The G protein-coupled receptor (GPCR) superfamily represents one of the largest and most diverse families of proteins in mammals and is found in nearly all multicellular life
[1, 2]. These proteins are cell-surface receptors that play a major role in signal transduction and perception of and response to the environment. GPCRs are divided into five highly diverged families: Rhodopsin/class A, Secretin/class B, Adhesion/class B, Glutamate/class C and Frizzled/Taste2/class F
. GPCR sequences within these families can share less than 25% identity between species
. GPCRs bind a diverse array of ligands including proteins, lipids, neurotransmitters, calcium, odorants, and other small molecules
. In vertebrates, GPCR signaling networks are associated with neurotransmission, cellular metabolism, secretion, cellular differentiation and growth, inflammatory and immune responses, smell, taste and vision
. All GPCRs share a core seven transmembrane α-helical region with an extracellular ligand binding domain that is coupled intracellularly to a G protein heterotrimer composed of α, β and γ subunits. GPCR activation leads to the exchange of GDP for GTP by a G protein, and G protein subunits then interact and regulate effector molecules (e.g. calcium, adenylyl cyclase, phospholipase C, phosphodiesterases, protein kinases), activating further downstream signaling pathways such as the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase (PI3K)-Akt and NF-kappaB pathways that ultimately activate transcription factors that affect gene expression and regulation
[6, 7]. Many of these scaffolding and signaling proteins mediate signal transduction in other intracellular pathways in eukaryotes and thus are highly conserved. The importance of these receptors is exemplified by the fact that 3-4% of human genes code for GPCRs and that nearly 30% of all currently marketed drugs target these receptors
. Numerous endocrine and sensory-related diseases are associated with GPCR mutations in humans
Despite the crucial importance of GPCR signaling in metazoa, the prevalence and function of these proteins in non-model organisms such as unicellular photosynthetic eukaryotes is not well understood. Diatoms are a major class of eukaryotic phytoplankton found throughout the world’s oceans that play a crucial role in primary production and nutrient cycling and serve as a base for marine food webs
. Diatoms are also responsible for forming large phytoplankton blooms that in some cases can be toxic to humans, marine mammals and seabirds
[11, 12]. While the molecular mechanisms by which diatoms perceive and respond to their surrounding environment have not been resolved, previous findings suggest a role for cell surface receptors linked to intracellular signaling pathways. For example, exposure to osmotic, shear or nutrient (iron) stress in culture leads to changes in cytosolic Ca2+ concentrations in the diatom Phaeodactylum tricornutum. The presence of a chemical-based defense system in P. tricornutum and Thalassiosira weissflogii has also been reported in which these diatoms respond to challenge via diatom-derived aldehydes triggering Ca2+ and nitric oxide release
. This “stress surveillance system” may function in cell-cell communication across diatom populations to detect damaged or stressed cells resulting from phytoplankton competitors and other ecological or physical stressors. These findings are important when considering environmental perception and response as alterations in Ca2+ homeostasis are a hallmark of signal transduction activation throughout the eukaryotes
. Levels of the second messenger cAMP have also been shown to change in cultures of P. tricornutum following exposure to elevated carbon dioxide levels
. While there is sequence evidence for putative GPCR signaling pathway proteins in the Thalassiosira pseudonana[17–19] and P. tricornutum genomes, the role GPCR signaling may play in regulating environmental perception and response in diatoms warrants more detailed investigation.
Here we use an in silico approach to probe the genomes of Pseudo-nitzschia multiseries [http://genome.jgi-psf.org/Psemu1/Psemu1.home.html], T. pseudonana, P. tricornutum and Fragilariopsis cylindrus [http://genome.jgi-psf.org/Fracy1/Fracy1.home.html] for translated nucleotide sequences with similarity to known GPCR signaling pathway proteins. We also probe expressed sequence tag (EST) libraries for each diatom to determine whether these genomic sequences are actively expressed in laboratory isolates. Our rationale for emphasizing sequence comparisons between diatoms and higher eukaryotes is three-fold. First, the GPCR signaling pathway is well-characterized in mammals compared to less well-studied, non-model organisms and thus the functions of putative homologs are better understood in this system. While model organisms such as yeast provide valuable insight into potential GPCR signaling mechanisms in mammalians, yeast and humans are found in the same eukaryotic supergroup, and thus other unicellular systems with different evolutionary histories found outside this supergroup would allow for further comparative analyses of GPCR signaling pathway diversity. Secondly, diatoms must rapidly sense and respond to multiple environmental changes, many of which are likely mediated by receptor-based signaling pathways. As major contributors to ocean productivity and carbon cycling, diatoms may play a critical role in the changing ecosystems of the future ocean, and thus understanding the breadth of their ability to sense and respond to environmental changes may be crucial to predicting their future success. Lastly, from a human health perspective, a better understanding of GPCR conservation and functionality in other organisms may provide further insight into the importance of these receptors as extracellular or environmental sensors and as pharmacological and human disease relevant targets.
The goal of this study is thus to provide a comprehensive analysis of the GPCR signaling repertoire and its potential functionality in sequenced diatoms by using a suite of bioinformatic tools aimed at annotating the genomes of non-model organisms. We hypothesize that the conservation of this pathway in diatoms may reflect shared mechanisms of environmental response related to GPCR signaling across the eukaryotes.