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Molecular Endocrinology, doi:10.1210/me.2004-0167
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Molecular Endocrinology 18 (11): 2790-2804
Copyright © 2004 by The Endocrine Society

Genomic Organization of Mouse Orexin Receptors: Characterization of Two Novel Tissue-Specific Splice Variants

Jing Chen and Harpal S. Randeva

Molecular Medicine Group, Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Address all correspondence and requests for reprints to: Dr. Harpal S. Randeva, Molecular Medicine Research Group, Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: hrandeva{at}bio.warwick.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2{alpha}R (443 aa) and mOX2ßR (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2{alpha}R, and the mOX2ßR revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP3) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTP{gamma}S revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP3 production at all concentrations in the mOX2ßR variant. Orexin-A, however, showed no difference in IP3 production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2{alpha}R mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2ßR gene expression, compared with both mOX1R and mOX2{alpha}R. This potentially implicates a fundamental physiological role for these splice variants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
OREXIN-A and -B were cloned from rat hypothalamus in 1998 by two independent groups (1, 2) using substractive cDNA cloning or orphan receptor technologies. Both peptides are derived from a common 130-amino acid (aa) precursor peptide, prepro-orexin, by proteolytic cleavage. Orexin-A, a 33-aa peptide and orexin-B, a 28-aa peptide, share 46% homology. The amino acid sequences of vertebrate orexin-A and -B reveal 100% and 93% interspecies identity, respectively. These peptides were initially shown to stimulate food consumption (1). However, in addition to playing a role in energy homeostasis (3, 4, 5), intracerebroventricular administration of orexin-A or -B, has been reported to exert divergent actions (6, 7, 8, 9) including the regulation of the sleep-wake cycle (10, 11).

Orexins orchestrate their actions by binding and activating two types of G protein-coupled receptors, orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), which display 64% homology in their amino acid level (1). The rat and human receptors display 94% and 95% homology for OX1R and OX2R, respectively (1). The OX1R preferentially binds orexin-A, whereas OX2R binds both orexin-A and -B, apparently with similar affinity. In recombinant systems, activation of either receptor results in a receptor-operated Ca2+ influx and elevation of intracellular Ca2+ concentrations (1, 12); OR-A has been reported to lead to a dose-dependent increase in IP3 (13). In addition, in rat hypothalamic cultures orexins cause protein kinase C-mediated Ca2+ influx (14). Furthermore, there is evidence suggesting that the orexin receptors can couple to the adenylate cyclase-cAMP system (13, 15, 16). However, the regulation by orexins of cAMP production may be tissue dependent (14).

Orexin receptors were originally shown to be present only in the hypothalamus (1), but now their presence has been noted in peripheral tissues of both the rat and in humans (17), including the myenteric plexus of the enteric nervous system and the endocrine cells of the gut (18) and the adrenal gland (13, 19), implicating orexins and their receptors in an increasing number of physiological responses. Similarly, in the mouse, orexins have also been shown to have divergent actions, including regulating feeding and metabolism, lowering blood glucose, and their expression being shown to be altered by food deprivation (5, 21, 22). Interestingly, although orexin receptors have been characterized in the rat and in humans, and display high homology, suggesting that they are highly conserved between mammalian species (1), to date these receptors have not been characterized in the mouse. More importantly, it is unclear in the mouse as to which receptors are involved in the physiological actions of orexins, and whether there is tissue specificity of these receptors.

In the present study, we therefore aimed to: 1) clone and characterize orexin receptors in the mouse, 2) determine their tissue distribution, and 3) determine their signaling characteristics. In this study, we describe for the first time the structure of mouse orexin receptors (mOX1R and mOX2R). Our findings reveal two variants of the mOX2R, with tissue-specific expression. These alternatively spliced variants differ in their C terminus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the mOX1R and mOX2R mRNA
mOX1R.
The 5' and 3' of the mOX1R cDNA were obtained by RACE (rapid amplification cDNA ends; Fig. 1AGo). The 5' and 3' of the mOX1R cDNA amplified to 480 bp and 750 bp, respectively. To clone the full-length mOX1R cDNA, PCR was performed with Marathon-Ready cDNA (mouse brain) as the template, using specific primers based on the 5'- and 3'RACE sequence (Fig. 1BGo); the product amplified at 1.4 kb.



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  		Fig. 1. Identification of mOX1R, mOX2{alpha}R, and mOX2ßR

A, 3'- and 5'RACE products were identified using mouse brain cDNA and through the use of rat OX1R-specific primers; bands of the 1-kb DNA ladder are indicated on the left. The corresponding band on the left of the DNA ladder is the 3'RACE product and that on the right of the ladder the 5'RACE product. B, To clone the full-length mOX1R cDNA, PCR was performed using Marathon-Ready cDNA (mouse brain) as the template, with specific primers based on the 5'- and 3'RACE sequence. C, To study the intact untranslated cDNA 3'end of the OX2R gene, a Primer 1S and a nested primer 2S were used for the first and second amplifications, respectively, plus AP1, as referred to in the text. Two bands of 0.65 kb and 1.2 kb in length were demonstrated by gel electrophoretic analysis; the resultant products cloned and sequenced, revealing two distinct isoforms of the mOX2R, termed mOX2{alpha}R (0.65 kb) and mOX2ßR (1.2 kb). D, As for mOX1R, 5'RACE was performed using a Marathon-Ready cDNA (mouse brain, CLONTECH). Rat OX2R primer was used, as mentioned in the text. The corresponding band on the right of the DNA ladder is the 5'RACE product. E, To amplify full-length cDNA for mOX2{alpha}R and mOX2ß two primers for each cDNA, as referred to in the text, were designed using 5'- and 3'-untranslated sequences (RACE) and amplification was performed with Marathon-Ready cDNA (mouse brain, CLONTECH). Specific amplification products were subcloned into the pGEM-T vector and sequenced. The products amplified at 1.3 kb (mOX2{alpha}R) and 1.8 kb (mOX2ßR).

 
mOX2R.
As for the mOX1R, the 3' and 5' of the mOX2R cDNA were obtained by RACE (Fig. 1Go, C and D, respectively). To determine a functional polyadenylation signal in the mOX2R, 3'RACE experiments were performed. As shown in Fig. 1CGo, amplified products of 650 bp and 1.2 kb in length were noted, and subsequently subcloned into pGEM-T vector. Sequence analysis indicated that the major functional polyadenylation signal was the typical AATAAA motif located 142 bp downstream from the stop codon. This suggested the existence of at least two subtypes of mOX2R. Specific primers for the full-length mOX2R were based on the 3'RACE sequence; this revealed two subtypes (Fig. 1EGo).

