing the ␤ 3 subtype and the newly described ␥ 4 , ␥ 10 , and ␥ 11 subtypes, to promote interaction of the same ␣ i subunit with the ␣ 2A -adrenergic receptor.Consistent with the steadily increasing number of G protein 1 ␤ and ␥ subtypes that has been revealed in recent years (1), in vivo studies have indicated a role for this structural diversity in the specificity of signaling. In this regard, antisense studies by Kleuss et al. (2,3) have demonstrated a specific requirement for the ␤ 1 and ␥ 3 subunits in the somatostatin receptor signaling pathway in rat pituitary GH 3 cells, with a similarly specific requirement for the ␤ 3 and ␥ 4 subunits in the muscarinic receptor signaling pathway. Also, a ribozyme study by Wang et al. (4) has shown a specific involvement of the ␥ 7 subunit in the ␤-adrenergic receptor signaling pathway in human kidney 293 cells. Taken together, these in vivo studies indicate that the composition of the ␤␥ dimer has important ramifications for the fidelity of signaling that is probably manifested at the level of the receptor.A growing body of in vitro evidence supports a direct interaction between the receptor and the ␤␥ dimer (5). In particular, direct interaction of transducin ␤␥ with rhodopsin has been shown with a fluorescence energy transfer technique (6). This association was blocked by a synthetic peptide derived from the carboxyl-terminal tail of rhodopsin, suggesting a site of direct contact between ␤␥ and rhodopsin. Moreover, cross-linking studies have confirmed a receptor contact site on the ␤ subunit. A synthetic peptide derived from the carboxyl-terminal portion of the putative third cytoplasmic loop of the ␣ 2A -AR could be cross-linked to the carboxyl-terminal region of the ␤ subunit (7).To date, in vitro studies examining the contribution of a limited number of ␤␥ dimers to the specificity of receptor coupling have not yielded the same high degree of discrimination shown in the in vivo studies cited above (8, 9). The present study extended this analysis to the ␣ 2A -adrenergic receptor and to ␤␥ dimers that represent the most extensive degree of structural diversity examined to date. Since baculovirus expression has been shown to be an effective means for producing functional G protein subunits (10 -13) as well as G protein-coupled receptors (8,9,14,15)...

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

“…Because this ␥ subunit is widely expressed (69), these observations suggest that cells containing ␥ 11 could use dimers containing ␥ 11 to couple ␣ subunits to receptors, allowing receptors to activate ␣ subunits without inducing effects on downstream ␤␥ targets. Taken further, this argues that receptor activation may release specific ␤␥ dimers, a concept that gains experimental support from biochemical experiments (31,(71)(72)(73), antisense mRNA experiments (74,75), small interfering RNA experiments (76), and data obtained with knockout mice (77).…”

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

“…The molecular mechanisms that lie at the basis of this process are still not fully identified. Evidence has accumulated for all three subunits -α, β and γ -of the G protein playing a role in this process [1][2][3][4][5][6][7][8][9]. However, the specific roles that these subunits play in the activation process are not entirely clear.…”

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

“…Extensive analyses of the βγ-dimer formation potential had indicated unrestricted dimer formation for β 1 and β 4 (i.e., dimers with all γ-subunits are found), restricted dimer formation of β 2 and β 3 (e.g., no dimers with γ 1 or γ 11 ), and no or only weak dimer formation for β 5 (6,7). Although a comprehensive analysis of Gαβγ complex formation is missing, it is likely that most of the Gαβγ combinations are capable of forming a functional complex (8)(9)(10). Taking into account the βγ-dimers believed to be nonexistent, this restriction still results in a number of ∼700 potential Gαβγ combinations.…”

“…Besides tissue-specific expression (4), it has quickly become clear that GPCRs display specificity for G-protein coupling and biased agonism (9,11,12). Although a variety of structures have been solved for G proteins and, more recently, for GPCRs (13)(14)(15), the only structural snapshot of the interaction between a GPCR and a G protein is provided by the structure of the complex between β 2 adrenergic receptor and Gα sS β 1 γ 2 (16).…”