The G Protein β Subunit Is a Determinant in the Coupling of Gs to the β1-Adrenergic and A2a Adenosine Receptors

Abstract

The signaling specificity of five purified G protein ␤␥ dimers, ␤ 1 ␥ 2 , ␤ 2 ␥ 2 , ␤ 3 ␥ 2 , ␤ 4 ␥ 2 , and ␤ 5 ␥ 2 , was explored by reconstituting them with G s ␣ and receptors or effectors in the adenylyl cyclase cascade. The ability of the five ␤␥ dimers to support receptor-␣-␤␥ interactions was examined using membranes expressing the ␤ 1 -adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the ␤ isoform. The ␤ 4 ␥ 2 dimer demonstrated the highest coupling efficiency to either receptor. The ␤ 5 ␥ 2 dimer coupled poorly to each receptor, with EC 50 values 40 -200-fold higher than those observed with ␤ 4 ␥ 2 . Strikingly, whereas the EC 50 of the ␤ 1 ␥ 2 dimer at the ␤ 1 -adrenergic receptor was similar to ␤ 4 ␥ 2 , its EC 50 was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the ␤␥ dimers were measured. ␤␥ dimers containing G␤ 1-4 were able to stimulate AC2 similarly, and ␤ 5 ␥ 2 was much less potent. ␤ 1 ␥ 2 , ␤ 2 ␥ 2 , and ␤ 4 ␥ 2 inhibited AC1 equally; ␤ 3 ␥ 2 was 10-fold less effective, and ␤ 5 ␥ 2 had no effect. These data argue that the ␤ isoform in the ␤␥ dimer can determine the specificity of signaling at both receptors and effectors.Signal transduction involving heterotrimeric G proteins 1 is a universal mechanism for the integration of extracellular stimuli such as hormones, neurotransmitters, odorants, and light (1, 2). The components involved in this signaling cascade are diverse, including a large number of receptors, G protein ␣ and ␤␥ subunits and effectors. Even though the diversity of the proteins in this system could potentially account for the known specificity of signaling in differentiated cells, the mechanisms for determining specificity are not completely defined. The ␤-adrenergic receptor is one of the most well characterized seven transmembrane spanning receptors, and provides an excellent example of selective coupling to a particular ␣ subunit, G s . When activated, G s can stimulate all nine adenylyl cyclase isoforms (3, 4). The G protein ␤␥ dimer, when released after receptor activation, is also able to regulate adenylyl cyclase (5). However, the regulation of the various isoforms of adenylyl cyclase by the ␤␥ dimer is much more selective; apparently, only AC2, AC4 (6, 7), and AC7 (8) are stimulated by ␤␥, whereas the neuronal-specific AC1 (4) and possibly AC5 and AC6 are inhibited by the dimer (9). Moreover, there is evidence that AC2 does not respond well to dimers composed of certain ␤ and ␥ subunits (10) or to dimers containing the phosphorylated ␥ 12 subunit (11). Thus, to understand fully the regulation of adenylyl cyclase by a G s -coupled receptor, one needs to know which ␤␥ dimers are most likely to support receptor G protein coupling and the effects of ␤␥ dimers on the various isoforms of adenylyl cyclase.The number of functionally distinct ␤␥ dimers is potentially very large, with seven G protein ␤ isoforms (including two splice ...

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

“…The purified ␣ subunits were concentrated to a volume of 100 -200 l, separated into aliquots, and stored at Ϫ80°C. Each preparation of G protein ␣ subunit used in this study was functional based on its ability to couple to recombinant receptors and ␤␥ subunits when reconstituted into Sf9 cell membranes (29,31,36). SDS gels documenting the purity of the G␣ subunits prepared by this procedure are published elsewhere (29,31).…”

“…The procedures used for purification of the ␣ subunits of G i , G q , G s , and G o were very similar; our methods for purification of G s and G q have been described in detail (29,31). Briefly, the desired G␣ subunit was overexpressed in Sf9 insect cells, with ␤ 1 and ␥ 2 subunits engineered to have hexahistidine and FLAG tags at their N termini (33).…”

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.…”

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…α i2 and α i3 G-protein subunits were purchased from Calbiochem (La Jolla, CA) and were used without further purification. FLAG-his tagged βγ subunits (β 1 γ 2 or β 4 γ 2 ) were prepared and purified as described previously [13,20]. The subunits were stored at −80°C in 2-5µL volume aliquots depending on stock concentration (3-7µM).…”

“…The f/p ratios were ≈ 2:1 for both Texas Red and fluorescein labeled proteins (hereafter Gα i3 fl ). G protein βγ dimers were prepared and purified as previously described [20,23]. Beads coated with ≈ 4 million M2 antiFLAG antibodies were prepared as previously described [22].…”