Molecular Basis for Interactions of G Protein βγ Subunits with Effectors

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Science  22 May 1998:
Vol. 280, Issue 5367, pp. 1271-1274
DOI: 10.1126/science.280.5367.1271


Both the α and βγ subunits of heterotrimeric guanine nucleotide–binding proteins (G proteins) communicate signals from receptors to effectors. Gβγ subunits can regulate a diverse array of effectors, including ion channels and enzymes. Gα subunits bound to guanine diphosphate (Gα-GDP) inhibit signal transduction through Gβγ subunits, suggesting a common interface on Gβγ subunits for Gα binding and effector interaction. The molecular basis for interaction of Gβγ with effectors was characterized by mutational analysis of Gβ residues that make contact with Gα-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-β2, and β-adrenergic receptor kinase revealed the Gβ residues required for activation of each effector and provides evidence for partially overlapping domains on Gβ for regulation of these effectors. This organization of interaction regions on Gβ for different effectors and Gα explains why subunit dissociation is crucial for signal transmission through Gβγ subunits.

Upon receptor activation, G proteins dissociate into free Gα and Gβγ subunits that can activate various effectors (1). Effector proteins of the Gβγ complex include phospholipases (2), adenylyl cyclases (3), ion channels (4), G protein–coupled receptor kinases (5) and phosphoinositide 3-kinases (6). Other potential Gβγ effectors include dynamin I and the nonreceptor protein tyrosine kinases Btk and Tsk (7). GDP-bound Gα subunits (Gα-GDP) can compete with Gβγ effectors and deactivate Gβγ-dependent signaling, suggesting that Gβγ may use a common binding surface for interaction with Gα and with its diverse effectors. Two regions on Gβγ that interact with Gα have been defined by the crystal structures of heterotrimeric Gαβγ (8), the switch interface (Gβ residues 57, 59, 98, 99, 101, 117, 119, 143, 186, 228, and 332) and the NH2-terminal interface (Gβ residues 55, 78, 80 and 89). Each of these residues on retinal Gβ (Gβ1) was substituted with alanine, and each Gβ1 mutant was expressed with either Gγ1 or Gγ2, two isoforms of the Gγ subunit. All mutated Gβ1γ1 dimers were folded properly, were post-translationally modified appropriately, and were expressed at similar amounts as in the wild type (9). The Gβγ mutants were tested for their ability to assemble into heterotrimers with Gα, to be activated by rhodopsin, and to interact with Gβγ downstream signaling partners: β-adrenergic receptor kinase (βARK), phospholipase C-β2 (PLC-β2), adenylyl cyclase 2 (AC2), muscarinic potassium channel (GIRK1/GIRK4), and the calcium channel α1B subunit (CCα1B).

To determine whether purified Gβ1H6γ1 mutants could form heterotrimers, we measured the ability of the Gβγ mutants to facilitate pertussis toxin–catalyzed adenosine diphosphate (ADP) ribosylation of transducin Gα-GDP (Gtα) (10). All mutants could support some level of ADP ribosylation, although Gβ mutants Ile80 → Ala80 (I80A), K89A, L117A, and W332A (11) showed reduced ability to form heterotrimers (Fig.1A).

Figure 1

Effects of Gβ1H6γ1 on heterotrimer assembly and receptor interaction. The data are the normalized percentage of wild-type (WT) recombinant Gβγ activity. (A) The ability of recombinant Gβ1H6γ1 and Gβ1 mutants to assemble into heterotrimers with Gtα was determined by testing whether pertussis toxin could ADP-ribosylate Gtα with [32P]nicotinamide adenine dinucleotide (10). The Gβ residue mutated to alanine is indicated by a number beneath each bar in the figure. Clear bar (C) represents the basal amount of ADP-ribosylation of Gtα that occurred in absence of Gβγ. (B) The ability of recombinant Gβ1H6γ1 and Gβ1 mutants to bind Gtα and interact with rhodopsin was determined by the amount of [35S]GTP-γ-S binding catalyzed by light-activated rhodopsin (12). Clear bar (C) is the basal amount of [35S]GTP-γ-S binding to Gtα in the presence of urea-washed rod outer segment membranes (50 nM) without added Gβγ. The data represent the mean ± SEM of duplicate determinations in three independent experiments. Alanine mutants that have a distinguishable activity from the wild type are indicated by gray bars; those with activity similar to the wild type are indicated by black bars (11).

