Identification of the Salmon Somatolactin Receptor, a New Member of the Cytokine Receptor Family
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内分泌学杂志 2005年第5期
Northwest Fisheries Science Center (H.F., P.S., W.W.D.), National Marine Fisheries Service, Seattle, Washington 98112; Division of Marine Bioscience (Y.O., S.A., K.Y., A.H.), Graduate School of Fisheries Sciences, Hokkaido University, Hokkaido 041-8611, Japan; School of Aquatic and Fishery Sciences (A.L.P., W.W.D.), University of Washington, Seattle, Washington 98195; and Center for Reproductive Biology (P.S.), Washington State University, Pullman, Washington 99164
Address all correspondence and requests for reprints to: Walton W. Dickhoff, School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195. E-mail: walton.w.dickhoff@noaa.gov; address reprint requests to: Haruhisa Fukada, Faculty of Agriculture, B200 Monobe, Nankoku, Kochi 783-8502, Japan.
Abstract
Somatolactin (SL) is a pituitary hormone of the GH/prolactin (PRL) family that so far has been found only in fish. Compared with GH and PRL, the primary structure of SL is highly conserved among divergent fish species, suggesting it has an important function and a discriminating receptor that constrains structural change. However, SL functions are poorly understood, and receptors for SL have not yet been identified. During cloning of GH receptor cDNA from salmon, we found a variant with relatively high (38–58%) sequence identity to vertebrate GH receptors and low (28–33%) identity to PRL receptors; however, the recombinant protein encoding the extracellular domain showed only weak binding of GH. Ligand binding of the recombinant extracellular domain for this receptor confirmed that the cDNA encoded a specific receptor for SL. The SL receptor (SLR) has common features of a GH receptor including FGEFS motif, six cysteine residues in the extracellular domain, a single transmembrane region, and Box 1 and 2 regions in the intracellular domain. These structural characteristics place the SLR in the cytokine receptor type I homodimeric group, which includes receptors for GH, PRL, erythropoietin, thrombopoietin, granulocyte-colony stimulating factor, and leptin. Transcripts for SLR were found in 11 tissues with highest levels in liver and fat, supporting the notion that a major function of SL is regulation of lipid metabolism. Cloning SLR cDNA opens the way for discovery of new SL functions and target tissues in fish, and perhaps novel members of this receptor family in other vertebrates.
Introduction
SOMATOLACTIN (SL), WHICH is a relatively recently discovered member of the GH/prolactin (PRL)/placental lactogen family, is produced by the pars intermedia of the pituitary gland of fish. SL was first isolated from Atlantic cod Gadus morhua (1) and more recently identified in several species of teleosts (2, 3), sturgeon, and lungfish (4). Thus, SL is present in the lineage leading to tetrapods, but so far it has been found only in fish. The structure of SL is similar to vertebrate GH and PRL, with an average amino acid sequence identity of 24% (5); however, it is more highly conserved than GH and PRL even among divergent lineages of fish (4). Physiological studies have suggested that SL, like vertebrate GH and PRL, may have a multitude of biological functions. The measurement of plasma SL levels in salmonids suggests that it might be involved in gonadal maturation (5, 6), smoltification (7), and stress responses (8). Other physiological effects of SL seen in teleosts include immune function (9), acid base balance (10), background adaptation (11), energy mobilization (8, 12), gonadal steroid biosynthesis (6), and phosphate (13), sodium (14), and calcium (15) metabolism. Genetic evidence from medaka indicates a role for SL in pigment cell proliferation (3). Recently, it has been suggested that the dominant role of SL is controlling lipolysis during and after the transition from the growing to the nongrowing season of temperate fish (16).
Despite these observed physiological effects of SL, the precise target cells and biological actions of SL remain unclear, and it is not known whether SL has a receptor distinct from GH and PRL. Thus far, there is no information about the SL receptor (SLR) either from classical ligand-binding studies or gene cloning. Understanding the biochemical nature of the SLR would pave the way for new studies of specific actions of SL at the cellular level. In the course of cloning the salmon GH receptor (GHR) cDNA, we found two cDNAs with high sequence homology to fish GHRs. The putative proteins of the two cDNAs differed from each other and revealed a structure typical of the cytokine receptor family. The deduced amino acid sequences indicated relatively high identity to other vertebrate GHRs and lower identity to other vertebrate PRL receptors (PRLR). Ligand-binding studies using the recombinant protein encoding the extracellular domain (ECD) of the receptor revealed one of the two receptors was the salmon GHR (17). The ECD of the other GHR-like gene showed weak binding to GH; however, the specific GH binding did not increase in a concentration-dependent manner as would be expected for a GHR (our unpublished data). These data suggested that the ligand for the other GHR-like cDNA might be the SLR.