The mOX1R cDNA encodes a 416-aa receptor, which has 94% and 99% homology with reported human and rat OX1Rs, respectively (Fig. 2AGo). The mOX2{alpha}R is a 443-aa receptor and the OX2ßR encodes 460 aa, with the latter having a longer C terminus. The amino acid of the mOX2{alpha}R shows 95% homology with both the reported human and rat sequence (Fig. 2BGo). Of note, the mOX2ßR shows a 92% and 98.7% homology with human and rat OX2Rs, respectively (Fig. 2BGo). These results indicate that these receptor genes are highly conserved between mammalian species.



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  		Fig. 2. Alignment of Amino Acid Sequence for the Mouse, Rat, and Human OX1R and OX2R

A, Deduced amino acid sequences of mouse (m), rat (r), and human (h) OX1R. Amino acid residues that are identical in all three sequences are marked by asterisks (*). The full-length nucleotide sequences of rat and human of OX1R(s) cDNA have been published; the full-length nucleotide sequence of mouse OX1R cDNA have been submitted to GenBank (see Appendix: accession no. AY336083). B, Deduced amino acid sequences of m, r, and h OX2R. The mOX2R splice variants are shown as mOX2{alpha}R and mOX2ßR. Amino acid residues that are identical in all four sequences are marked by asterisks (*). The full-length nucleotide sequences of rat and human of OX2R(s) cDNA have been published; the full-length nucleotide sequence of mouse OX2{alpha}R and OX2ßR cDNAs have been submitted to GenBank (see Appendix: accession nos. Y336084 and AY336085, respectively).

 
Genomic Structure mOXR Genes
mOX1R Gene.
In the present study, the full-length of the mouse OX1R cDNA has been cloned. The mOX1R mRNA sequence was used to search the BLAST genome National Center for Biotechnology Information database. The corresponding mOX1R gene located on the contig AL 606925 and spanning approximately 9 kb, was found to map to chromosome 4. The genomic sequence of the mOX1R (Fig. 3Go) consists of nine exons; exon-3 through to exon-9 contain the coding region of 416 aa (GenBank accession no. AY336083) as determined by aligning the cDNA sequence to genomic sequence. The size of the exons and introns of the mOX1R gene, along with the splice donor and splice acceptor junction sequence, are summarized in Table 1Go. The N-terminal extracellular domain of the receptor is encoded by the first three exons. Transmembrane (TM) II, IV, V, VI, and VII are entirely within exons 4, 5, 6, 7, and 8, respectively, whereas TM domains I and III are each interrupted by a single intron. Intracellular domains one and two are either intronless (1i and 2i) or have a single intron located 1 aa (3 bp) from the intracellular domain/TM junction (4i). The third intracellular domain (3i) is interrupted by an intron located 9 aa from TM V.



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  		Fig. 3. Schematic Representation of the Structure of the mOX1R Gene

Functional transcripts are shown below: 5'- and 3'-UTRs are indicated by hatched boxes, and solid boxes represent coding regions.

 

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  		Table 1. Exon-Intron Organization of the Mouse Orexin Receptor Type 1 Gene

 
mOX2R Gene.
To determine the structure of the coding sequence of the mOX2R, and whether the mOX2R gene gives rise to alternative splice variants, we performed long distance PCR with mouse genomic DNA and primers to mOX2R cDNA sequence, to systematically walk up and down the entire coding sequence of the mOX2R gene. By sequencing these products, we were able to confirm the mOX2R cDNA sequence and identify the locations and sizes of introns.

Alignment of these genomic PCR products demonstrated that coding region of the mOX2R gene spans more than 40 kb of DNA and is interrupted by seven introns and eight exons. Table 2Go shows the size of introns and the intron-exon splice junction sequences in the coding region of the mOX2R gene. As shown in Table 2Go, all the splice acceptor and donor sequence agree with the GT/AG consensus sequence (23). It appears that, like the mOX1R, the mOX2R splice variants maintain their 7-TM domain characteristics (Fig. 4Go).


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  		Table 2. Exon-Intron Organization of the Mouse Orexin Receptor Type 2 Gene

 


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  		Fig. 4. Prediction of TM Helices in Proteins of mOX1R, mOX2{alpha}R, and mOX2ßR Using the TMHMM Server Version 2.0 (Center for Biological Sequence, Lyngby, Denmark)

These reveal that all receptors maintain 7-TM domain characteristics.

 
The N terminus of the OX2R is encoded by the first exon. TMs I, II, V, VI, and VII are within exons 1, 2, 4, 5, and 6, respectively, whereas TM III and IV are shared with exon 3. Intracellular domains 1, 2, and 3 are either intronless (1i and 2i), or have a single intron located 10 aa (30 bp) from the intracellular domain/TM junction (3i). The fourth intracellular domain (4i) is interrupted by two introns, the first intron located 4 aa (12 bp) from TM VII, and the last intron located in the cytoplasmic tail of OX2R. The mOX2{alpha}R extends from exon VIIa for 6 bp (which we have designated exonVIIb; Fig. 5Go). A polyadenylation site is predicted at the end of this sequence and the expressed sequence tags are observed to terminate at a stop codon, 142 bp before the poly-adenylation site. For the mOX2ßR, this alternate termination replaces the coding region of exon VIIIa with 17 aa encoded by 3'-untranslated region (UTR).



