Functional phylogenetics reveals contributions of pleiotropic peptide action to ligand-receptor coevolution

The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of functions, a phenomenon known as pleiotropy. Here we examine whether pleiotropic actions of peptide genes might influence ligand-receptor coevolution. Four test groups of neuropeptides characterized by conserved C-terminal amino acid sequence motifs and their cognate receptors were examined in the red flour beetle (Tribolium castaneum): 1) cardioacceleratory peptide 2b (CAPA); CAPAr, 2) pyrokinin/diapause hormone (PK1/DH); PKr-A, -B, 3) pyrokinin/pheromone biosynthesis activating hormone (PK2/PBAN); PKr-C, and 4) ecdysis triggering hormone (ETH); ETHr-b. Ligand-receptor specificities were established through heterologous expression of receptors in cell-based assays for 9 endogenous ligands. Based on ligand-receptor specificity analysis, we found positive pleiotropism exhibited by ETH on ETHR-b and CAPAr, whereas PK1/DH and CAPA are more highly selective for their respective authentic receptors than would be predicted by phylogenetic analysis. Disparities between evolutionary trees deduced from receptor sequences vs. functional ligand-receptor specificities lead to the conclusion that pleiotropy exhibited by peptide genes influences ligand-receptor coevolution.