The Ancient Link between G-Protein-Coupled Receptors and C-Terminal Phospholipid Kinase Domains

Abstract

Sensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling. Heterotrimeric G-protein subunits activated by G-protein-coupled receptors (GPCRs) are typical stimulators of phospholipid signaling enzymes such as phosphatidylinositol phosphate kinases (PIPKs) or phospholipase C (PLC). However, a direct connection between the two pathways likely exists in oomycetes and slime molds, as they possess a unique class of GPCRs that have a PIPK as an accessory domain. In principle, these so-called GPCR-PIPKs have the capacity of perceiving an external signal (via the GPCR domain) that, via PIPK, directly activates downstream phospholipid signaling. Here we reveal the sporadic occurrence of GPCR-PIPKs in all eukaryotic supergroups, except for plants. Notably, all species having GPCR-PIPKs are unicellular microorganisms that favor aquatic environments. Phylogenetic analysis revealed that GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. In addition to GPCR-PIPKs, we identified five hitherto-unknown classes of GPCRs with accessory domains, four of which are universal players in signal transduction. Similarly to GPCR-PIPKs, this enables a direct coupling between extracellular sensing and downstream signaling. Overall, our findings point to an ancestral signaling system in eukaryotes where GPCR-mediated sensing is directly linked to downstream responses.IMPORTANCE G-protein-coupled receptors (GPCRs) are central sensors that activate eukaryotic signaling and are the primary targets of human drugs. In this report, we provide evidence for the widespread though limited presence of a novel class of GPCRs in a variety of unicellular eukaryotes. These include free-living organisms and organisms that are pathogenic for plants, animals, and humans. The novel GPCRs have a C-terminal phospholipid kinase domain, pointing to a direct link between sensing external signals via GPCRs and downstream intracellular phospholipid signaling. Genes encoding these receptors were likely present in the last common eukaryotic ancestor and were lost during the evolution of higher eukaryotes. We further describe five other types of GPCRs with a catalytic accessory domain, the so-called GPCR-bigrams, four of which may potentially have a role in signaling. These findings shed new light onto signal transduction in microorganisms and provide evidence for alternative eukaryotic signaling pathways.

Keywords: G-protein-coupled receptors; Phytophthora; cell signaling; oomycetes; phospholipid-mediated signaling.

FIG 1

Consensus cladogram of selected eukaryotes, with lineages and genera with species having one or more GPCR-PIPKs highlighted in bold. The size of the circles is proportional to the average number of GPCR-PIPKs per taxon. The tree is based on phylogenies described by Keeling et al. and Koonin (67, 68), and the placement of the Apusozoa is based on data reported by Paps et al. (69). Dashed polytomies indicate unresolved relationships.

FIG 2

Degree of conservation of all GPCR-PIPKs included in this study (top) and sequence logo of the newly identified LRxGI motif (bottom). The degree of conservation was determined by the use of a sliding moving average over 50 positions. The approximate positions of three conserved PIPK motifs (DLGKS, MDYSL, and KK) are indicated. Color coding of amino acid residues in the sequence logo is shown according to chemical properties as follows: red, acidic; blue, basic; black, hydrophobic; purple, neutral; green, polar.

FIG 3

(a) Typical domain organization in the four PIPK types. The regions contained in the dashed box were used for constructing the tree. Protein and domain lengths are not to scale. (b) Phylogenetic tree of type I, II, III, and IV PIPKs. The outer circle shows the classification of PIPKs based on domain composition, with orange representing type I, II, and III PIPKs and lime type IV PIPKs (i.e., GPCR-PIPKs). Split-color bars indicate truncated gene models. The color coding of the species names corresponds to the coding of the supergroup colors described for Fig. 1. The numbers at the nodes represent bootstrap support percentages from RAxML (500 replicates); bootstrap values of <50 are omitted. Clades encompassing oomycete proteins are collapsed and classified based on homology with P. infestans PIPK classes (16). Dashed lines are truncated for simplification and indicate arbitrary branch lengths. A tree with noncollapsed clades that includes codes of the gene models is provided in Fig. S2.

FIG 4

Schematic representation of the domain organization of GPCR-bigrams in oomycetes and their predicted catalytic activities. Blue cylinders represent transmembrane helices. PIPK, phosphatidylinositol-4-phosphate 5-kinase; INPP, inositol polyphosphate phosphatase; PIP, phosphatidylinositol; AC, adenylyl cyclase; PDE, phosphodiesterase; DEP, Dishevelled, Egl-10, and pleckstrin; AP, aspartic protease.