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  		Fig. 5. Schematic Representation of the Structure of the mOX1R Gene

Functional transcripts are shown below for the mOX2{alpha}R and mOX2ßR splice variants; 5'- and 3'-UTRs are indicated by hatched boxes, and solid boxes represent coding regions.

 
Interestingly, analysis of the amino acid sequence suggests that these 17 aa confer regions for an additional casein kinase II phosphorylation site (T456) and the presence of an N-myristoylation site (G448) in the mOX2Rß splice variant (SWISS-PROT database). The C terminus of the mOX2Rß splice variant also contains a proline residue (P450), which can influence the structure of the receptor C terminus. Additionally, there is a cluster of serine residues in the extra 17 aa encoded by the mOX2Rß splice variant.

Binding Characteristics of the mOXRs (Displacement Studies)
To characterize the binding properties of the mouse orexin receptors, mOX1R, mOX2{alpha}R, and mOX2ßR cDNAs were subcloned into stably transfected into human embryonic kidney (HEK) 293 cells. Orexin-A and -B were able to displace their respective radiolabeled ligands from their binding sites in a concentration-dependent manner in stably transfected HEK-293-OX1R cDNA (Fig. 6AGo). The concentration required to induce half-maximum response (EC50) was 20 ± 1.9 (SD) nM and 2500 ± 80 nM for orexin-A and orexin-B, respectively. Competitive displacement studies of 125I-orexin-A and 125I-orexin-B by orexin-A and -B (cold), showed no differences in pharmacological characteristics of the mOX2{alpha}R and mOX2ß (Fig. 6Go, B and C, respectively). The EC50 was 30 ± 2.1 nM and 31.1 ± 2.6 nM for orexin-A for mOX2{alpha}R and mOX2ß (P = 0.87) respectively, and the EC50 was 28 ± 1.9 nM, for both splice variants when challenged with orexin-B. The pharmacological specificity of all the mOXRs receptors was assessed by use of CRH (0.01–10 µM), and this was found to have no effect in the radioreceptor assay in displacing 125I-Orexin-A and 125I-Orexin-B (data not shown). No specific binding was detected in cells transfected with the pcDNA 3.1 vector alone. Scatchard analysis of orexin-A and -B binding for the mOX2{alpha}R and mOX2ßR was consistent with the presence of a single population of high-affinity receptors, which displayed identical high-affinity binding for orexin-A and -B (data not shown). The maximum binding site concentrations were found to be similar for both receptors (2.8 ± 0.6 and 3.0 ± 0.7 nmol/mg protein for mOX2{alpha}R and mOX2ßR, respectively), confirming that transfection efficiencies were not different for the two splice variants, and hence receptor densities were similar.



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  		Fig. 6. Binding Characteristics of Mouse Orexin Receptors

A, Competitive displacement curves of 125I-OR-A and 125I-OR-B in the presence of increasing concentrations of unlabeled orexin-A and -B in membranes prepared from HEK-293 cells transfected with the mOX1R receptor. Results are mean ± SEM of four experiments. B, Competitive displacement curves of 125I-OR-A in the presence of increasing concentrations of unlabeled orexin-A in membranes prepared from HEK-293 cells transfected with the mOX2{alpha}R receptor. Results are mean ± SEM of four experiments. C, Competitive displacement curves of 125I-OR-B in the presence of increasing concentrations of unlabeled orexin-B in membranes prepared from HEK-293 cells transfected with the mOX2ßR receptor. Results are mean ± SEM of four experiments.

 
Functional Analysis of Intracellular Second-Messenger Generation by Orexins
To determine their functional analysis, mouse orexin receptors—mOX1R, mOX2{alpha}R, and mOX2ßR cDNAs were subcloned into the expression vector pcDNA 3.1 and stably transfected into HEK-293 cells.

cAMP
Results from four independent experiments, showed that the mOX1R, mOX2{alpha}R, and mOX2ßR (cells) demonstrated no cAMP response to orexin-A and -B challenge (with concentrations up to 1 µM). In all types of cells, the integrity of adenylate cyclase system was tested by the use of forskolin and GTP{gamma}S, which revealed a dose-dependent increase in cAMP (data not shown).

Inositol Triphosphate (IP3)
For HEK-293-OX1R cell line tested, significant dose-dependent (10–6 to 10–11 M) IP3 production was observed (Fig. 7AGo), with a maximum response at 1000 nM with orexin-A (464 ± 25% of basal; P < 0.001). Orexin-B showed a similar profile, but with a smaller response than orexin-A at all time points (P < 0.01; AUC); however, like orexin-A, the maximum response was at 1000 nM with orexin-B (250 ± 18% of basal; P < 0.05).