In this study, the complete primary sequence of a masu salmon SLR was cloned. The ligand specificity of SLR was confirmed by ligand-binding assays using a recombinant protein encoding the ECD of the receptor. In addition, the tissue distribution and abundance of SLR mRNA in various tissues was determined by real-time quantitative RT-PCR.
Materials and Methods
Experimental animals and blood and tissue samples
The fish used for cDNA cloning were 2-yr-old masu salmon (Oncorhynchus masou), which were reared at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University (Hokkaido, Japan). Fish were held in outdoor concrete ponds supplied with a continuous flow of river water at ambient temperature and photoperiod. They were fed commercial trout food (Oriental Yeast Co. Ltd., Tokyo, Japan) twice per day. Livers were collected from fish after euthanasia with 0.1% 2-phenoxyethanol (Kanto Kagaku, Tokyo, Japan) and then stored at –80 C.
The tissue distribution of SLR gene expression was determined using 1-yr-old coho salmon (Oncorhynchus kisutch), reared in recirculated fresh water at 11 C at the Northwest Fisheries Science Center (Seattle, WA). Fish of the same age were used for cloning of the partial cDNA of the coho salmon hepatic SLR. The fish received a commercial pellet diet (Biodiet Grower; Bioproducts Inc., Warrenton, OR) ad libitum once per day. After euthanasia with 0.1% tricane methanesulfonate (MS-222; Argent Chemical Laboratories, Redmond, WA), tissues were collected and then stored at –80 C. Animal-use protocols were approved by the University of Washington Institutional Animal Care and Use Committee.
Cloning of masu salmon SLR cDNA and partial cloning of coho salmon SLR cDNA
Total RNA of the liver was isolated using Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. RT was carried out with 3 μg total RNA, 50 ng random hexamers, and 200 U Maloney murine leukemia virus reverse transcriptase, RNase H minus, point mutant (Promega, Madison, WI). The cDNA fragment of masu salmon SLR was obtained by PCR using first-strand cDNA of masu salmon liver as a template. Degenerate primers were designed on the basis of conserved amino acid sequences of GHRs available in the GenBank database. The forward primer was 5'-GAC TGG AA(A/G) GA(A/G) TGT CCG GAT TAC-3' (designed from DWKECPDY sequence in the ECD), and the reverse primer was 5'-AAG GA(C/T) GA(C/T) GA(C/T) TCG GGG CGC GC-3' (designed from MDFYAQV sequence in the intracellular domain). PCR amplification was performed in a 50-μl volume using Hotstar Taq DNA polymerase (QIAGEN, Valencia, CA), and 35 cycles were run as follows after incubation for activation of Taq polymerase (95 C, 15min): 94 C for 30sec, 55 C for 30 sec, and 72 C for 1 min. Finally, the temperature was held at 72 C for 11 min. After electrophoresis with 1.5% agarose gel, a single band was observed, which was extracted from the agarose using QIAEXII (QIAGEN). The purified PCR product was ligated to an Easy T-Vector (Promega) and transfected into XL-I blue Escherichia coli cells (Novagen, Madison, WI). After incubation of colonies, plasmid vectors were purified and sequenced by the dye-termination method using ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA).
Full-length SLR was obtained by 5' rapid amplification of cDNA ends (5'-RACE) and 3'-RACE using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA). The first-strand cDNA of masu salmon liver was used as a template. Two reverse gene primers were designed for 5'-RACE as follows: GSP-1, 5'-CAA GGT ATC GTA GGT TTT GTT CTG GCT-3' (nucleotide position 355–381), and GSP-2, 5'-CGT CAA GTC ATT CTT CTT CCA GTA CTG-3' (nucleotide position 217–243). First PCR was done using GSP-1 and universal primer mix A (UPM). To obtain more of the upstream coding sequence, GSP-2 and UPM were used in a second PCR. The primers for 3'-RACE were as follows: GSP-3, 5'-CTG GAG GTC CCA TGC CCC GGG CTC CAG-3' (nucleotide position 1282–1308), and GSP-4, 5'-CGC CTT CTG CCG AGA GCA AGC CCC ACC AG-3' (nucleotide position 1682–1710). The first PCR of 3'-RACE was done using GSP-3 and UPM. To obtain more of the downstream coding sequence, GSP-4 and UPM were used in PCR. Finally, the open reading frame was amplified by PCR using SLR 5' untranslated region 5'-TGA CAT CTT TGT GTT AGC AAA GGA AAG C-3' (nucleotide position –38 to –10) and SLR 3' untranslated region 5'-ATG ACA AAT GGC TTC CAA CCT CTA CG-3' (nucleotide position 2006–2031) with High Fidelity Platinum Taq polymerase (Invitrogen, Carlsbad, CA). All PCR products were subcloned into the pGEM Easy T-vector and sequenced by the dideoxy chain termination method (ABI 310).
