Tracing the Origins of the Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP)

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) is a well-conserved neuropeptide characteristic of vertebrates. This pluripotent hypothalamic neuropeptide regulates neurotransmitter release, intestinal motility, metabolism, cell division/differentiation, and immunity. In vertebrates, PACAP has a specific receptor (PAC₁) but it can also activate the Vasoactive Intestinal Peptide receptors (VPAC₁ and VPAC₂). The evolution of the vertebrate PACAP ligand - receptor pair has been well-described. In contrast, the situation in invertebrates is much less clear. The PACAP ligand - receptor pair in invertebrates has mainly been studied using heterologous antibodies raised against mammalian peptides. A few partial PACAP cDNA clones sharing >87% aa identity with vertebrate PACAP have been isolated from a cnidarian, several protostomes and tunicates but no gene has been reported. Moreover, current evolutionary models of the peptide and receptors using molecular data from phylogenetically distinct invertebrate species (mostly nematodes and arthropods) suggests the PACAP ligand and receptors are exclusive to vertebrate genomes. A basal deuterostome, the cephalochordate amphioxus (Branchiostoma floridae), is the only invertebrate in which elements of a PACAP-like system exists but the peptides and receptor share relatively low sequence conservation with the vertebrate homolog system and are a hybrid with the vertebrate glucagon system. In this study, the evolution of the PACAP system is revisited taking advantage of the burgeoning sequence data (genome and transcriptomes) available for invertebrates to uncover clues about when it first appeared. The results suggest that elements of the PACAP system are absent from protozoans, non-bilaterians, and protostomes and they only emerged after the protostome-deuterostome divergence. PACAP and its receptors appeared in vertebrate genomes and they probably shared a common ancestral origin with the cephalochordate PACAP/GCG-like system which after the genome tetraploidization events that preceded the vertebrate radiation generated the PACAP ligand and receptor pair and also the other members of the Secretin family peptides and their receptors.

Keywords: deuterostomes; early metazoan; evolution; neuropeptide; protostomes; receptor.

FIGURE 1

Current evolutionary model for Secretin peptide family members. The exons of the peptide coding region are represented. PACAP-like family members are proposed to have evolved from a common ancestor exon early in the chordate radiation via exon and gene/chromosome duplication events (Sherwood et al., 2000; Cardoso et al., 2007a, 2010; Ng et al., 2012; Hwang et al., 2013). The same ancestral molecule is also suggested to originate the glucagon(GCG)-like peptides but for simplicity this is not represented in the figure. Boxes represent exons and lines introns and the peptide coding exons are indicated by the peptide abbreviation. Dashed lines indicate undefined evolutionary pathways. PACAP and PRP and VIP and PHI share the same gene precursor and GHRH and SCT are encoded by a single exon. The predicted position of each gene block in the Gnathostome Ancestral Genome (GAC) linkage groups are indicated (Hwang et al., 2013). The genome tetraploidization events (1R and 2R) are represented.

FIGURE 2

Sequence conservation of the invertebrate PACAP mature peptides. The mature sequence of the invertebrate peptides were extracted and compared with PACAP from the human (P18509) and two Salmonidae fish the river trout (Salmo trutta, XM_029756051.1) and the Siberian giant trout (Hucho taimen, HAGJ01147357.1) peptide homologs. The post-translational internal cleavage–amidation site (Gly²⁸-Lys²⁹-Arg³⁰) that generates the shortest peptide isoform (PACAP-27) in human, which is predicted in the other peptide sequences is underlined. The percentage of amino acid sequence identity (%ID) for the human (H), river trout (T), and Siberian giant trout (GT) mature PACAP-38 peptides is given. The vertebrate, hydra, protostome and invertebrate deuterostome mature peptide sequences were used to interrogate the protozoan, non-bilaterian, protostome and invertebrate deuterostome genomes and transcriptomes for homologs. Accession numbers of the non-vertebrate peptides are: cockroach (Periplaneta americana, AB083652), crab (Eriocheir japonica, AB121765), squid (Sepioteuthis lessoniana, AB083651), planarian (Dugesia japonica, AB083649), and hydra (Hydra magnipapillata, AB083650). The tunicate Chelyosoma productum (Chelyosoma-1 and Chelyosoma_2, were obtained from McRory and Sherwood, 1997) and the shrimp (Litopenaeus vannamei) from Lugo et al. (2013). Complete amino acid conservation is annotated with “*”, partial conservation with “.” and the position of the consensus amino acids conserved in the greatest number of sequences is indicated with “:”.

