Elsevier

Cellular Signalling

Volume 21, Issue 5, May 2009, Pages 737-744
Cellular Signalling

3 forms distinct dimers with specific Gγ subunits and preferentially activates the β3 isoform of phospholipase C

https://doi.org/10.1016/j.cellsig.2009.01.018Get rights and content

Abstract

Heterotrimeric G proteins regulate multiple effectors of which some are mediated via the Gβγ dimers. There is evidence to suggest that the functions of Gβγ dimers are not shared by all possible permutations of Gβγ complexes. Here, we report our efforts in defining the formation of distinct Gβγ dimers and their functional differences in activating phospholipase Cβ (PLCβ) isoforms. Co-immunoprecipitation assays using Cos-7 cells transiently expressing 48 different combinations of Gβ(1–4) and Gγ(1–5, 7–13) subunits showed that Gβ1 and Gβ4 could form dimers with all known Gγ subunits, whereas several dimers could not be observed for Gβ2 and Gβ3. All Gβ1γ and Gβ2γ dimers significantly stimulated PLCβ isoforms (PLCβ2  PLCβ3 > PLCβ1), but Gβ3γ and Gβ4γ dimers were poor activators of PLCβ1 and exhibited preference for PLCβ3 and PLCβ2, respectively. All Gβ subunits revealed to date contain the previously identified PLCβ2-interacting residues, but their neighboring residues in the proposed 3-D structures are different. To test if differences in these neighboring residues affect the interactions with PLCβ isoforms, we generated several Gβ3 mutants by replacing one or more of these residues with their Gβ1 counterparts. One of these mutants (M120I, S140A and A141G triple mutant) acquired enhanced PLCβ2-activating functions when co-expressed with different Gγ subunits, while the corresponding stimulation on PLCβ3 was not altered. Taken together, our results show that the exact composition of a Gβγ dimer can determine its selectivity for activating PLCβ isoforms, and certain residues in Gβ3 may account for the preferential stimulation of PLCβ3 by Gβ3γ dimers.

Introduction

Signal transduction by G protein-coupled receptors (GPCRs) is primarily mediated through the actions of Gα and Gβγ subunits. The molecular mechanism by which Gαβγ heterotrimers transduce signals to their downstream effectors is well established [1]. However, our understanding on how signaling specificity by individual GPCR is achieved remains rudimentary. Accumulating evidences have suggested that GPCR signaling specificity is determined by distinct combinations of Gα, Gβ and Gγ subunits. By using antisense oligonucleotides against different G protein subunits, specificities of SST2 and M4 receptors for Gαo2β3γ3 and Gαo1β1γ4, respectively, to inhibit L-type Ca2+ channels have been demonstrated [2]. At least 20 Gα, 6 Gβ and 12 Gγ subunits have been identified to date [3], thereby giving rise to a host of distinct combinations of Gαβγ heterotrimers. Although not all theoretical combinations of Gαβγ subunits can be formed in vivo [4], the shear number of different possible Gαβγ heterotrimers poses a formidable barrier in discerning discrete functions of G protein subunits. Functional variations of the same GPCR between different cell types may arise from the differential availability of distinct Gαβγ heterotrimers, as well as the expression of effector molecules.

Numerous studies have been directed at delineating the Gα-mediated regulation of adenylyl cyclase (AC) and phospholipase Cβ (PLCβ) [1]. Despite their widespread involvement in different signaling events [5], much less are known with regard to the functional specifications of Gβγ subunits. The tightly associated Gβγ complex is an obligatory requirement for receptor activation of G proteins [6] and recombinant Gβγ dimers have been shown to directly modulate the functions of PLCβ [7], PLCη [8], ACs [9], G protein-gated K+ channels [10] and Ca2+ channels [11].

It has been proposed that both Gβ [12] and Gγ subunits [13] are important for determining the efficacy of Gβγ dimer formation. Interestingly, different Gβ and Gγ subunits have varying affinities for one another in dimer formation [14]. Early studies have shown that Gβ2 and Gγ1 do not dimerize with each other, whereas Gβ1 and Gγ2 can form a dimer readily [15], [16]. Dimerization studies using in vitro translated Gβ and Gγ subunits have been performed [12]. However, the exact combinations of Gβγ subunits that exist in mammalian tissues have not yet been fully characterized. Gβ(1–4) subunits are co-expressed in various tissues. In contrast, the distributions of Gγ subunits are highly diverse [17]. To fully comprehend the contribution of Gβγ-dependent signals in mammalian cells, it is critical to define the possible combinations of Gβγ dimers.