Cloning of the partial cDNA encoding the coho salmon SLR was done to provide sequence design of primers and probes for real-time quantitative RT-PCR. Total RNA was isolated from the liver using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) followed by the Molecular Research Center protocol, and the cDNA was synthesized as described above. The PCR was done using the following primers: 5'-CAC TGT GCA ATG AGG GCC TTC AA-3' (forward; nucleotide position 655–677) and 5'-GAC CCA GCA GTC TCT GTA AGT CTG AAC-3' (reverse; nucleotide position 1052–1078). The PCR products were sequenced by the dideoxy chain termination method (ABI 3100).
Structural and phylogenetic tree analyses
Predictions of a signal peptide, transmembrane region, and potential N-glycosylation sites were performed using prediction server of the Center for Biological Sequence Analysis (www.cbs.dtu.dk) based on the deduced amino acid sequences. Conserved peptide domains (fibronectin type III domain and cytokine receptor domain) were predicted by prsBLAST from the National Center for Biotechnology Information database (NCBI) (www.ncbi.nlm.nih.go) and by InterProscan from European Molecular Biology Laboratory-European Bioinformatics Institute (www.ebi.ac.uk), respectively. Multiple amino acid sequence alignments were constructed using the ClustalW from the DNA Data Bank of Japan website (www.ddbj.nig.ac.jp) (18). After manual correction of the alignments, the amino acid sequences were subjected to the ClustalW analysis to construct a phylogenetic tree using a neighbor-joining method (19). The ClustalW analysis was performed using default settings except for gaps that were treated as missing characters. Relative branch support was evaluated by bootstrap analysis. NJ plot software (http://pbil.univ-lyon1.fr/software/njplot.html) was used to prepare a graphical view of the phylogenetic tree (20).
Expression, refolding, and purification of the recombinant ECD of SLR (SLR-ECD)
The fragment encoding the ECD of SLR was amplified by PCR using the full-length SLR cDNA as the template. Primers for SLR-ECD were as follows: SLR-ECD (F; nucleotide position 61–83), 5'-TCC TCG CTG ATG GAC CCT GGC TC-3', and SLR-ECD (R; nucleotide position 721–745), 5'-CGT TGA CTC TTT ATT GGG AAT CTC-3'. PCR was done using proofreading Taq polymerase (Advantage 2 polymerase; Clontech). PCR products were cloned to the pCR-T7 vector (Invitrogen) and transfected to Top-10F' E. coli cells. Vectors from positive clones were purified and transformed into the BL21(DE3) pLysS E. coli strain for protein expression.
Transfected cells were grown in 100 ml of LB-broth medium containing 20 μg/ml ampicillin at 37 C in a 250-ml flask. When the absorbance at 600 nm reached 0.4, isopropylthiol-?-D-galactoside was added to the tube at a final concentration of 0.4 mM. Cells were grown for an additional 4 h and then collected by centrifugation at 5000 x g for 15 min and frozen at –30 C. Inclusion bodies were purified using BugBuster protein extraction reagent (Novagen). Refolding and purification of histidine-tagged SLR-ECD (His-SLR-ECD) were done as described in our previous report (17). Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL) using BSA as a standard.
Electrophoresis
SDS-PAGE was carried out according to Laemmli using a 15% acrylamide gel. The gel was stained with Bio-Safe colloidal Coomassie brilliant blue G-250 (Bio-Rad, Hercules, CA). Sizes of recombinant proteins were estimated using the following standards (Amersham Bioscience Corp, Piscataway, NJ): -lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), albumin (67 kDa), and phosphorylase b (94 kDa).
Hormones
Native GH and PRL were purified from pituitaries of coho salmon according to the methods of Jackson et al. (21). Purification of native SL was performed according to the method of Rand-Weaver et al. (1). The purity of the proteins obtained by this method was estimated to be more than 95% by SDS-PAGE. This is similar to that obtained using the same method for purification of GH, PRL, and SL from many species of fish (see Ref. 22 for example).
Iodination of SL
Iodination of SL was performed by the chloramine T method according to the method of Rand-Weaver et al. (5). The specific activity of labeled hormone was determined by a self-displacement method (23) using recombinant His-SLR-ECD (specific activity: 10.2 μCi/μg).