Because Gβγ is essential for functional heterotrimer interaction with activated receptors that catalyze the exchange of GDP for guanosine triphosphate (GTP) on the Gα subunit, we also measured the ability of the Gβ mutants to support light-activated rhodopsin-catalyzed nucleotide exchange on the α subunit of transducin (Gtα) (12). All switch interface mutants (except K57A and Y59A) and the NH2-terminal interface mutants I80A and K89A were defective in formation of functional heterotrimers (Fig. 1B). Some Gβ mutants were impaired in both assays, indicating that residues 80, 89, 117, and 332 of Gβ are the major determinants of binding to Gtα. The switch interface mutants (S98A, W99A, M101A, N143A, and D186A) were normal in heterotrimer assembly, but were impaired functionally in supporting receptor-catalyzed nucleotide exchange on Gtα. This observation indicates that Gβγ may actively participate in receptor-catalyzed nucleotide exchange, rather than being simply a passive binding partner in receptor–G protein interactions.

Gβγ mediates translocation of G protein–coupled receptor kinases from the cytosol to the membrane, in order that these kinases can phosphorylate activated G protein–coupled receptors and initiate receptor internalization (13). The Gβ mutants varied in their ability to associate with βARK1 (Fig.2A) (14). Alanine mutations at Gβ residues 117 and 143 resulted in decreased binding to βARK1. In contrast, alanine mutations at Gβ residues 57, 59, 89, 186, and 332 of Gβ led to increased binding to βARK1. The mutations that resulted in decreased binding are found on the left side of the Gβ surface (Fig. 3) and likely form the βARK binding interface, whereas those mutations that led to increased binding were clustered together at the middle of the structure (Gβ residues 57, 59, and 332) or are at the right side of the surface (Gβ residue 89).

Figure 2

Gβγ-dependent interactions with βARK, PLC-β2, AC2, muscarinic potassium channel, and CCα1B-containing calcium channel. (A) The amount of Gβ1 present in βARK immune complexes was detected as described (14). Light orange bars show those mutations that decrease Gβγ/βARK interaction, while dark orange bars show mutations that increase the interaction. The data contained in the bar graph represent duplicate determinations in two independent experiments. (B) Gβ1γ1-dependent activation of PLC-β2 was determined as described (17). Clear bar (C) represents the basal PLC-β2 activity in the absence of Gβγ. Light purple bars show those mutations that decrease Gβγ-mediated PLCβ2 activation, while dark purple bars indicate mutations that determine enhanced activation. The data are presented as the normalized percentage of wild-type recombinant Gβγ activity and represent the mean ± SEM of duplicate determinations in three independent experiments. (C) Gβ1γ2-dependent activation of AC2 was determined by reconstituting membranes as described (18). Data represent duplicate determinations from two independent experiments. (D) Gβ1γ2-dependent activation of GIRK potassium channel was determined as described (19). Protein immunoblotting showed that all mutants were expressed in equal amounts. Clear bar (C) represents control oocytes injected with GIRK1, GIRK4, and Gγ2 RNAs. (E) Gβ1γ2-dependent inhibition of CCα1B calcium channels was determined as described (21). Protein immunoblotting revealed that all mutants were expressed in similar amounts. The amount of Gβγ-dependent inhibition was calculated as a ratio of the mean prepulse facilitation (MPF) in either the absence or presence of Gβγ. MPF is the statistically averaged relief of the channel inhibition by use of a large depolarizing prepulse. Clear bar (C) represents channel activity in absence of Gβγ. The light yellow bars indicate the Gβ1 mutations that decrease channel modulation and lead to calcium currents that are statistically indistinguishable from the basal calcium current (P < 0.1 for K78A and W332A mutants and P < 0.01 for M101A, N119A, T143A, and D186A mutants). The dark yellow bars indicate Gβ1 mutants that have increased inhibitory activity (P < 0.001 for L55A mutant and P < 0.01 for I80A mutant).