FIGURE 3

A dendrogram representing the phylogenetic relationship of non-bilaterian phyla. A partial PACAP cDNA was isolated from the Hydra magnipapillata. In other non-bilaterian phyla the existence of PACAP remains to be established (-). The evolutionary relationship between the species was based on (Brunet and King, 2017).

FIGURE 4

A dendrogram representing the phylogenetic relationships of the main Ecdysozoan phyla. Molecular and expression data (IHC) available for putative PACAP and VIP is presented (El-Salhy et al., 1983; Andries et al., 1984; Andriès et al., 1991). The tissues where putative PACAP or VIP were found are also indicated. - no data available; ^∗ partial cDNA.

FIGURE 5

A dendrogram representing the phylogenetic relationships of the main Lophotrochozoan phyla. Available molecular and expression data (IHC) for putative PACAP or VIP is presented (Reglödi et al., 2000; Somogyvári-Vigh et al., 2000; Hernádi et al., 2008; Boros et al., 2010; Pirger et al., 2010a). The tissues where putative PACAP or VIP were found are also indicated. - no data available; * partial cDNA; + heterologous antibody.

FIGURE 6

A dendrogram representing the phylogenetic relationship of the invertebrate deuterostome phyla. Available molecular and expression data (PCR and IHC) for putative PACAP and VIP is presented (McRory and Sherwood, 1997). The tissues where putative PACAP or VIP were found are indicated. - no data available; * full-length cDNA.

FIGURE 7

Multiple sequence alignment of the cephalochordate PACAP-like peptides. Comparison of the human mature peptide sequence with the predicted cephalochordate PACAP-like peptides. The percentage of identity (%ID) with the human mature PACAP-38 peptide is given. Complete residue conservation is annotated with a “*” and highlighted in bold, partially conserved residues are denoted by “.” and the position of the consensus amino acids present in the greatest number of sequences are indicated with “:”. Residues in the vertebrate peptides important for receptor activation are annotated in bold and italics.

FIGURE 8

Evolutionary relationships of the nematode and arthropod family B1 GPCRs with the human homologs. The six nematode and arthropod receptor subfamilies are represented in different colors and the human gene homologs are indicated. The phylogenetic tree was modified from Cardoso et al. (2014).

FIGURE 9

Proposed evolutionary model for the Ecdysozoans and Lophotrochozoan family B1 GPCRs. The main metazoan receptor subfamily gene clusters are represented by full colored circles according to their proposed common origin in the bilaterian ancestral genome. Four genes precursors for family B GPCR subfamilies arouse from gene duplication events in the bilaterian ancestral genome. These genes subsequently evolved under distinct evolutionary pressures in the protostome and deuterostome lineages (for more details see Cardoso et al., 2014). Species-specific gene duplications/deletions within each receptor family are not represented. The two rounds of genome duplication (1R and 2R) in the deuterostome radiation are represented. The phylogeny of Cluster B is represented in Supplementary Figure 1. The rest of the data was obtained from Cardoso et al. (2006; , unpublished; On et al., 2015). The figure was adapted from Cardoso et al. (2014) and is not drawn to scale.

FIGURE 10

Molecular data for PACAP and PACAP receptors in metazoan. The peptide is represented by pentagons and the receptors by circles. The major events associated with gene family evolution are indicated. In lamprey, a adcyap1 peptide gene precursor and adcyap1R1/vipr1 receptor gene were isolated (Ng et al., 2012). In the tunicate Chelyosoma productum two PACAP precursors (pacap1 and pacap2) were isolated but their existence remains to be confirmed, as do the PACAP precursors in the related species, Ciona intestinalis and in the scheme are represented by a dashed lined pentagon (McRory and Sherwood, 1997; Cardoso et al., 2010). The cephalochordate PACAP-like peptides and receptors are represented by striped pentagons and circles, respectively, as they are hybrids of the vertebrate PACAP/GCG peptide and receptor system (On et al., 2015). The genome duplication events that occurred earlier during the vertebrate radiation (1R, 2R) and the teleost specific (3R) are also annotated. The teleost peptide and receptor genes that resulted from 3R are represented by a and b. The major evolutionary events that explain existing molecular data are mapped with boxes with a flesh colored background. The main events associated with the appearance of the metazoan nervous system are also represented. The evolutionary relationship between the species represented was based on (Brunet and King, 2017). A cross (X) means not found in molecular databases (TSA and WGS) and likely to be absent in the species represented. ?- unknown existence.