Binding assay
Liver membranes from coho salmon were prepared according to Yao and Le Bail (24). Purified recombinant His-SLR-ECD or liver membranes and 125I-labeled SL were used for the binding tests. Recombinant His-SLR-ECD (25 pmol) in assay buffer (10 mM Tris-HCl, pH 8.0, containing 0.1% BSA, 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride) was incubated with 125I-labeled SL (40,000 cpm) in duplicate in the absence (total binding) or presence (nonspecific binding) of 2 μg of unlabeled SL in a final volume of 250 μl. After incubation at room temperature for 20 h, free 125I-labeled SL was separated according to the method of Sandowski et al. (25). Radioactivity of the pellets was measured in a -counter (3 min/tube). Specific binding was calculated by subtracting the nonspecific binding from the total binding. Affinity constants (Ka) were estimated using GraphPad Prism software version 4.0 (GraphPad Software Inc., San Diego, CA).
Real-time quantitative RT-PCR for SLR
Total RNA was extracted from tissues using TriReagent described as above. Integrity of the RNA was verified by an OD absorption ratio of OD 260 nm/OD 280 nm greater than 1.9, and 300 ng of total RNA was used for the synthesis of the first-strand cDNA. RT was performed in a reaction mixture containing 0.5 mM dNTPs, 112.5 μg random hexamers (Promega), 6 U RNase inhibitor (ABI), 10 mM dithiothreitol, 37.5 U Maloney murine leukemia virus reverse transcriptase (SuperScript II; Invitrogen), and 1x RT buffer for SuperScript II in a final volume of 15 μl. RT was performed for 10 min at 25 C, followed by 60 min at 48 C, and finally for 5 min at 95 C.
For design of specific primers and probes for the assay of coho salmon SLR, SLR cDNA was partially cloned from liver of coho salmon. Primers and probe for real-time quantitative PCR were designed by primer express program (ABI) as follows: forward primer, 5'-CAG CAT TGC TTA AGA AGG GAA AG-3'; reverse primer, 5'-TGG AGA GCC CGC ATA CCA-3'; and probe, 5'-FAM-CCA CTC AGG CTG AAG TTC AGC TCG TCC A-TAMRA-3'. The primers and probe were designed to a region in the intracellular domain. The predicted length of the amplicon is 73 bp. The forward primer spans a predicted intron/exon boundary to avoid amplification of genomic DNA. For normalization of data, an 18S ribosomal gene was used. Primers and probes for 18S were purchased from ABI. PCR (25 μl) contained 12.5 μl TaqMan Universal PCR Master Mix (ABI), 3 μl of the first-strand cDNA for SLR assay, or 2 μl of a 1/100 dilution for the first-strand cDNA for the 18S assay, 0.9 μM forward and reverse primers, and 0.2 μM probe. Amplification and detection of samples were performed with the ABI 7700 system using the following thermal cycling conditions: 50 C for 2 min, 95 C for 15 sec, and 60 C for 1 min (40 cycles). Four serial dilutions of cDNA were run to determine PCR efficiency. The efficiencies (E) were calculated from the slope of the relationship log input cDNA vs. the threshold cycle (Ct) for the serial dilution of a sample: E = 10–1/slope. Steady-state SLR mRNA levels were calculated relative to the 18S gene as in the method of Pfaffl (26). Relative expression was calculated from the Ct for SLR and 18S from a given sample and PCR efficiencies of the SLR (ESLR) and 18S (E18S) amplifications: relative expression = [(E18S + 1) x Ct18S]/[(ESLR + 1) x CtSLR].
Statistics
Comparison of SLR gene expression in various tissues from male and female coho salmon was done using a two-way ANOVA followed by Bonferroni post hoc tests with Prism version 4.0. Differences between groups were considered significant at P < 0.05.
Results
Cloning and phylogenetic analysis of SLR
The SLR cDNA was cloned and sequenced to reveal that it consists of 2589 bp encoding 657 amino acid residues (Fig. 1). The deduced amino acid sequence of SLR is composed of a signal peptide (20 amino acids), an extracellular domain (228 amino acids), a single transmembrane region (23 amino acids), and an intracellular domain (386 amino acids). The amino acid sequence shows relatively high identity to GHRs (37–60% to mature GHR) and low identity to PRLRs across all species (28–33% to mature PRLR) (Table 1). In general, the deduced SLR protein has higher identity to nonsalmonid GHRs than it does to salmonid GHRs. The partial cDNA for the coho salmon SLR (371 bp) was 98% identical to the masu salmon SLR (data not shown). SLR has common features of a GHR including a FGEFS motif, three pairs of cysteine residues in the ECD, a single transmembrane region, and Box 1 and Box 2 regions in the intracellular domain (Fig. 1 and Table 2). A cytokine receptor domain (amino acid position 38–139) and fibronectin type III domain (amino acid position 137–230) were found in the ECD.
