OX-A and OX-B are encoded by the HCRT gene. Structures of the human gene [from UCSB genome browser (http://genome.ucsc.edu); intronic sequence is shown at 1/10th scale of exon sequence], mRNA, and protein gene products shown. IC₅₀ values for radioligand binding by OX-A and OX-B are depicted with the exception of the affinity of OX-B for OX₁ receptors (420 nM, not shown), which is ∼10-fold less than for OX₂ receptor (36 nM) (Sakurai et al., 1998).

Amino acid divergence in the structures of OX₁ and OX₂ receptors among human, rat, and canine sequences. Homology models were created using MOE software (Chemical Computing Group, Montreal, QC, Canada) based on the crystal structure of carazolol binding to the β₂-adrenergic receptor (Protein Date Bank code 2rh1) (Cherezov et al., 2007; Rosenbaum et al., 2007) used as the template. Sequence alignment of the transmembrane helices and the extracellular loop 2 (ECL2) of OX₁ and OX₂ receptors with the 2rh1 structure was performed according to Malherbe et al. (2010) and the best model selected from 10 intermediates. Backbone coordinates remained identical to the crystal structure such that no minimization was performed (structural data comparison and figure generated by M. Katharine Holloway, Ph.D., Chemical Modeling and Informatics, Merck Research Laboratories). The predicted structure of OX₁ (A) and OX₂ (B) receptors along with their ligand binding sites (lower panels) showing the peptide backbone (green) for sequence conserved among human, rat, and dog. Space-filling residues exhibiting sequence divergence are shown in magenta and the amino acid position and sequence substitutions are indicated in the lower panel (human/dog/rat amino acids at these positions). TM, transmembrane helices. The binding site is occupied by carazolol (yellow) used in homology modeling to the β₂-adrenergic receptor.

Orexin receptor phylogeny and evolutionary conservation. Human OX₁ and OX₂ receptor sequences were used in BLASTP algorithm searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify orthologous orexin receptor sequences from other species as well as nonorexin receptor sequences sharing sequence identity and homology. Human NPFF receptor 1, NPFF receptor 2, and GPR103 protein sequences were then used to identify orthologous versions of these sequences in other species. Sequences were then aligned, and phylogenetic trees generated by using the Cobalt Phylo Tree tools at NCBI (http://www.ncbi.nlm.nih.gov/tools/cobalt), using human rhodopsin as the out-group. Note: human NPFF receptor 1 and NPFF receptor 2 sequences share greater identity with orexin receptors, whereas GPR103 (QRFP receptor) shares greater sequence similarity (residues of similar property) such that phylogenetic comparison, including GPR103 sequences from multiple species places them nearer orexin receptor sequences.

The sleep-promoting effects of DORA-12 are absent in OX_1/2 receptor double-knockouts. Electrocorticogram and electromyogram were monitored in wild-type (A) and age-matched mice with targeted ablation of both OX₁ and OX₂ receptors (B) by radiotelemetry to determine mean time spent in the indicated sleep stages as described previously (Winrow et al., 2012). At times indicated by arrows, vehicle [20% vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate), by mouth, closed symbols] or DORA-12 at 60 or 100 mg/kg (open symbols) were administered in a balanced 5-day crossover paradigm. Treatment occurred during the late active phase, approximately 4 h before the onset of the inactive phase (10:00/10:30 AM; zeitgeber time, 08:00/08:30; black arrows). Closed and open bars below each plot represent dark (active) and light (inactive) phases, respectively. Plotted points are the mean time spent in each sleep state during 30-min intervals after treatment over 5 days of consecutive treatment as determined by automated scoring and analysis as described previously (Winrow et al., 2012). Error bars (where visible) depicting the S.E.M. for each point are included, and time points exhibiting significant differences between vehicle and DORA-12 responses are indicated by gray vertical lines and tick marks (short, p < 0.05; medium, p < 0.01; long, p < 0.001; linear mixed effects model for repeated measures applied t test).