Functional Pairing of Class B1 Ligand-GPCR in Cephalochordate Provides Evidence of the Origin of PTH and PACAP/Glucagon Receptor Family

Abstract

Several hypotheses have been proposed regarding the origin and evolution of the secretin family of peptides and receptors. However, identification of homologous ligand-receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand-receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates.

Keywords: GPCR; PACAP/glucagon; PTH; amphioxus.

Fig. 1.

Phylogenetic tree of bfPTHR-like and vertebrate PTHR, PACAP and Glucagon receptor families. The maximum likelihood tree was generated using Mega 6.06. The trees were calculated by maximum likelihood method with the JTT +I, +G, +F, model. One thousand bootstrap simulations were used to test the reliability of branching. The bfPTHR-like genes are highlighted in red boxes. CALCR and CRHR subfamilies were used as outgroups. Accession numbers of the sequences used are listed in supplementary table S3, Supplementary Material online. The uncompressed version of this phylogenetic tree is showed in supplementary figure S6, Supplementary Material online. Species abbreviation: coelacanth (co), elephant shark (el), lamprey (lp), tunicate (Tu) and Branchiostoma floridae (bf). Number of sites used in this phylogenetic reconstruction is 277.

Fig. 2.

Alignment of (A) bfPTH-like and bfPACAP/GLUCs with vertebrate PTH and (B) bfPTH-like and bfPACAP/GLUsC with human and zebrafish secretin peptide family. The alignment was generated using the ClustalW and displayed by BOXSHADE software (version 3.2, by ExPASy Bioinformatics Resource Portal, http://www.ch.embnet.org/). Black background indicates conserved residues, while the gray background indicates similar residues. bf, Branchiostoma florida; ef, elephant shark; hu, human; xe, Xenopus; ze, zebrafish.

Fig. 3.

Phylogenetic analysis of bfPTH-like and bfPACAP/GLUCs peptide precursors with vertebrate PTH, GCG and PACAP families. Full-length precursors amino acid sequences from vertebrates anole lizard (an), chicken (ch), elephant shark (el), human (hu), mouse (mo), Xenopus (xe), zebrafish (ze), and invertebrate Branchiostoma floridae (bf) were used to generate a maximum likelihood tree using Mega 6.06 (with JTT model and combined with +I, +G, +F). One thousand bootstrap simulations were used to test the reliability of branching. Vertebrate PACAP, GCG, and PTH subfamilies are highlighted in different colors. Predicted mature peptides within bfPTH1, bfPTH2, and bfPACAP/GLUCs precursors are labeled next to the gene names. Number of sites used in this phylogenetic reconstruction is 57.

Fig. 4.

cAMP response of bfPTHR-like receptors to various peptides. Intracellular cAMP accumulation in response to 100 nM amphioxus and vertebrate superfamily peptides was tested on CHO-K1 cells transiently transfected with (A) bf98C, (B) bf95, (C) bf98A, (D) bf98B, (E) bf173, and (F) pcDNA3.1, negative control. Peptide species: Branchiostoma floridae (bf), coelacanth (co), goldfish (gf), human (hu), sea lamprey (lp), and zebrafish (ze). Fold changes in cAMP were obtained by comparing with the control (no peptide treatment). The red lines represent the control value. Data are means ± SEM from at least three experiments performed in duplicate. All data were analyzed by one-way analysis of variance (ANOVA) and followed by Dunnett’s test using the computer software PRISM (GraphPad Software Inc., San Diego, CA). *, P ≤ 0.001 and #, P ≤ 0.05.

Fig. 5.

cAMP response of bf98C and bf95 to graded concentrations of peptides. Effects of (A) bfPTH-like, (B) vertebrate PTH and PTHrP, and (C) vertebrate PACAP or VIP on graded peptide concentrations assessed as intracellular cAMP accumulation in bf98C-expressing cells. (D) Measurement of intracellular cAMP elevation in CHO-K1 cells transiently expressing bf95 in response to graded concentrations of endogenous ligands, bfPACAP/GLUC peptides (bfPACAP/GLUCa, bfPACAP/GLUCb, and bfPACAP/GLUCc). Data are means ± SEM from at least three experiments performed in triplicate.

Fig. 6.

Synteny of PACAP/GCG and PTH peptide and receptor subfamily in amphioxus with spotted gar (Lepisosteus oculatus), Xenopus, and human. Chromosome numbers are on the right side of the indicated gene, and the gene locations (megabase) on the chromosome are shown on the left hand side. In vertebrates, paralogs (or orthologs) of each gene on different paralogons were aligned on the same row. Orthologous genes are labeled with green colors. PTH/PTHrP and PACPA/GLUC receptor family, including the putative amphioxus PTHR-like and PACAP/GLUC receptor, are highlighted in red. White boxes represent other class B1 GPCR. The GPCRs that missing in mammals are represented by white boxes with dash line. The table summarized all the connection of the orthologous genes between amphioxi and vertebrates. The strong connection between amphioxi and vertebrates syntenic loci implies that ancestral class B1 GPCRs were probably located on the same locus before the emergence of amphioxus.