FIG. 1. Nucleotide and deduced amino acid sequences of masu salmon SLR. Nucleotides and amino acids are positively numbered beginning with the initiation methionine. The putative regions are as follows: signal peptide (underlined), conserved cysteine residues in the extracellular domain (circled), potential N-glycosylation sites (boxed in open squares), site B (bold), FGEFS motif (underlined with a dotted line), single transmembrane domain (underlined with two solid lines), Box 1 and Box 2 (boxed in shaded rectangles), tyrosine residues in the intracellular domain (triangles), and stop codon (*).
TABLE 1. Amino acid sequence identities of masu salmon SLR with GHRs and PRLRs
TABLE 2. Comparison of conserved regions among SLR, GHRs, and PRLR in teleosts
The deduced amino acid sequences of the SLR cDNA, and representative vertebrate and available teleost GHR and PRL sequences were used to infer a phylogeny of SLR (Fig. 2). This analysis clearly divided the sequences into GHR and PRL groups and also separated teleost GHR and PRLR groups from those of tetrapod vertebrates. SLR was included in the GHR group, and it was especially close to marine fish species and relatively distant from salmon and eel GHRs based on high bootstrap values.
FIG. 2. Phylogenetic analysis of masu salmon SLR sequences including other major vertebrate and all available teleost GHRs and PRLRs. The analysis used the mature proteins excluding the signal peptide. Numbers shown at each branch are bootstrap values. Branch lengths indicate proportionality to amino acid changes on the branch. Accession numbers of those proteins in National Center for Biotechnology Information database are shown in Table 1. Scale bar shows substitutions per site.
Expression of His-SLR-ECD in E. coli
The recombinant His-SLR-ECD protein was expressed in BL21 (DE3) pLysS E. coli cells, refolded, and purified. Most of His-SLR-ECD protein eluted from the diethylaminoethyl-52 column with 0.2 M NaCl. Purified His-SLR-ECD subjected to SDS-PAGE with or without ?-mercaptoethanol revealed a single band in reducing conditions (31.5 kDa) and two bands in nonreducing conditions (65 and 31 kDa) (Fig. 3).
FIG. 3. Results of 15% SDS-PAGE of purified SLR-ECD under reducing (1 ) and nonreducing conditions (2 ). The gel was stained with Coomassie Brilliant Blue. Numbers show the molecular weight (MW) of standards (M)
Saturation and competition experiments
To examine specific binding, a constant amount of His-SLR-ECD or solubilized liver membrane was incubated with increasing concentrations of 125I-labeled SL. Specific binding increased in a concentration-dependent manner and approached saturation at high concentrations of 125I-labeled SL (Fig. 4, A and B). The Scatchard plot showed a better fit in a single-binding-site model (R2 = 0.962) compared with a two-binding-site model (R2 = 0.693). The Ka of recombinant His-SLR-ECD was 2.60 x 109 M–1, and the binding capacity was 56.70 nM (Fig. 4C). The Ka and binding capacity of native SLR in solubilized liver membrane were similar to the purified recombinant His-SLR-ECD (Ka = 2.01 x 109 M–1 and 26.12 fmol/mg protein) (Fig. 4D). Specific binding was displaced by SL (ED50 = 3.9 ng/tube; 15.6 ng/ml) (Fig. 5) and higher concentrations of GH (ED50 = 31 ng/tube; 124 ng/ml) and PRL (ED50 = 3000 ng/tube; 12,000 ng/ml).
FIG. 4. A representative binding saturation curve using increasing amounts of 125I-labeled coho SL and either SLR-ECD (A) or coho salmon liver membrane (B). Each point represents the mean of duplicate determinations. Scatchard plots (C and D) were obtained from the data in A and B, respectively. Ka, Affinity constant; Bmax, maximum binding capacity.
FIG. 5. Competition of unlabeled coho salmon SL (), GH (), and PRL (). Each point represents the mean of duplicates. Recombinant SLR-ECD was used for this experiment.
Tissue distribution and abundance of SLR
The level of SLR mRNA in various tissues of coho salmon was detected and quantified by real-time RT-PCR (Fig. 6). Data were normalized to 18S mRNA levels; the Ct of 18S was similar among various tissues. No significant differences in the relative abundance of SLR mRNA in various tissues were found between males and females. All tested tissues expressed SLR, with the highest expression levels in liver and visceral fat. SLR mRNA abundance was lowest in kidney and spleen.
