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Identification of G Protein-Coupled Receptor Signaling Pathway Proteins

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Identification of G Protein-Coupled Receptor Signaling Pathway Proteins Port et al. BMC Genomics 2013, 14:503 http://www.biomedcentral.com/1471-2164/14/503 RESEARCH ARTICLE Open Access Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics Jesse A Port1, Micaela S Parker2, Robin B Kodner2, James C Wallace1, E Virginia Armbrust2 and Elaine M Faustman1* Abstract Background: The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. Results: The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. Conclusions: The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of evidence for some GPCR pathway proteins in one or more of the diatoms, such as the Gγ-subunit, may be due to differences in genome completeness and genome coverage for the four diatoms. The high divergence of putative diatom GPCR sequences to known class C GPCRs suggests these sequences may represent another, potentially ancestral, subfamily of class C GPCRs. Keywords: Cell signaling, Diatom, Environment, G protein-coupled receptor, Human health, Ocean * Correspondence: [email protected] 1Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, WA, USA Full list of author information is available at the end of the article © 2013 Port et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Port et al. BMC Genomics 2013, 14:503 Page 2 of 15 http://www.biomedcentral.com/1471-2164/14/503 Background cytosolic Ca2+ concentrations in the diatom Phaeodactylum The G protein-coupled receptor (GPCR) superfamily tricornutum [13]. The presence of a chemical-based defense represents one of the largest and most diverse families of system in P. tricornutum and Thalassiosira weissflogii has proteins in mammals and is found in nearly all multicellu- also been reported in which these diatoms respond to lar life [1,2]. These proteins are cell-surface receptors that challenge via diatom-derived aldehydes triggering Ca2+ and play a major role in signal transduction and perception of nitric oxide release [14]. This “stress surveillance system” and response to the environment. GPCRs are divided into may function in cell-cell communication across diatom five highly diverged families: Rhodopsin/class A, Secretin/ populations to detect damaged or stressed cells resulting class B, Adhesion/class B, Glutamate/class C and Frizzled/ from phytoplankton competitors and other ecological Taste2/class F [3]. GPCR sequences within these families or physical stressors. These findings are important when can share less than 25% identity between species [4]. considering environmental perception and response as GPCRs bind a diverse array of ligands including proteins, alterations in Ca2+ homeostasis are a hallmark of signal lipids, neurotransmitters, calcium, odorants, and other transduction activation throughout the eukaryotes [15]. small molecules [1]. In vertebrates, GPCR signaling Levels of the second messenger cAMP have also been networks are associated with neurotransmission, cellular shown to change in cultures of P. tricornutum following metabolism, secretion, cellular differentiation and growth, exposure to elevated carbon dioxide levels [16]. While inflammatory and immune responses, smell, taste and there is sequence evidence for putative GPCR signaling vision [5]. All GPCRs share a core seven transmembrane pathway proteins in the Thalassiosira pseudonana [17-19] α-helical region with an extracellular ligand binding and P. tricornutum [20] genomes, the role GPCR signaling domain that is coupled intracellularly to a G protein may play in regulating environmental perception and re- heterotrimer composed of α, β and γ subunits. GPCR sponse in diatoms warrants more detailed investigation. activation leads to the exchange of GDP for GTP by Here we use an in silico approach to probe the genomes a G protein, and G protein subunits then interact and of Pseudo-nitzschia multiseries [http://genome.jgi-psf.org/ regulate effector molecules (e.g. calcium, adenylyl cyclase, Psemu1/Psemu1.home.html], T. pseudonana [17], P. phospholipase C, phosphodiesterases, protein kinases), tricornutum [20] and Fragilariopsis cylindrus [http://gen- activating further downstream signaling pathways ome.jgi-psf.org/Fracy1/Fracy1.home.html] for translated nu- such as the mitogen-activated protein kinase (MAPK), cleotide sequences with similarity to known GPCR phosphoinositide-3 kinase (PI3K)-Akt and NF-kappaB signaling pathway proteins. We also probe expressed se- pathways that ultimately activate transcription factors quence tag (EST) libraries for each diatom to determine that affect gene expression and regulation [6,7]. Many whether these genomic sequences are actively expressed in of these scaffolding and signaling proteins mediate laboratory isolates. Our rationale for emphasizing sequence signal transduction in other intracellular pathways in comparisons between diatoms and higher eukaryotes is eukaryotes and thus are highly conserved. The importance three-fold. First, the GPCR signaling pathway is well- of these receptors is exemplified by the fact that 3-4% of characterized in mammals compared to less well-studied, human genes code for GPCRs and that nearly 30% of all non-model organisms and thus the functions of putative currently marketed drugs target these receptors [8]. homologs are better understood in this system. While Numerous endocrine and sensory-related diseases are model organisms such as yeast provide valuable insight associated with GPCR mutations in humans [9]. into potential GPCR signaling mechanisms in mam- Despite the crucial importance of GPCR signaling in malians, yeast and humans are found in the same metazoa, the prevalence and function of these proteins eukaryotic supergroup, and thus other unicellular systems in non-model organisms such as unicellular photosyn- with different evolutionary histories found outside this thetic eukaryotes is not well understood. Diatoms are a supergroup would allow for further comparative ana- major class of eukaryotic phytoplankton found through- lyses of GPCR signaling pathway diversity. Secondly, out the world’s oceans that play a crucial role in primary diatoms must rapidly sense and respond to multiple production and nutrient cycling and serve as a base for environmental changes, many of which are likely mediated marine food webs [10]. Diatoms are also responsible for by receptor-based signaling pathways. As major contribu- forming large phytoplankton blooms that in some cases tors to ocean productivity and carbon cycling, diatoms can be toxic to humans, marine mammals and seabirds may play a critical role in the changing ecosystems of the [11,12]. While the molecular mechanisms by which future ocean, and thus understanding the breadth of diatoms perceive and respond to their surrounding their ability to sense and respond to environmental environment have not been resolved, previous findings sug- changes may be crucial to predicting their future success. gest a role for cell surface receptors linked to intracellular Lastly, from a human health perspective, a better under- signaling pathways. For example, exposure to osmotic, standing of GPCR conservation and functionality in other shear or nutrient (iron) stress in culture leads to changes in organisms may provide further insight into the Port et al. BMC Genomics 2013, 14:503 Page 3 of 15 http://www.biomedcentral.com/1471-2164/14/503 importance of these receptors as extracellular or environ- 25-50% sequence identity to human GPCR pathway mental sensors
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