Though the heterotrimeric G-proteins signaling system is one of the best studied in eukaryotes, its provenance and its prevalence outside of model eukaryotes remains poorly understood. We utilized the wealth of sequence data from recently sequenced eukaryotic genomes to uncover robust G-protein signaling systems in several poorly studied eukaryotic lineages such as the parabasalids, heteroloboseans and stramenopiles. This indicated that the G alpha subunit is likely to have separated from the ARF-like GTPases prior to the last eukaryotic common ancestor. We systematically identified the structure and sequence features associated with this divergence and found that most of the neomorphic positions in Ga form a ring of residues centered on the nucleotide binding site, several of which are likely to be critical for interactions with the RCS domain for its GAP function. We also present evidence that in some of the potentially early branching eukaryotic lineages, like Trichontonas, G alpha is likely to function independently of the G beta gamma subunits. We were able to identify previously unknown G gamma subunits in Naegleria, suggesting that the trimeric version was already present by the time of the divergence of the heteroloboseans from the remaining eukaryotes. Evolution of G alpha subunits is dominated by several independent lineage-specific expansions (LSEs). In most of these cases there are concomitant, independent LSEs of RGS proteins along with an extraordinary diversification of their domain architectures. The diversity of RGS domains from Naegleria in particular, which has the largest complement of G alpha and RGS proteins for any eukaryote, provides new insights into RGS function and evolution. We uncovered a new class of soluble ligand receptors of bacterial origin with RGS domains and an extraordinary diversity of membrane-linked, redox-associated, adhesion-dependent and small molecule-induced G-protein signaling networks that evolved in early-branching eukaryotes, independently of parallel systems in animals. Furthermore, this newly characterized diversity of RCS domains helps in defining their ancestral conserved interfaces with G alpha and also those interfaces that are prone to extensive lineage-specific diversification and are thereby responsible for selectivity in G alpha-RCS interactions. Several mushrooms show LSEs of G alpha s but not of RGS proteins pointing to the probable differentiation of Gas in conjunction with mating-type diversity. When combined with the characterization of the 7TM receptors (GPCRs), it becomes apparent that, through much of eukaryotic evolution, cells contained both 7TM receptors that acted as GEFs and those as GAPs (with C-terminal RCS domains) for G alpha s. Only in some lineages like animals and stramenopiles the 7TM receptors were restricted to GEF only roles, probably due to selection imposed by the rate-constants of the G alpha s that underwent lineage-specific expansion in them. In the alveolate lineage the 7TM receptors occur independently of heterotrimeric G-proteins, suggesting the prevalence of G-protein-independent signaling in these organisms. Published by Elsevier B.V.