FIG. 6. Steady-state levels of SLR transcripts measured by real-time quantitative RT-PCR relative to 18S RNA in various tissues from coho salmon. The black column shows male and the stippled column shows female values. Each column represents mean ± SE (n = 4).
Discussion
In this study, the full-length SLR cDNA from masu salmon was cloned and found to be structurally similar to GHR and PRLR. The SLR shares common features of the class I cytokine receptors including a single transmembrane domain, extracellular N-terminal and intracellular C-terminal domains, a cytokine receptor region, a fibronectin type III domain, and conserved cysteine residues in the ECD (27). The SLR protein shares common features of a GHR including Box 1, Box 2, and FGEFS motif. However, the region of SLR that corresponds to site B of GHR differs from all related teleost GHRs. Site B of GHR has been suggested to be part of the GH-binding site (28). Thus, based on these comparisons coupled with the results of our ligand-binding studies (discussed below), the cloned cDNA appears to encode the previously undescribed SLR of teleosts.
Analysis of the evolution of the SL protein, the cognate ligand for SLR, shows clearly that SL evolved from a common ancestor of GH and PRL (29, 30). Although SL appears equally distinct from GH and PRL, in our analyses the SLR is clearly more closely related to the GHR than the PRLR. The absence of SL in tetrapods coupled with the presence of GH and PRL in all vertebrates indicates that GH and PRL and their receptors, by association, evolved early in the evolution of vertebrates and before the rise of tetrapods. Our analysis of the SLR suggests that it evolved from the GHR rather than the PRLR, although more SLR sequences from other species are needed to reveal these evolutionary relationships.
Recombinant His-SLR-ECD was used to determine ligand specificity, particularly whether it was simply another variant of the GHR or PRLR. The His-SLR-ECD revealed high-affinity, saturable binding to 125I-labeled SL. Although members of this receptor family often have two hormone binding sites, Scatchard analysis of the His-SLR-ECD indicated a best fit with a single-binding-site model. In most cases of interactions between ligand and receptor(s), the 1:2 complex rapidly dissociates into the 1:1 complex as shown in rat, rabbit, cattle, and man (31). Therefore, the Scatchard plot of the His-SLR-ECD might appear as the single-site binding-site model similar to what was found for the rabbit GHR (32) and the trout PRLR (33). The affinity of SL binding to His-SLR-ECD (Ka = 2.6 x 109 M–1) was similar to that observed for SL binding to salmon liver membranes (Ka = 2.01 x 109 M–1), GH binding to trout GHR [0.7 x 109 M–1 (24); 2.4 x 109 M–1 (34)], and PRL binding to tilapia PRLR [1.7 x 109 M–1 (35)]. This similar binding affinity suggests that the recombinant His-SLR-ECD was expressed and refolded correctly. The binding capacity of His-SLR-ECD was 56.70 nM, which accounts for approximately half of the added SL, and indicated that only 57% of the recombinant His-SLR-ECD was active. Analysis of the recombinant His-SLR-ECD by SDS-PAGE revealed a singe band of 31.5 kDa under reducing conditions and bands of 31.5 and 65 kDa under nonreducing conditions. Presumably, the larger band is a dimer formed by a disulfide bond between unpaired cysteine residues, which could not be removed by our purification methods. Therefore, the lower than expected binding capacity is probably because of this dimerization of the His-SLR-ECD.
Competitive binding analysis of the SLR showed that SL was most effective in displacing labeled SL. The 50% displacement of labeled SL by GH was achieved at a 7.9-fold higher concentration of GH compared with SL, whereas PRL required a 769-fold higher concentration for equivalent displacement. In addition to verifying that this putative SLR is not just a variant form of GHR, these results suggest that SLR is relatively less specific for differentiating between SL and GH compared with the GHR, which is highly specific for GH (17). Thus, it is possible that under normal physiological conditions, some GH may bind the SLR and that SL and GH may functionally interact through the SLR. For example, during fasting, plasma GH levels increase to 100 ng/ml, whereas SL levels remain relatively low (around 10 ng/ml) (Ref. 36 ; our unpublished data). After many weeks of fasting, it is possible that GH may have significant interaction with the SLR. The relative differences in specificity between GH and SL for GHR and SLR are reminiscent of the salmon gonadotropins and their receptors. The FSH receptor binds both salmon FSH and LH, whereas the LH receptor is highly specific for LH (37).