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  		Fig. 7. Orexin-Induced IP3 Accumulation from Mouse Orexin Receptors

A, Orexin-A/B induced IP3 accumulation from mOX1R. For the IP3 stimulation assay, cells were plated in six-well plates and subcultures in DMEM until 95% confluency. After incubation with inositol-free DMEM with [3H]myo-inositol (3 µCi/ml) for 20 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 30 mM LiCl for 30 min at 37 C. Different concentrations of h/m orexin A or B were used to stimulate phosphoinositide turnover in the presence of 30 mM LiCl for 30 min at 37 C. This was followed by extraction of inositol phosphates and neutralization. IP3 levels were determined by competitive binding assay. Results are expressed as the mean ± SEM of four independent experiments. *, P < 0.001 and **, P < 0.05 compared with basal; +, P < 0.05 and ++, P < 0.01 when OR-A compared with OR-B. B, Orexin-A induced IP3 accumulation from cells transfected with mOX2{alpha}R and mOX2ßR splice variants. For the IP3 stimulation assay, cells were plated in six-well plates and subcultures in DMEM until 95% confluency. After incubation with inositol-free DMEM with [3H]myo-inositol (3 µCi/ml) for 20 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 30 mM LiCl for 30 min at 37 C. Different concentrations of h/m orexin A were used to stimulate phosphoinositide turnover in the presence of 30 mM LiCl for 30 min at 37 C. This was followed by extraction of inositol phosphates and neutralization. IP3 levels were determined by competitive binding assay. Results are expressed as the mean ± SEM of four independent experiments. *, P < 0.01; **, P < 0.001 compared with basal for both receptor splice variants; +, P = 0.15 when mOX2ßR compared with mOX2{alpha}R. C, Orexin-B induced IP3 accumulation from cells transfected with mOX2{alpha}R and mOX2ßR splice variants. Experimental procedures were as for orexin-A above. Results are expressed as the mean ± SEM of four independent experiments. *, P < 0.01; **, P < 0.001; and +, P < 0.05 compared with basal for both receptor splice variants; ++, P < 0.01 when mOX2ßR compared with mOX2{alpha}R.

 
In HEK-293-OX2{alpha}R cells, orexin-A and -B elicited a dose-dependent (10–6 to 10–11 M) increase in IP3 (Fig. 7Go, B and C), with a maximum effect observed at concentrations of 1000 nM for both orexin-A (453 ± 32% of basal; P < 0.001: Fig. 7BGo) and orexin-B (250 ± 21% of basal; P < 0.05: Fig. 7CGo); orexin-A being significantly more potent than orexin-B (P < 0.01). Similarly, in HEK-293-OX2ßR cells, there was a dose-dependent (10–6 to 10–11 M) increase in IP3 in response to orexin-A and -B (Fig. 7Go, B and C), with a maximum effect at 1000 nM for both orexin-A (490 ± 35% of basal; P < 0.001: Fig. 7BGo) and orexin-B (393 ± 30% of basal; P < 0.001; Fig. 7CGo). Like the mOX2{alpha}R, HEK-293-OX2ßR cells were more responsive to orexin-A than -B in terms of IP3 release. It is of interest to note that both orexin-A and -B showed more IP3 production in HEK-293-OX2ßR cells than the HEK-293-OX2{alpha}R cells, although failing to reach significance with orexin-A (P = 0.15); however, orexin-B was significantly more potent (P < 0.01). The IP3 results described are from four independent experiments.

Tissue Distribution of OX1R, OX2{alpha}R, and OX2ßR
Figure 8Go reveals the tissue distribution of OX1R, OX2{alpha}R, and OX2ß, as investigated by PCR. As in the rat, all receptors were detected in the brain, consistent with the original observations that orexins are regulatory peptides that function within the central nervous system. Orexin receptors are not confined to the central nervous system and have been described in peripheral tissues of humans (13, 16, 24) and the rat (25). Here we show that orexin receptors are also widely distributed in the mouse. However, unlike OX1R and OX2ß mRNAs, OX2{alpha}R mRNA was undetectable in skeletal muscle and kidney. These preliminary findings suggest that OX1R, OX2{alpha}R, and OX2ß appear to be widely distributed in the mouse and exhibit tissue specificity.



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  		Fig. 8. Tissue Distribution of mOX1R, mOX2{alpha}R, and mOX2ßR as Determined by PCR Amplification

Lanes M, DNA marker; 1, brain; 2, spleen; 3, lung; 4, liver; 5, muscle; 6, kidney; 7, testis; 8, embryo-7 d; 9, embryo-11 d; 10, embryo-15 d; 10, embryo-17 d.

 
Quantitative Analysis of Orexin Receptor Gene Expression in the Hypothalamus: Effect of Food Deprivation
Real-time PCR was performed to investigate the effects of 24-h food deprivation on orexin receptor gene expression in the hypothalamus. Figure 9Go illustrates the differential effects of food deprivation on orexin receptor subtypes. The melting curve analysis shows a single melting maximum of 90 C for OX1R (Fig. 9AGo), 89 C for the OX2{alpha}R (Fig. 9AGo), 89 C for the OX2ßR (Fig. 9AGo), and 90 C for the ß-actin (data not shown), thus confirming product specificity. The OX1R melting peak corresponded to a 219-bp fragment, OX2{alpha}R to a 215-bp fragment, the OX2ßR to a 266-bp fragment and the ß-actin to a 198-bp fragment. Quantification data were analyzed using the Light Cycle analysis software (Roche Molecular Biochemicals, Mannheim, Germany).



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  		Fig. 9. Quantitative Analysis of Mouse Orexin Receptor(s) mRNA from Hypothalami of Fed and Food-Deprived Male Mice

A, Melting curve analysis of mOX1R, mOX2{alpha}R and mOX2ßR genes, where (1) and (2) correspond to the mouse OXR genes from hypothalami of fed and food-deprived animals, respectively. The PCR product for each of the amplification reactions is identified as a sharp peak centered at the melting temperature of the product, i.e. 90 C (mOX1R), 89 C (mOX2{alpha}R), and 89 C (mOX2ßR) and 92 C for ß-actin (data not shown). Nonspecific amplification products tend to melt at much lower temperatures and over a broad range. The area under the overall melting peak is related to the linear phase of gene amplification. The differences in mouse OXR gene(s) expression were noticeable even when the PCR products from the Light-Cycler were resolved on a 1.6% agarose gels, as shown, whereas ß-actin expression was identical (data not shown). B, Diagrammatic representation of the effects of food deprivation on mouse orexin receptor gene expression in the hypothalamus. mOX1R, mOX2{alpha}R and mOX2ßR genes in food deprived animals normalized to ß-actin mRNA levels are expressed relative to fed animals (control). Results are the means ± SEM of four independent experiments. *, P < 0.01; **, P, < 0.001 comparing food deprived with fed animals.