Figure 3

A schematic representation of the regions of Gβ involved in interactions with effectors and Gα subunit. The crystal coordinates of Gβ1γ1 [Protein Databank entry 1tbg; (8, 26)] were used to generate a surface model of the dimer in GRASP. Gβ is gray, and Gγ is pink. The pale green surface is the area on Gβγ that is covered by Gα in the G protein heterotrimer crystal structure. The effector-interacting residues on Gβ are circled with a different color for each effector: orange, βARK; magenta, PLC-β2; teal, AC2; blue, potassium channel; and yellow, calcium channel. Gα-GDP, when bound to Gβγ, covers all these distinct yet partially overlapping effector interaction regions on Gβ and, thus, blocks Gβγ regulation of all the effectors. This figure and other more detailed figures showing residues interacting with individual effectors can be viewed at

Gβγ is an important modulator of various isoforms of phospholipase C–β (2, 15) and adenylyl cyclase (16), effectors that regulate intracellular concentration of second messengers inositol 1,4,5-triphosphate and cyclic adenosine 3′,5′-monophosphate. The ability of the Gβγ mutants to stimulate the activity of PLC-β2 was determined by quantitating the amount of inositol 1,4,5-triphosphate produced by the purified enzyme in the presence of the Gβγ mutants (17). Some 13 of the 15 Gα-interacting residues of Gβ we tested were important for Gβγ-dependent activation of PLC-β2, suggesting that Gα and PLC-β binding regions on Gβ are overlapping (Fig. 2B). Mutants W99A and D228A no longer activated PLC-β2 and mutants I80A, K89A, M101A, L117A, N119A, T143A, D186A, and W332A were less effective than wild-type Gβγ. Mutants L55A and S98A activated PLC-β2 to a greater extent than wild-type Gβγ. These residues are circled in magenta on the Gβγ surface (Fig. 3). The effects of the Gβγ mutants on AC2 activation were determined in vitro (18) in the presence of constitutively activated Gsα that has glutamine at residue 227 mutated to leucine (Q227L). All the Ala mutations of Gβ residues, except I80A and T143A, had decreased ability to activate AC2 (Fig. 2C); their locations on the Gβγ structure are indicated in teal (Fig. 3).

We also measured K+ currents in Xenopus laevisoocytes injected with RNAs for GIRK1/GIRK4 and Gβγ mutants (19). The ability of Gβγ to increase conductance through the muscarinic potassium channel GIRK1/GIRK4 was disrupted by alanine mutations at Gβ residues 55, 78, 80, 89, 99, and 228 (Fig. 2D). All these Gβ residues except W99 and D228 cluster within the NH2-terminal interface of Gβ (Fig. 3; blue lines).

Gβγ inhibits the activity of certain calcium channels (20). We measured the ability of Gβγ mutants to inhibit the conductance of Ca2+ channels in HEK 293 cells expressing CCα1B-containing Ca2+ channels and Gβγ mutants (21). Alanine mutations of Gβ residues 55 and 80, which lie close together at the top of Gβ, had enhanced ability to inhibit current through CCα1B-containing Ca2+ channels (Fig. 2E). Alanine mutations of Gβ residues 78, 101, 119, 143, 186, and 332 were no longer able to inhibit current through calcium channels.

Our results demonstrate that many of the Gα-interacting residues of Gβ are important in interactions between Gβγ and its signaling partners, and show the functional importance of individual amino acids in the signal transfer from Gβγ to effector activation. Alanine mutation of these Gβ residues may increase, decrease, or abolish Gβγ-dependent interactions. It was unexpected to find Gβγ mutants that are better than the wild type at stimulating the activity of PLC-β2 and inhibiting Ca2+ channels. One possible reason for such “gain-of-function” mutations is that turn-off of Gβγ-mediated signaling requires Gα-GDP competition with effectors. The normal side chains at these positions may be destabilizing to effector interaction resulting in a lower affinity in order to allow reassembly of heterotrimeric Gαβγ.

Each signaling partner for Gβ relies on a different subset of Gβ residues for its interaction and hence, creates a set of unique “footprints” on Gβ (Fig. 3). These results are consistent with studies that have suggested a common effector binding surface on Gβγ located near the region of residues 70 to 145 of Gβ (22). These data raise an interesting issue of how G proteins and their effectors are oriented with respect to the membrane and whether their orientations change during subunit dissociation and activation (8, 23). The Gα-binding surface on Gβγ may not be the only region of effector interaction. Other Gβγ regions of effector interactions that have been implicated are the coiled-coil interface at the NH2-termini of Gβ and Gγ (24) and the COOH-terminal region of Gβ (25).

The alanine mutations of Gα-interacting Gβ residues provide an initial framework to determine how Gβγ subunits interact with and regulate so many different effectors that have so little structural similarity. Our studies show that the effector interaction regions are clustered on Gβ such that they partially overlap one another (Fig.3). This mode of clustering allows for one key regulator (Gα) to regulate Gβγ signal transmission to multiple effectors.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: h-hamm{at}


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