Huang Y et al. (2019), Molecular characterization of G-protein-coupled...

ECB-ART-49887

PeerJ 2019 Jan 01;7:e8044. doi: 10.7717/peerj.8044.

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Molecular characterization of G-protein-coupled receptor (GPCR) and protein kinase A (PKA) cDNA in Perinereis aibuhitensis and expression during benzo(a)pyrene exposure.

Huang Y , Sun J , Han P , Zhao H , Wang M , Zhou Y , Yang D , Zhao H .

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BACKGROUND: G-protein-coupled receptors (GPCRs) are one of the most important molecules that transfer signals across the plasma membrane, and play central roles in physiological systems. The molecular architecture of GPCRs allows them to bind to diverse chemicals, including environmental contaminants. METHODS: To investigate the effects of benzo(a)pyrene (B(a)P) on GPCR signaling, GPCR and the protein kinase A (PKA) catalytic subunit of Perinereis aibuhitensis were cloned. The expression patterns of these two genes during B(a)P exposure were determined with real-time fluorescence quantitative PCR. The PKA content in P. aibuhitensis under B(a)P exposure was examined. RESULTS: The full-length cDNAs of PaGPCR and the PaPKA catalytic subunit were 1,514 and 2,662 nucleotides, respectively, encoding 338 and 350 amino acids, respectively. Multiple sequence alignments indicated that the deduced amino acid sequence of PaGPCR shared a low level of similarity with the orphan GPCRs of polychaetes and echinoderms, whereas PaPKA shared a high level of identify with the PKA catalytic subunits of other invertebrates. B(a)P exposure time-dependently elevated the expression of PaGPCR and PaPKA. The expression of both PaGPCR and PaPKA was also dose-dependent, except at a dose of 10 μg/L B(a)P. The PKA content in concentration group was elevated on day 4, with time prolonging the PKA content was down-regulated to control level. DISCUSSION: These results suggested that GPCR signaling in P. aibuhitensis was involved in the polychaete's response to environmental contaminants.

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	Figure 1. Nucleotide sequence and deduced amino acid sequence of GPCR from Perinereis aibuhitensis.Initiation codon (ATG) and termination codon (TAA) are highlighted in red boxes. The seven-transmembrane (7TM ) domains (TM I to TM VII) are underlined with red lines. The E/DRY and NPXXY motifs are in shadow.
	Figure 2. Multiple alignment analysis of PaGPCR with other GPCR protein.Amino acid residues that are conserved in at least of 50% sequence are shaded and similar amino acids are shaded in dark. The GenBank accession number for these proteins are as follows: (Platynereis dumerilii orphan G protein coupled receptor, 56AKQ63061.1; Strongylocentrotus purpuratus galanin receptor type 2-like, XP_003727596.1; Acanthaster planci galanin receptor type 2-like, XP022098630.1; Apostichopus japonicus putative galanin receptor type 2-like, PIK48567.1).
	Figure 3. Analysis of transmembrane region of PaGPCR.The whole sequence is labeled as inside (blue line) or out side (pink line), and the transmembrane region was labeled with red line.
	Figure 4. The three dimensional structure of PaGPCR.The helix is colored by blue, the sheet is colored by magenta, and the loop is colored by salmon.
	Figure 5. Nucleotide sequence and deduced amino acid sequence of PaPKA.Initiation codon (ATG) and termination codon (TGA) are highlighted in red boxes; conservative phosphorylation site, DFG triplet and APE motif are highlighted in green boxes; the glycine-rich loop GTGSFGRV (50–57aa), Ser/Thr active site RDLKPEN (165–171aa), PKA-regulatory-subunit-binding site LCGTPEY (198–204aa) are underlined with red.
	Figure 6. Multiple alignment of PaPKA with other PKA.Amino acid residues that are conserved in at least of 50% sequence are shaded and similar amino acids are shaded in dark. The GenBank accession number for these proteins are as follows: Aplysia califormica catalytic subunit of PKA, NP_001191420.1; Xenopus tropicalis cAMP depedent protein kinase catalytic subunit, NP_001164667.1; Branchiostoma floridae cAMP depedent protein kinase, XP_002600447.1; Danio rerio cAMP depedent protein kinase catalytic subunit, NP_001030148.1; Octopus bimaculoides cAMP depedent protein kinase catalytic subunit, XP_014777153.1; Lingula anatina cAMP depedent protein kinase catalytic subunit, XP_013409439.1; Crassostrea gigas cAMP depedent protein kinase catalytic subunit, XP_011439335.1; Biomphalaria glabrata cAMP depedent protein kinase catalytic subunit, XP_013072294.1; Salmo salar cAMP depedent protein kinase catalytic subunit, XP_014071121.1; Gallus gallus cAMP depedent protein kinase catalytic subunit, XP_015146370.1.
	Figure 7. The three dimensional structure of PKA from P. aibuhitensis.The helix is colored by blue, the sheet is colored by magenta, and the loop is colored by salmon, DFG triplet is labeled in magenta.
	Figure 8. Phylogenetic analysis of PaGPCR related to GPCR of other invertebrates and vertebrates.The information of other GPCR are same as the information in Fig. 2; the tree topologies were evaluated with 1,000 replicates.
	Figure 9. Phylogenetic analysis of PaPKA related to PKA of other invertebrates and vertebrates.The information of other PKA sequence are as the information in Fig. 6; the tree topologies were evaluated with 1,000 replicates.
	Figure 10. The relative expression level of PaGPCR and PaPKA cDNAs under various B(a)P concentration exposure.(A) represents PaGPCR, (B) represents PaPKA. Different lowercase letters indicate significant difference (P < 0.05). all data as mean + SD. N = four worms.
	Figure 11. The PKA content under various B(a)P concentration exposure.Different lowercase letters indicate significant difference (P < 0.05). All data as mean + SD. N = three worms.

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