 
Using this analysis, and correcting over the ß-actin gene expression, it was shown that food deprivation led to a significant increase in gene expression of all mouse orexin receptors in the mouse hypothalamus (Fig. 9BGo). Compared with the fed state, the increase in orexin receptor mRNA expression after food deprivation was as follows: 1) OX1R-45% (P < 0.01); 2) OX2{alpha}R-62% (P < 0.01); and 3) OX2ßR-156% (P < 0.001). These findings were also visible when the PCR products were resolved on a 1.6% agarose gel.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we report for the first time the full-length cloning and sequencing of cDNA encoding 7-TM domain orexin receptors from mouse brain termed mOX1R and mOX2R. In addition, compared with the rat and human orexin receptors, we identified for the first time the existence of at least two subtypes of mOX2R. Comparison of the complete amino acid sequence for all the mOXR cDNAs to that of the human and rat homologs, revealed a high degree of sequence identity, indicating that these receptor genes are highly conserved between these species.

We show that the structure of the coding region of the mOX2R gene, including its 3'-flanking region, provides evidence for the presence of alternative splice variants of the mOX2R mRNA in its C terminus. The coding region of the mOX2R gene is composed of eight exons and seven introns, and spans more than 40 kb of genomic DNA. It is evident from our findings that the N-terminal extracellular domain, intra- and extracellular loops and the 7-TM domains are identical for both mOX2{alpha}R and mOX2ßR, both displaying similar intron-exon organization. The difference between these two mOX2R splice variants appears from exon 7, which extends into the 3' noncoding region of the mOX2R gene, as mOX2R{alpha}. However, exon 8, which is confined to mOX2ßR, encodes an additional 17 aa in the C-terminal cytoplasmic tail of the mOX2R, and extends into the 3'-UTR region of the mOX2R gene, enabling it to function differently than the mOX2{alpha}R variant.

It is known that the size, structure, and function of cytoplasmic tails vary across the G protein-coupled receptor superfamily. The largest numbers of splice variants are situated in the C terminus of 7 TM receptors, and consequently can lead to different signaling characteristics. For example, the 13 residues of the proximal cytoplasmic tail of the human endothelin receptor are required for endothelin-1-dependent increases in calcium (26). A further interesting example is the thromboxane A2 receptor (TXR), which has two variants in the C terminus, termed TXR{alpha} and TXRß (27). These receptor variants do not differ in ligand binding or phospholipase C activation, but whereas TXR{alpha} stimulates adenylate cyclase, TXRß inhibits it, demonstrating the importance of the C terminus length and sequence in determining receptor-G protein activation and hence receptor function. With regard to the two-mOX2R splice variants, we found no differences in the binding characteristics with orexin-A and -B, and both splice variants failed to stimulate cAMP production. However, our current findings in HEK-293 cells, of no cAMP production with orexins, do not in any way suggest that mouse orexin receptors do not couple to the adenylyl cyclase-signaling pathway. It is likely that orexin signaling pathways are cell type specific; for example, it has been shown that orexins activate the adenylate cyclase-dependent signaling cascade in the adrenal gland (13, 16).

Of more interest is the observation that orexin-B was more potent in stimulating IP3 production in HEK-293 cells transfected with the mOX2Rß splice variant than mOX2R{alpha}, although there was no statistical difference in IP3 production between mOX2R{alpha} and mOX2Rß upon stimulation with orexin-A. We have described similar ligand-specific increase in IP3 with relation to the CRH receptor (28), where we were able to demonstrate that urocortin was more potent than CRH in activating the Gq/phospholipase C/IP3/protein kinase C pathway. Given that the binding characteristics were similar for both mOX2R splice variants, our findings suggest that the sequences in the distal residues of the C terminus may be particularly important for specific coupling to phospholipase C for mouse orexin receptors, rather than influencing the binding of orexin-A and -B. Studies from other C terminus splice variants have also revealed different effects on the signaling pathway used by a receptor (26, 27, 29). Moreover, a number of processes are involved in the regulation and function of 7TM receptors (30, 31, 32, 33, 34, 35), including receptor desensitization, which may also apply to the mOX2R splice variants. However, the IP3 response observed between the mOX2R splice variants was transient and reached maximal for both variants after 3 min of incubation with orexins. The short timing of incubation (3 min) leads us to hypothesize that the differences detected may be due to the difference in the ability to couple to the cognate G proteins, rather than a difference in receptor desensitization. Currently, studies are underway to address these issues.

Our findings that alternative splicing of the mOX2R mRNA can produce mOX2R transcripts with heterogenous 3'-UTR sequences prompted us to examine the tissue-specific nature of this phenomenon. PCR analysis demonstrated differential expression of the mOX2R splice variants, with particular tissue-specific expression for liver, muscle, and kidney. However, currently there are no means of quantitatively detecting these C terminus splice variants at protein level. Splice variants for several 7TM receptors are differentially distributed (36, 37), including those with C terminus variants (38, 39, 40), and may suggest tissue-specific functions. There is also evidence that splice variants have a temporal difference and may have potential physiological implications, such as postnatal development (41). It is possible that different expression patterns may occur owing to distinct orexin receptor promoters that are situated upstream of the 5' end sequence of the mOX2R gene. However, the physiological relevance and the possible pathological implications of the tissue-specific expression of mOX2R splice variants remain to be determined.

