Eukaryotic G protein signaling evolved to require G protein-coupled receptors for activation

Abstract

Although bioinformatic analysis of the increasing numbers of diverse genome sequences and amount of functional data has provided insight into the evolution of signaling networks, bioinformatics approaches have limited application for understanding the evolution of highly divergent protein families. We used biochemical analyses to determine the in vitro properties of selected divergent components of the heterotrimeric guanine nucleotide-binding protein (G protein) signaling network to investigate signaling network evolution. In animals, G proteins are activated by cell-surface seven-transmembrane (7TM) receptors, which are named G protein-coupled receptors (GPCRs) and function as guanine nucleotide exchange factors (GEFs). In contrast, the plant G protein is intrinsically active, and a 7TM protein terminates G protein activity by functioning as a guanosine triphosphatase-activating protein (GAP). We showed that ancient regulation of the G protein active state is GPCR-independent and "self-activating," a property that is maintained in Bikonts, one of the two fundamental evolutionary clades containing eukaryotes, whereas G proteins of the other clade, the Unikonts, evolved from being GEF-independent to being GEF-dependent. Self-activating G proteins near the base of the Eukaryota are controlled by 7TM-GAPs, suggesting that the ancestral regulator of G protein activation was a GAP-functioning receptor, not a GEF-functioning GPCR. Our findings indicate that the GPCR paradigm describes a recently evolved network architecture found in a relatively small group of Eukaryota and suggest that the evolution of signaling network architecture is constrained by the availability of molecules that control the activation state of nexus proteins.

Fig. 1. Distribution of G protein components among eukaryotes

(A) The indicated taxa are representative genomes. The presence of G protein elements in the indicated species or lineages is represented by red, blue, green, and yellow dots for genes encoding Gα, Gβγ, Opisthokont GPCRs, and RGS proteins, respectively. Lack of a dot signifies that those genes were not found. We organized the eukaryotes into six supergroups: Opisthokonta (containing C. owczarzaki and H. sapiens), Amoebozoa (containing D. discoideum), Archaeplastida (containing A. thaliana), Excavata (containing T. vaginalis), Chromalveolata (containing E. siliculosus), and Rhizaria. (B) Regulation of G protein activation in animals. Ligand-bound GPCR accelerates the dissociation of GDP from the G protein α subunit by changing the orientation of its helical domain. Gα hydrolyzes GTP, thereby inactivating itself. GTP hydrolysis is promoted by an RGS or other GAP protein. Nonreceptor GEFs, such as the protein Ric8 (resistance to inhibitors of cholinesterase), act as noncanonical and cytosolic GEFs. (C) Regulatory model of G protein signaling in Arabidopsis. The Arabidopsis Gα protein AtGPA1 rapidly releases its GDP as a result of spontaneous fluctuations between its Ras domain and helical domain. AtGPA1 slowly hydrolyzes its bound GTP; however, the membrane-localized 7TM-RGS protein AtRGS1 constitutively promotes GTP hydrolysis or acts as a GDI. (D) Frequency of TM helices in RGS domain–containing sequences among the 5169 sequences queried (see Materials and Methods). The TM helices were predicted with the membrane prediction software program SOSUI. (E) Distribution of 7TM-RGS proteins in Eukaryotes by a maximum-likelihood (ML) tree of 7TM-RGS proteins. Individual trees of the 7TM and RGS domains are shown in fig. S4 and were generated as described in Materials and Methods. The single genus Naegleria has 50 7TM-RGS proteins.

Fig. 2. Analysis of nucleotide exchange and hydrolysis by representative Gα subunits indicates that fast nucleotide exchange is an ancestral property

(A) Phylogeny showing the rates of nucleotide exchange and GTP hydrolysis of representative Gα subunits from each of the eukaryotic supergroups: T. vaginalis (Excavata), D. discoideum (Amoebozoa), C. owczarzaki (non-animal Opisthokonta), H. sapiens (Opisthokonta), and A. thaliana (Archae-plastida). See Fig. 1 and figs. S1 to S4 for the genes encoding GPCRs and 7TM-RGS proteins that are found in each clade. No information is available for Rhizaria because the genome was only recently released. Note that the origin of the nucleotide exchange–limited G cycle appears to have come after the split between the Amoebozoa and Opisthokonta and the Rhizaria, Chromalveolata, and Archaeplastida. The rates ± SE are computed from more than 12 data points shown in (B) to (F). A cutoff value of 1.0 in the k_obs/k_cat indicates the rate-limiting step. (B to F) Superimposed time courses of [³⁵S]GTPγS binding and single-turnover [γ-³²P]GTP hydrolysis in room temperature reactions containing 500 nM Gα proteins from C. owczarzaki, D. discoideum, A. thaliana, E. siliculosus, and H. sapiens. The [γ-³²P]GTP hydrolysis data from the H. sapiens Gα protein are presented as means ± SEM of four independent experiments. The [³⁵S]GTPγS binding data for T. vaginalis Gα proteins are presented as means ± SEM of three (TvGα1, TvGα2, and TvGα4) or seven (TvGα5) independent experiments. Nucleotide exchange and hydrolysis data for EsGα5, HsGα_i1, AtGPA1, and CoGα3 and hydrolysis data for TvGα5 are calculated from means of at least two replicates, and the variation is expressed as SD.

