Agnathan VIP, PACAP and their receptors: ancestral origins of today's highly diversified forms

Abstract

VIP and PACAP are pleiotropic peptides belonging to the secretin superfamily of brain-gut peptides and interact specifically with three receptors (VPAC(1), PAC(1) and VPAC(2)) from the class II B G protein-coupled receptor family. There is immense interest regarding their molecular evolution which is often described closely alongside gene and/or genome duplications. Despite the wide array of information available in various vertebrates and one invertebrate the tunicate, their evolutionary origins remain unresolved. Through searches of genome databases and molecular cloning techniques, the first lamprey VIP/PACAP ligands and VPAC receptors are identified from the Japanese lamprey. In addition, two VPAC receptors (VPACa/b) are identified from inshore hagfish and ligands predicted for sea lamprey. Phylogenetic analyses group these molecules into their respective PHI/VIP, PRP/PACAP and VPAC receptor families and show they resemble ancestral forms. Japanese lamprey VIP/PACAP peptides synthesized were tested with the hagfish VPAC receptors. hfVPACa transduces signal via both adenylyl cylase and phospholipase C pathways, whilst hfVPACb was only able to transduce through the calcium pathway. In contrast to the widespread distribution of VIP/PACAP ligands and receptors in many species, the agnathan PACAP and VPAC receptors were found almost exclusively in the brain. In situ hybridisation further showed their abundance throughout the brain. The range of VIP/PACAP ligands and receptors found are highly useful, providing a glimpse into the evolutionary events both at the structural and functional levels. Though representative of ancestral forms, the VIP/PACAP ligands in particular have retained high sequence conservation indicating the importance of their functions even early in vertebrate evolution. During these nascent stages, only two VPAC receptors are likely responsible for eliciting functions before evolving later into specific subtypes post-Agnatha. We also propose VIP and PACAP's first functions to predominate in the brain, evolving alongside the central nervous system, subsequently establishing peripheral functions.

Figure 1. Amino acid alignment of VIP and PACAP-27 peptide sequences and C-terminal processing sites from various chordates.

The amino acid alignment of (A) VIP and (B) PACAP-27 were generated using the default settings of Mega 5.0 software. Predicted sequences are denoted by “*” and retrieved from Ensembl or PreEnsembl online genome databases. The tribasic processing sites are boxed. Identical residues to that of human VIP and PACAP are denoted by “.”. Residues indicated important for selectivity and interaction based on mammalian studies are denoted by “–” , , , –.

Figure 2. Phylogenetic analysis of PHI/VIP and PRP/PACAP hormone precursors.

The tree was constructed by the PAM Matrix (Dayhoff) model by Maximum-Likelihood method, MEGA 5.0 software. Predicted sequences from the Ensembl genome database are denoted by “*”. The deduced sequences from this study are boldfaced. The numbers above each branch indicate the percentage of bootstrap replications in which that branch was found based on 500 replications. Proglucagon sequences were used as the outgroup.

Figure 3. Phylogenetic analysis of vertebrate VIP/PACAP receptors (VPAC₁, VPAC₂ and PAC₁).

The tree was constructed based on the PAM Matrix (Dayhoff) model by Maximum-Likelihood method, MEGA 5.0 software. The monophyletic groups are indicated on the right. Cloned receptor sequences from this study are boldfaced. Predicted sequences from the Ensembl genome database are denoted by “*”. The numbers above each branch indicate the percentage of bootstrap replications in which that branch was found based on 500 replications. Glucagon, GLP-1, GLP-2 and GIP receptor sequences were used as the outgroup.

Figure 4. Functional characterization of hfVPACa and hfVPACb.

Intracellular cAMP accumulation in response to 100 nM vertebrate superfamily peptides on COS-7 cells transiently transfected with (A) hfVPACa and (B) hfVPACb. Peptide species: slp, sea lamprey; gf, goldfish; h, human; o, ovine; zf, zebrafish; x, Xenopus; ct, catfish and cp, carp. Data represent the mean ± S.E.M. of at least 4 experiments performed in duplicates, p<0.01 is denoted by “*”. Effects of graded concentrations of (C) agnathan and (D) mammalian VIP and PACAP peptides on COS-7 cells transiently expressing hfVPACa. Data are expressed as the mean ± S.E.M. of at least 6 experiments performed in duplicates. Measurement of intracellular calcium elevation in CHO-K1 cells transiently expressing (E) hfVPACa and (F) hfVPACb in response to graded concentrations of sea lamprey PACAP. Data are expressed as the mean ± S.E.M. of at least 4 experiments. RFU, relative fluorescence units. (G) Shown are representative of confocal fluorescence images of CHO-K1 cells expressing (i) hfVPACa-pEYFP, (ii) hfVPACb-pEYFP and (iii) pEYFP-N1.

Figure 5. Transcript expression profile of agnathan VPAC receptors and PACAP.

Tissue distribution patterns of (A) hfVPACa and (B) hfVPACb; a relative abundance of 1 was set arbitrarily for the mRNA expressed in the brain. (C) The expression level of hfVPAC receptors in the hagfish brain was calculated from respective standard curves. (D) Tissue distribution pattern of jlpPACAP; a relative abundance of 1 was set arbitrarily for the mRNA expressed in the brain. Data are expressed as the mean ± S.E.M. of four experiments performed in duplicates.

Figure 6. In situ hybridization analysis of hfVPAC mRNAs in the brain of E.burgeri.

(A) Summary of relative abundance of hfVPACa and hfVPACb receptor expressions in various brain regions: High (+++), moderate (++), low (+), very low (–).The images and schematic diagrams show the distribution of hagfish mRNA in collateral sections of several brain regions (B–G). From left to right of the schematic diagrams, hfVPACa and hfVPACb images inclusive of their negative controls using a 1∶30 ratio of DIG-labeled anti-sense probe and unlabeled anti-sense probe and positive signals using specific complementary probes are shown. Fast green was used for counterstaining. Scale bars, 0.25mm for B and D; 0.15mm for C, E–G.

Figure 7. An evolutionary scheme of the VIP/PACAP ligands and receptors in vertebrates.

The boxes denote exons for the ligands and genes for the receptors. Unknown or unclear events are denoted by dotted lines or question marks. The phylogenetic timeline for the events are not to scale.