The meritorious work by Sakurai et al. (1) revealed that food deprivation up-regulated expression of orexin and its receptors in the hypothalamus, which in turn coordinates behavioral, autonomic, and neuroendocrine responses to biochemical and metabolic changes. Like Sakurai, we noted that both mOX1R and mOX2Rs were up-regulated after food deprivation, with mOX2Rß showing the greatest increment. Interestingly, food restriction is known to disrupt the sleep-wake pattern, and as compared with OX1R, the OX2R has been shown to play a greater and more important role in the sleep/wake cycle (10). Therefore, given that food deprivation may alter circadian rhythms, such as the sleep/wake cycle (10), it is possible that the up-regulation of orexin receptors in food-deprived mice may be induced by alterations in circadian rhythms as well. Collectively, in addition to the functional differences of the mOX2R splice variants observed by us, the greater increase in mOX2ßR gene expression compared with mOX2{alpha}R, may suggest a more important role for this splice variant in arousal cycles, sleep/wake (circadian rhythms) or feeding. However, it is beyond the scope of this paper to speculate on the full biological significance of the regulation of these receptor splice variants.

In conclusion, we describe novel data in the form of the structure of orexin receptors in the mouse, and report the full-length cloning and sequencing of mRNA encoding these 7TM domain receptors. The intron-exon organization of both mOXRs is shown, and we provide evidence for the first time the presence of orexin receptor splice variants in any animal species. The alternative splicing of the mOX2R gene, in the C terminus, reveals tissue-specific nature of this phenomenon and signaling characteristics of the mOX2R{alpha} and mOX2Rß splice variants. Finally, it is clear that more work is necessary, particularly in the area of physiological and pathophysiological relevance of our findings, and the use of specific splice variant knockout mice and/or short interfering RNA will no doubt help to understand the function of these mOX2R splice variants in more detail.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Human/mouse (h/m) orexin-A, orexin-B and radioiodinated orexins, 125I-orexin-A (rat/h/m) and 125I-orexin-B (rat/m), were obtained from Phoenix Pharmaceuticals Inc. (Belmont, CA). The mammalian expression vector pcDNA 3.1 was obtained from Invitrogen Life Technologies (Paisley, Scotland, UK). Waters Sep-Pak C18 columns were obtained from Millipore Corp. Ltd. (Watford, Hertfordshire, UK). [3H] myo-inositol, and the cAMP assay kits were obtained from NEN Life Science Products (Hertfordshire, UK). SV Total RNA Isolation System and pGEM-T vector (Promega, Southampton, UK). Marathon-Ready cDNA, multiple-tissue cDNA panels, mouse genomic DNA, and GenomeWalker kit were obtained from (CLONTECH, Oxford, UK). PCR and cloning reagents were purchased from Invitrogen Life Technologies. Synthetic oligonucleotide primers and enzymes were purchased from Invitrogen Life Technologies. DMEM, inositol-free DME/Ham’s F-12 medium and Lipofectamine reagent were obtained from Invitrogen Life Technologies QIAquick Gel Extraction kit was obtained from QIAGEN (Valencia, CA).

5'RACE
mOX1R.
The 5'-end of mOX1R was obtained by 5'RACE using the commercially prepared Marathon-Ready cDNA system (CLONTECH) in accordance with the manufacturer’s recommendations. An uncloned library of the adapter-ligated cDNA prepared from mouse brain was used to amplify the 5'-end of the OX1R cDNA. First-round PCR was performed using a rat OX1R-specific primer (5'-GTACTGCTTCGGATAGAGATAATC-3') and the adapter primer (AP1: 5'-CCATCCAATACGACTCACTATAGGGC-3') (CLONTECH). The products of the first PCR served as a template for the second round of amplification reaction. A nested PCR was then performed using the AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3') and a second rat cDNA-specific primer (5'-GAGGAACTCGTCCTCATAGTCTGG-3').

The products of the second PCR were analyzed by using 1.0% agarose gel electrophoresis, purified by using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) and ligated using T4 DNA Ligase Kit (Invitrogen Life Technologies) into the pGEM-T vector (Promega) for sequence analysis. Positive isolated clones were sequenced in an automated DNA sequencer and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information.

mOX2R.
As for mOXR1, 5'RACE was performed using a Marathon-Ready cDNA (CLONTECH). The cDNA was initially amplified using the kit supplied AP1 and the rat OX2R primer (5'-GATGAGAGCCACGACGAACACGATG-3'). The reaction products were then further amplified using a supplied nested AP2 and a nested rat OX2R-specific primer (5'-CGGCTCTTGAGTTTCATTCAGCTC-3'). The products of the PCRs were analyzed as for the mOXR1.

Analysis of 3'-UTR by 3'RACE
mOX1R.
3'RACE was performed to detect functional polyadenylation signals in the 3'-UTR of the mouse orexin-R1. Specific primers designed from the rat OX1R were used in combination with nonspecific primers purchased commercially (CLONTECH), to obtain the 3'end of the mOX1R cDNA. The two OX1R-specific primers used for nested PCR were: 5'-GTTTGGGATGTTTCGCCAAGCCAGC-3' and 5'-GCCAGACACAAGTCCTTGTCCTTG-3'.

mOX2R.
To study the intact untranslated cDNA 3'end of the OX2R gene, a primer 1S (5'-CGGATTAGCGCTGTTGCTGCTGAG-3') and a nested primer 2S (5'-CGGATGCTCATGGTTGTACTTCTG-3') were used for the first and second amplifications, respectively, plus AP1 supplied by CLONTECH. Two bands of 0.65 and 1.2 kb in length were demonstrated by gel electrophoretic analysis (see Results). The resultant products were cloned into the pGEM-T vector and the clones were sequenced, revealing two distinct isoforms of the mOX2R, termed mOX2{alpha}R (0.65 kb) and mOX2ßR (1.2 kb).