Since GPCR-mediated activation of G proteins concomitantly releases the Gβγ dimer, and because many pathways are regulated by Gβγ dimers [3], it is important to establish if different Gβγ dimers share the same ability to modulate the various effectors or have unique signaling capabilities. There is indeed evidence to suggest that specific Gβγ combinations can regulate distinct effectors. It has been demonstrated that Gβ2γ2 stimulates both PLCβ2 and c-Jun N-terminal kinases, while Gβ5γ2 activates the former only [18]. Although the activities of all four isozymes of PLCβ (PLCβ1–4) are stimulated in the presence of GTP-bound Gα subunits of the Gq family, only PLCβ1–3 are Gβγ-responsive [19]. Purified Gβγ dimers have been shown to activate PLCβ isozymes in the order of PLCβ3  PLCβ2 > PLCβ1 in vitro [7], and many Gβγ dimers (e.g., Gβ1γ1–3 and Gβ2γ2) share the ability to stimulate PLCβ2 and PLCβ3 [15]. However, not all Gβγ dimers are expected to stimulate PLCβ isoforms effectively because otherwise activation of any G protein will result in an increase of inositol trisphosphate production.

In this report, we determined the formation of Gβγ dimers by co-immunoprecipitation of 48 possible combinations of Gβγ complexes from Cos-7 cells transiently co-expressing Gβ(1–4) and Gγ(1–5, 7–13). The functionality of individual Gβγ dimers was assessed by their ability to stimulate PLCβ(1–3) isozymes, certain residues which may determine preferential activation of PLCβ3 by Gβ3γ dimers have also been mapped. Our results support the notion that different permutations of Gβγ dimers can differentially activate PLCβ isozymes, and thus contribute towards the diversity of GPCR signaling in various cellular environments.

Section snippets

Materials

Cos-7 kidney fibroblasts were purchased from American Type Culture Collection (Manassas, VA). The cDNAs encoding PLCβ1, PLCβ2 and PLCβ3 were obtained from Dr. Richard Ye (University of Illinois at Chicago). Flag-tagged human Gβ1, Gβ2, Gβ3, Gβ4 and HA-tagged human Gγ1, Gγ2, Gγ3, Gγ4, Gγ5, Gγ7, Gγ8, Gγ9, Gγ10, Gγ11, Gγ12 and Gγ13 cDNA constructs were obtained from UMR cDNA Resource Center (Rolla, MO), while cDNAs encoding Gβ and Gγ subunits (without Flag-/HA-tag) were obtained from the same

Formation of various Gβγ dimers in Cos-7 cells

Much effort has been directed to investigate if in vitro translated Gβ and Gγ subunits are capable of forming functional dimers [12], [21]. These studies provide insights for the possible interactions between individual Gβ and Gγ subunits in vitro. Since certain intracellular functions (e.g. proteins governing selective assembly) may only be retained in intact cells [22], we transiently expressed different combinations of Gβ and Gγ subunits in Cos-7 cells and then assessed Gβγ dimer formations

Discussion

Of the 6 Gβ subunits (Gβ1–5 and Gβ5L) identified to date, Gβ1 to Gβ4 are similar in terms of their primary sequences, whereas Gβ5 and Gβ5L show less homology with the others [17]. It has been suggested that different Gβ and Gγ subunits may have varying affinities for one another. The Gβ subunit is folded into a propeller-like structure containing seven WD motifs [26], while the Gγ subunit is plastered on one side of the toroidal structure of the Gβ subunit, with the N-terminal of Gβ forming a

Acknowledgements

We thank the kind donation of various cDNAs of PLCβ isoforms from Dr. Richard Ye (University of Illinois at Chicago). We also thank Dr. Maurice Ho, Winnie Lau and Wendy Yeung for the technical support and helpful discussion.

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    This work was supported in part by the Research Grants Council of Hong Kong (HKUST 660107), the University Grants Committee (AoE/B-15/01), and the Hong Kong Jockey Club. YHW was a recipient of the Croucher Senior Research Fellowship.

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