Fig. 3. Specific regulation of Gα subunits by a 7TM-RGS supports the notion of coevolution of Gα subunits with receptor GAPs

(A) Steady-state hydrolysis of GTP by the indicated wild-type T. vaginalis Gα (TvGα) subunits. Purified Gα subunits (250 or 500 nM) were incubated with 10 μM [γ-³²P]GTP for the indicated times before hydrolyzed ³²PO₄ was extracted with charcoal and quantified. All of the wild-type T. vaginalis Gα subunits displayed relatively slow rates of intrinsic hydrolysis. Data show the relative amounts of GTP hydrolyzed per mole of Gα subunit from two independent experiments. (B to F) Effects of RGS proteins on the GTP hydrolysis rates of TvGα subunits. The indicated Gα subunit (250 nM) and the indicated RGS proteins (500 nM) were incubated over a (B and D) 2-hour or (C and E) 3-hour period. Steady-state [γ-³²P]GTP hydrolysis rates were calculated for (B) TvGα1, (C) TvGα2, (D) TvGα4, and (E) TvGα5 in the presence of 500 nM RGS1, RGS2, or glutathione S-transferase (GST). (F) Summary. Enhancement of the GTPase activities of the indicated TvGα subunits by RGS1 or RGS2 relative to those in the presence of the GST control. GAP activity was only seen with RGS2 on TvGα5. *P < 0.01, analysis of variance (ANOVA) followed by Tukey’s test. All other combinations led to statistically insignificant changes in GTPase activity. All data are representative of at least two experiments. Error bars represent SD.

Fig. 4. Nucleotide binding kinetics of wild-type and chimeric Gα subunits support a monophyletic relationship for the property of fast nucleotide exchange

(A to D) Cartoon representations of wild-type and chimeric G proteins with the Ras and helical domains indicated: (A) the T. vaginalis Gα5 subunit structure (green), (B) the T. vaginalis TvGα5^{αi hel} chimera containing the T. vaginalis Ras (green) and human helical (red) domains, (C) the human chimera HsGα_i1^{α5 hel} containing the human Ras (red) and the T. vaginalis helical (green) domains, and (D) the human Gα_i1 subunit (all red). (E and F) Purified (E) TvGα5 and TvGα5^{αi hel} subunits (400 nM) and (F) HsGα_i1^{α5 hel} and HsGα_i1 subunits (400 nM) were incubated at 25°C before nonhydrolyzable GTPγS was added, and the change in intrinsic fluorescence was monitored over time. GTP binding manifests as a positive change in intrinsic fluorescence. (G) Purified TvGα5, HsGα_i1, or HsGα_i1^{α5 hel} chimeric proteins (500 nM) were incubated with the radio-nucleotide [³⁵S]GTPγS for the indicated times before bound radionucleotide was quantified. (H and I) Single-turnover GTP hydrolysis assays in which the indicated purified Gα proteins (800 nM) were preloaded with hydrolyzable [γ-³²P]GTP for the indicated times and then incubated in an excess of non-hydrolyzable GTPγS before charcoal extraction and quantification of ³²PO₄ were performed. (J) Steady-state GTP hydrolysis of chimeric proteins. The procedure followed was the same as that for the wild-type TvGα proteins in Fig. 3. Note that wild-type HsGα_i1, TvGα5, and TvGα5^{αi1 hel} displayed slow steady-state hydrolysis, whereas HsGα_i1^{α5 hel} displayed faster steady-state hydrolysis. All data are representative of at least two experiments. Error bars represent SD. GTPγS binding and GTP hydrolysis data for HsGα_i1 and TvGa5 are as shown in Fig. 2.