Isolation of Full-Length Mouse Orexin Receptor cDNA by PCR
To amplify full-length cDNA for mOX1R, mOX2{alpha}R, and mOX2ß two primers for each cDNA were designed using 5'- and 3'-untranslated sequences (RACE) and amplification was performed with Marathon-Ready cDNA (mouse brain, CLONTECH). Specific amplification products were subcloned into the pGEM-T vector and sequenced.

OX1R: (sense) 5'-GAGCTCATTACTCTTCATCGTG-3'

(antisense) 5'-CAGCAGTCTTCCTGTGACTGCTG-3'

OX2{alpha}R: (sense) 5'-GAGACAAGCTTGCAGCACTGAG-3'

(antisense) 5'-TGAGTCGGGTATCCTCATCATAG-3'

OX2ßR: (sense) 5'-GAGACAAGCTTGCAGCACTGAG-3'

(antisense) 5'-GGTCGGTCAATGTCCAATGTTC-3'

To Identify Intron-Exon Junction Sequences of the Mouse Orexin Receptor Type 2 by Genomic Walking
To obtain intron-exon junction sequences, the genomic walking method was used. The sequence of the mouse orexin receptor type gene was PCR amplified using the mouse GenomeWalker kit (CLONTECH). Briefly, the supplied mouse genomic DNA, digested with various restriction enzymes and ligated at both 5' and 3' ends with an adapter DNA of known sequence, served as templates in PCR using a primer to the adapter sequence and a series of forward and reverse primers to mOX2R cDNA sequences. The PCR products were subcloned into the pGEM-T vector and sequenced.

PCR Determination of Intron Sizes of the OX2R
To obtain intron sizes, PCR was performed with the Elongase system and mouse genomic DNA (CLONTECH). The sizes and locations of introns within genomic DNA PCR products were determined by a combination of agarose gel electrophoresis and DNA sequencing. By comparing the sequence of the amplified genomic DNA to that of the mOX2R cDNA, we were able to identify intron-exon boundaries and thereby also determine the sizes of the introns and exons.

Orexin Receptor Gene Distribution in Mouse Tissues by PCR
Experiments were carried out with the Mouse Multiple Tissue cDNA Panel I kit (CLONTECH) according to the manufacturer’s recommendations. PCRs were performed using gene-specific primers with mouse multiple tissue cDNA. The set of primers for the amplification of orexin receptors were:

OX1R: 5'-CCAGACTATGAGGACGAGTTCCTC-3'(sense)

5'-GATGAAGCTGAGAGTCAGCACTGC-3'(antisense)

OX2{alpha}R/OX2ßR: 5'-GAGACAAGCTTGCAGCACTGAG-3' (sense)

OX2{alpha}R: 5'-TGAGTCGGGTATCCTCATCATAG-3' (antisense)

OX2ßR: 5'-GGTCGGTCAATGTCCAATGTTC-3' (antisense)

Functional Analysis of Orexin Receptors
Stable Cell Transfections.
The cDNA coding for mOX1R, mOX2{alpha}R, and mOX2ß were inserted into the expression vector pcDNA 3.1 (Invitrogen Life Technologies) and stably transfected into HEK-293 cells with Lipofectamine reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. The cells were grown in DMEM (Invitrogen Life Technologies), to select for transfected cells. After selection for stably transfected cells with the antibiotic G418, a number of these cell lines were selected for characterization of their binding and signaling properties. Cultures were maintained at 37 C in humidified atmosphere of 5% CO2 in air.

Membrane Preparation
When confluent, HEK-293 cells were washed with PBS, and then homogenized in extraction buffer (A) containing 10 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl 2, 0.1% BSA, and 0.1% bacitracin (pH 7.2). The homogenate was centrifuged at 600 x g for 30 min at 4 C and the supernatant was further centrifuged at 40,000 x g for 60 min at 4 C. The pellet was rinsed twice, resuspended in binding buffer (B) containing 10 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin (pH 7.2) and aliquoted (50 µg in 50-µl aliquots) in microtubes.

Radioligand Binding Assay
For competitive displacement studies, mOX1R, mOX2{alpha}R, and mOX2ßR cell membranes (100 µg of protein) were incubated with 1 nM of 125I-orexin-A in the presence or absence of unlabeled peptide (0.1–1000 nM). Nonspecific binding was measured in the presence of 1 µM of unlabeled orexin-A. For Scatchard analysis, membranes (100 µg of protein) were incubated with 125I-orexin-A (0.2–2 nM) and unlabeled orexin-A (1000 molar excess) in 50 µl of binding buffer. The tubes were incubated at 22 C for 120 min. The reaction was terminated by adding 1 ml/tube of ice-cold 20% polyethylene glycol. After centrifugation at 10,000 x g for 30 min, at 4 C, the pellets were washed once with 20% polyethylene glycol and radioactivity was measured in a {gamma}-counter (Packard Instruments, Meriden, CT). Experiments were also conducted for 125I-orexin-B, in the presence or absence of unlabeled peptide, using the same protocol. Four independent experiments were conducted with each ligand.

The binding data were analyzed using the computer program equilibrium binding data analysis (42), which provides initial estimates of equilibrium binding parameters by Scatchard and Hill analyses and then produces a file for the nonlinear curve-fitting program Ligand (43).

Second Messenger Studies
Measurement of cAMP.
HEK-293 cells stably transfected with cDNA coding for mOX1R, mOX2{alpha}R, and mOX2ß were cultured until 95% confluent. Before treatment, cells were washed once with DMEM, followed by preincubation with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthin for 20 min. Cells were then stimulated with h/m orexin A or B (0.1–1000 nM) for 15 min at 37 C. The reactions were terminated by addition of 0.1 M HCl. After an overnight freeze/thaw cycle, the cAMP levels were measured in whole cell extracts using RIA (Dupont-NEN, Hertfordshire, UK). Four independent experiments were conducted for each receptor with orexin-A, and similarly with orexin-B.