To shed light on possible functions of SL, levels of SLR mRNA were measured by real-time quantitative PCR in various tissues. The SLR gene was expressed in all tissues, with the highest levels in liver and visceral fat, which suggests that liver and visceral fat are major SL target organs. Although the proposed functions of SL are quite varied (see Introduction), recent work on Mediterranean fishes suggests that SL may play a role in seasonal variation in lipid metabolism (16). Furthermore, injection of SL into European sea bass (Dicentrarchus labrax) inhibits hepatic acetyl-coenzyme A carboxylase and decreases the respiratory quotient, suggesting activation of lipid catabolism (2). The involvement of SL in lipid metabolism has also been suggested by the study of cobalt rainbow trout, which lacks SL-producing cells in the pituitary and accumulates a large amount of ip fat tissue (38). Combined with the physiological information, the SLR mRNA distribution data support the concept that SL has a major function in regulating lipolysis in the liver and visceral adipose tissue. Furthermore, the presence of SLR transcripts in the kidney, spleen, and gonad is consistent with observed effects of SL on ion transport, immune function, and steroidogenesis.
In summary, a SLR cDNA was cloned for the first time from masu salmon, and structural analyses indicate that SLR is a new member of the class I cytokine-type receptor family. High levels of SLR transcripts in liver and visceral fat support the notion that a major function of SL is regulation of lipid metabolism. The availability of SLR sequence and methods to quantify SLR transcripts should provide opportunities to determine the precise biological actions of SL at the cellular level in fishes, and may lead to the discovery of novel receptors for this hormone family in other vertebrates.
Acknowledgments
We are grateful Dr. M. Rand-Weaver for the gift of SL antiserum. We also thank Dr. Munetaka Shimizu, Mr. J. T. Dickey, and Ms. K. Cooper for their assistance.
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Address all correspondence and requests for reprints to: Walton W. Dickhoff, School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195. E-mail: walton.w.dickhoff@noaa.gov; address reprint requests to: Haruhisa Fukada, Faculty of Agriculture, B200 Monobe, Nankoku, Kochi 783-8502, Japan.
Abstract
Somatolactin (SL) is a pituitary hormone of the GH/prolactin (PRL) family that so far has been found only in fish. Compared with GH and PRL, the primary structure of SL is highly conserved among divergent fish species, suggesting it has an important function and a discriminating receptor that constrains structural change. However, SL functions are poorly understood, and receptors for SL have not yet been identified. During cloning of GH receptor cDNA from salmon, we found a variant with relatively high (38–58%) sequence identity to vertebrate GH receptors and low (28–33%) identity to PRL receptors; however, the recombinant protein encoding the extracellular domain showed only weak binding of GH. Ligand binding of the recombinant extracellular domain for this receptor confirmed that the cDNA encoded a specific receptor for SL. The SL receptor (SLR) has common features of a GH receptor including FGEFS motif, six cysteine residues in the extracellular domain, a single transmembrane region, and Box 1 and 2 regions in the intracellular domain. These structural characteristics place the SLR in the cytokine receptor type I homodimeric group, which includes receptors for GH, PRL, erythropoietin, thrombopoietin, granulocyte-colony stimulating factor, and leptin. Transcripts for SLR were found in 11 tissues with highest levels in liver and fat, supporting the notion that a major function of SL is regulation of lipid metabolism. Cloning SLR cDNA opens the way for discovery of new SL functions and target tissues in fish, and perhaps novel members of this receptor family in other vertebrates.
Introduction
SOMATOLACTIN (SL), WHICH is a relatively recently discovered member of the GH/prolactin (PRL)/placental lactogen family, is produced by the pars intermedia of the pituitary gland of fish. SL was first isolated from Atlantic cod Gadus morhua (1) and more recently identified in several species of teleosts (2, 3), sturgeon, and lungfish (4). Thus, SL is present in the lineage leading to tetrapods, but so far it has been found only in fish. The structure of SL is similar to vertebrate GH and PRL, with an average amino acid sequence identity of 24% (5); however, it is more highly conserved than GH and PRL even among divergent lineages of fish (4). Physiological studies have suggested that SL, like vertebrate GH and PRL, may have a multitude of biological functions. The measurement of plasma SL levels in salmonids suggests that it might be involved in gonadal maturation (5, 6), smoltification (7), and stress responses (8). Other physiological effects of SL seen in teleosts include immune function (9), acid base balance (10), background adaptation (11), energy mobilization (8, 12), gonadal steroid biosynthesis (6), and phosphate (13), sodium (14), and calcium (15) metabolism. Genetic evidence from medaka indicates a role for SL in pigment cell proliferation (3). Recently, it has been suggested that the dominant role of SL is controlling lipolysis during and after the transition from the growing to the nongrowing season of temperate fish (16).
