1. Jon del Castillo1
  1. 1Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada L4A IK6
    2Department of Ecology and Evolution, University of Toronto, Toronto, Ontario, Canada
    3Department of Life Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada
  1. Correspondence should be addressed to D A Lovejoy; Email: david.lovejoy{at}
  1. Figure 1

    Human CRH paralogues. CRH and urocortin consist of one paralogous lineage, whereas urocortins 2 and 3 comprise the second paralogous lineage in chordates.

  2. Figure 2

    Scheme of the molecular interaction among CRH ligands, receptors and the binding protein in chordates and insects. See text for discussion.

  3. Figure 3

    A model for the CRH peptide and receptor co-evolution in chordates. A single ligand and receptor gene present in the protochordate genome was inherited by the early chordates. The first round of genome duplication led to the initial expansion of ligand and receptor into two paralogues. Over time, the receptors evolved into early R1 and R2 forms, whereas the two peptide genes evolved into either CRH/UI-like or Ucn2/Ucn3-like forms. The next genome duplication created the divergence of CRH-like peptides into CRH and UI/Ucn/SVG and Ucn2 and Ucn3 forms, whereas the receptors diverged into R1 and R2 forms. We postulate that the redundant R1 and R2 paralogues were subsequently lost and, therefore, only two CRH receptors were retained by modern chordates. CRH, corticotrophin-releasing hormone; UI, urotensin I; Ucn, urocortin. The black lines and arrows indicate the evolutionary direction and selection. Coloured arrows matching the ligand indicate the affinity for the receptors.

  4. Figure 4

    Major regions of CRH1 and CRH2 expression in rat brain. AM, amygdala; AN, arcuate nucleus; AO, accessory olfactory bulb; AP, anterior pituitary gland; BN, bed nucleus; CB, cerebellum; CP, caudate putamen; CT, cortex; DR, dorsal raphe nucleus; HF, hippocampal formation; IC, inferior colliculus; IP, intermediate lobe of the pituitary gland; OB, olfactory bulb; PB, parabrachial nucleus; PN, paraventricular nucleus; PO, pontine nuclei; SC, superior colliculus; SN, septal nucleus; ST, nucleus of the solitary tract; SU, substantia nigra; VMH, ventromedial hypothalamus; VT, ventral tegmental area.

  5. Figure 5

    Maximum-likelihood phylogeny of CRF-like proteins. CRH-like protein sequences were obtained via BLAST searches of the NCBI sequence database, aligned using ClustalW (Thompson et al. 1994, Larkin et al. 2007) and trimmed by hand to eliminate regions of uncertain alignment. The trimmed data set was then subjected to phylogenetic analyses using the maximum-likelihood and Bayesian methods (Guindon & Gascuel 2003, Ronquist & Huelsenbeck 2003, Guindon et al. 2010). The maximum-likelihood phylogenetic methods were implemented in the program PHYML 3.0 (Guindon & Gascuel 2003, Guindon et al. 2005), using the LG amino acid replacement matrix (Le & Gascuel 2008). For the likelihood analyses, node support was assessed using an approximate likelihood-ratio test (aLRT, Anisimova & Gascuel (2006)). Bayesian inference was performed in MrBayes 3.1.2, using a model that allows for jumping among fixed amino acid substitution rate matrices (Ronquist & Huelsenbeck 2003), with all of the protein sequence data in a single partition. Two Markov chain Monte Carlo runs were performed, with four chains each (three heated and one cold) for 1 million generations. Convergence was assessed using standard methods, including the average s.d. of split frequencies and the potential scale reduction factor (PSRF, Gelman & Rubin (1992)). The first 25% of trees sampled were discarded as burn-in, and the remaining trees were taken as representative of the posterior probability distribution (Fig. 4). Node is support indicated by likelihood aLRT values and Bayesian posterior probabilities (italics). CRH1 and CRH2 receptors cluster as discrete groups and represent together a sister lineage of the insect DH receptors. Species abbreviation, species name and accession name are indicated: ADOME: Acheta domesticus, Q16983.1; AAEGYI: Aedes aegypti, ABX57919.1; Ameiu: Ameiurus nebulosus, AAK01069; Anoli: Anolis carolinensis, XP0032211923; APISU A: Acyrthosiphon pisum, XP003244979.1; APISU B: Acyrthosiphon pisum, XP001944842.2; BMALA: Brugia malayi, XP 001899608.1; BMORI: Bombyx mori, XP004933474.1; Bos R1, R2: Bos taurus, NP776712 and NP001179474; BTERR: Bombus terrestris, XP003394723.1; Calli: Callithrix jacchus, XP002748148; CGIGA: Crassostrea gigas, EKC3340.1; CINTE: Ciona intestinalis, XP002123381.1; CQUIN: Culex quinquefasciatus, DAA06284.1; CSINE: Clonorchis sinensis, GAA51272.1; Danio: Danio rerio, XP696346; DMELA1, A2: Drosophila melanogaster, NP610960.1 and NP725175.3; DVIRIA, B: Drosophila virilis, XP002059297.1 and XP002050193.1; Gallu RI, R2: Gallus gallus, NP989652 and NP989785; Haplo: Haplochromis burtoni, ACV53954; ISCASP_ PU, _HP: Ixodes scapularis, XP002403968.1 and XP002403764.1; HomoR1, R2: Homo sapiens, NP001138618 and ABV59317; Macac: Macaca mulatta, EHH17404; MOCCI: Metaseiulus occidentalis, XP002123381.1; Monod R1, R2: Monodelphis domestica, XP001375959 and XM001373511.2; Mus: Mus musculus, Q60748; Muste: Mustela putorius furo, XP004762632; NLUGE: Nilaparvata lugens, CA625575.2; Octod: Octodon degus, XP00466578; Oncor: Oncorhynchus keta, CAC81754; Orcin R1, R2: Orcinus orca, XP004275734 and XP004269989; PHUMA; Pediculus humanus corporis, XP002424517.1; Ptero: Pteropus alecto, ELK12633; Rana: Rana catesbeiana, BAD36784; Rattu: Rattus norvegicus, NP112261; Salmi: Saimiri boliviensis boliviensis, XP003942463; SKOWA: Saccoglossus kowalevskii, NP001161520.1; SPURP: Strongylocentrotus purpuratus, XP790450.3; TCAST: Tribolium castaneum, NP001167548.1; TSPIR: Trichinella spiralis, XP003376880.1 and Tursi R1, R2: Tursiops truncatus, XP004318934 and XP004314441.

  6. Figure 6

    Evolution and structure–function relationships of CRH receptors. At the origin, only a single ligand, receptor and binding protein were present. Two rounds of gene expansion events in chordates led to the formation of four ligands, two receptors, but only a single binding protein.

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