Measurement of IP3
For the IP3 stimulation assay, cells were plated and subcultured in DMEM until 95% confluency. After incubation with inositol-free DMEM with [3H]myo-inositol (3 µCi/ml) for 20 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 30 mM LiCl for 3 min at 37 C. Different concentrations of h/m orexin A or B were used to stimulate phosphoinositide turnover in the presence of 30 mM LiCl for 5 min at 37 C. The reactions were stopped by addition of chloroform-methanol-hydrochloric acid (50:100:1) at specified time intervals. The supernatant was applied to prefilled poly-prep columns (AG 1-X8 resin 100–200 mesh chloride form, Bio-Rad Laboratories, Inc., York, UK) and [3H]IPs were resolved and quantified as previously described (20). The radioactivity was measured by a ß-counter. As with cAMP studies, four independent experiments were conducted for each receptor with orexin-A, and similarly with orexin-B.

Effect of Food Deprivation on Orexin Receptor Gene Expression in the Hypothalamus
Animal Preparation.
All procedures described were approved by the University of Warwick (Coventry, UK) on use and care of animals. Eight-week-old male TO mice (Bantin & Kingon, Hull, UK) were housed in groups of two in environmentally controlled conditions (22 ± 2 C, humidity 40–60%) under a 12-h light, 12-h dark schedule (lights on 0600 h). Mice were allowed unrestricted access to standard laboratory pellet rodent diet (CRM, Biosure, Cambridge, UK) and access to tap water, before being subjected to the study. After a week of habituation to these conditions, mice were randomly distributed into two groups. The first group was allowed to eat freely (ad libitum). The second group was food deprived for 24 h, beginning at the onset of the dark cycle (lights out 1800 h), before both groups of animals were killed by CO2 inhalation the following day at 1800 h. A total of 12 mice were killed. Hypothalamic were immediately removed, and immediately snap frozen in liquid nitrogen. Samples were then stored at –80 C until RNA extraction.

Total RNA Preparation and cDNA Synthesis.
Total RNA was prepared from mouse hypothalami (fed and food deprived) using SV Total RNA isolation system (Promega), according to the manufacturer’s guidelines. First strand cDNA synthesis was performed using RNase Reverse Transcriptase (Invitrogen Life Technologies), according to manufacturer’s recommendation.

Quantitative RT-PCR Analysis of Orexin Receptor Gene Expression
Quantitative PCR was performed on a Light Cycler system (Roche Molecular Biochemicals). PCRs were carried out in a reaction mixture consisting of 5.0-µl reaction buffer and 2.0 mM MgCl2 (Biogene, Kimbolton, UK), 1.0 µl of each primer (5 ng/µl), 2.5 µl cDNA, and 0.5 µl Light Cycler DNA Master SYBR Green I (Roche Molecular Biochemicals).

Protocol conditions consisted of denaturation at 95 C for 15 sec; followed by 40 cycles at 94 C for 1 sec, 58 C for 12 sec, and 72 C for 15 sec: followed by melting curve analysis. For analysis, quantitative amounts of OX1R, OX2{alpha}R, or OX2ßR gene expression were normalized against the housekeeping gene ß-actin. Quantitative data analysis was made possible through the use of OX1R, OX2{alpha}R or OX2ßR RNA from serially diluted hypothalamic cDNA, using 1-, 10-, 100-, and 1000-fold dilutions. Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.6% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Invitrogen Life Technologies) to estimate the band sizes. As a negative control for all the reactions, distilled water was used in place of the cDNA. Four independent experiments were conducted.

The primers used were: for OX1R, sense 5'-AGGACTCTCCTCAGCTGAAGTG-3', antisense 5'-ACCAAGGCTATGAGGAACACAG-3'; for OX2{alpha}R and OX2ßR, sense 5'-GGTTCATCATCGCCAAGGAGAC-3', antisense for OX2{alpha}R 5'-TGAGTCGGGTATCCTCATCATAG-3', and antisense for OX2ßR 5'-GTGAGATTCCATAAGGATGCTC-3'; and for ß-actin sense 5'-AAGAGAGGTATCCTGACCCT-3', antisense 5'-TACATGGCTGGGTGTTGAA-3'.

Sequence Analysis
The PCR products from hypothalamic samples were purified from the 1.6% agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). PCR products were then sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information, confirming the identity of our products.

Statistical Analysis
Data are shown as the mean ± SEM of each measurement. In each case, results were evaluated between groups by using two-tailed Student’s t test, with significance determined at the level of P < 0.05. Statistical analysis of variance was also performed measuring the difference in PCR products.

Appendix: Gene Accession Nos.
Mouse orexin receptor type-1: AY336083

Mouse orexin receptor type-2{alpha}: AY336084

Mouse orexin receptor type-2ß: AY336085

Mouse orexin type-2 receptor gene: AY339383 to AY339390


   ACKNOWLEDGMENTS
 
The authors are very grateful to Dr. E. Karteris for his immense help with editing of the manuscript. Also we thank Dr. Grammatopoulos and Prof. Hillhouse for their help. H.S.R. would like to thank his wife, Jaspal, for her immense support with this project.


   FOOTNOTES
 
This work was supported by Coventry General Charities and The British Heart Foundation (PG/03/131/16192).

Abbreviations: aa, Amino acid; AP, adapter primer; GRK, G protein-coupled receptor-specific kinase; HEK, human embryonic kidney; h/m, human/mouse; IP3, inositol triphosphate; mOX1R and mOX2R, mouse orexin receptor(s); OX1R, type-1 orexin receptor; OX2R, type-2 orexin receptor; RACE, rapid amplification of cDNA ends; TM, transmembrane; TXR, thromboxane receptor; UTR, untranslated region.

Received for publication April 20, 2004. Accepted for publication July 8, 2004.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
 MATERIALS AND METHODS
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