Despite these observed physiological effects of SL, the precise target cells and biological actions of SL remain unclear, and it is not known whether SL has a receptor distinct from GH and PRL. Thus far, there is no information about the SL receptor (SLR) either from classical ligand-binding studies or gene cloning. Understanding the biochemical nature of the SLR would pave the way for new studies of specific actions of SL at the cellular level. In the course of cloning the salmon GH receptor (GHR) cDNA, we found two cDNAs with high sequence homology to fish GHRs. The putative proteins of the two cDNAs differed from each other and revealed a structure typical of the cytokine receptor family. The deduced amino acid sequences indicated relatively high identity to other vertebrate GHRs and lower identity to other vertebrate PRL receptors (PRLR). Ligand-binding studies using the recombinant protein encoding the extracellular domain (ECD) of the receptor revealed one of the two receptors was the salmon GHR (17). The ECD of the other GHR-like gene showed weak binding to GH; however, the specific GH binding did not increase in a concentration-dependent manner as would be expected for a GHR (our unpublished data). These data suggested that the ligand for the other GHR-like cDNA might be the SLR.
In this study, the complete primary sequence of a masu salmon SLR was cloned. The ligand specificity of SLR was confirmed by ligand-binding assays using a recombinant protein encoding the ECD of the receptor. In addition, the tissue distribution and abundance of SLR mRNA in various tissues was determined by real-time quantitative RT-PCR.
Materials and Methods
Experimental animals and blood and tissue samples
The fish used for cDNA cloning were 2-yr-old masu salmon (Oncorhynchus masou), which were reared at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University (Hokkaido, Japan). Fish were held in outdoor concrete ponds supplied with a continuous flow of river water at ambient temperature and photoperiod. They were fed commercial trout food (Oriental Yeast Co. Ltd., Tokyo, Japan) twice per day. Livers were collected from fish after euthanasia with 0.1% 2-phenoxyethanol (Kanto Kagaku, Tokyo, Japan) and then stored at –80 C.
The tissue distribution of SLR gene expression was determined using 1-yr-old coho salmon (Oncorhynchus kisutch), reared in recirculated fresh water at 11 C at the Northwest Fisheries Science Center (Seattle, WA). Fish of the same age were used for cloning of the partial cDNA of the coho salmon hepatic SLR. The fish received a commercial pellet diet (Biodiet Grower; Bioproducts Inc., Warrenton, OR) ad libitum once per day. After euthanasia with 0.1% tricane methanesulfonate (MS-222; Argent Chemical Laboratories, Redmond, WA), tissues were collected and then stored at –80 C. Animal-use protocols were approved by the University of Washington Institutional Animal Care and Use Committee.
Cloning of masu salmon SLR cDNA and partial cloning of coho salmon SLR cDNA
Total RNA of the liver was isolated using Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. RT was carried out with 3 μg total RNA, 50 ng random hexamers, and 200 U Maloney murine leukemia virus reverse transcriptase, RNase H minus, point mutant (Promega, Madison, WI). The cDNA fragment of masu salmon SLR was obtained by PCR using first-strand cDNA of masu salmon liver as a template. Degenerate primers were designed on the basis of conserved amino acid sequences of GHRs available in the GenBank database. The forward primer was 5'-GAC TGG AA(A/G) GA(A/G) TGT CCG GAT TAC-3' (designed from DWKECPDY sequence in the ECD), and the reverse primer was 5'-AAG GA(C/T) GA(C/T) GA(C/T) TCG GGG CGC GC-3' (designed from MDFYAQV sequence in the intracellular domain). PCR amplification was performed in a 50-μl volume using Hotstar Taq DNA polymerase (QIAGEN, Valencia, CA), and 35 cycles were run as follows after incubation for activation of Taq polymerase (95 C, 15min): 94 C for 30sec, 55 C for 30 sec, and 72 C for 1 min. Finally, the temperature was held at 72 C for 11 min. After electrophoresis with 1.5% agarose gel, a single band was observed, which was extracted from the agarose using QIAEXII (QIAGEN). The purified PCR product was ligated to an Easy T-Vector (Promega) and transfected into XL-I blue Escherichia coli cells (Novagen, Madison, WI). After incubation of colonies, plasmid vectors were purified and sequenced by the dye-termination method using ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA).
Full-length SLR was obtained by 5' rapid amplification of cDNA ends (5'-RACE) and 3'-RACE using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA). The first-strand cDNA of masu salmon liver was used as a template. Two reverse gene primers were designed for 5'-RACE as follows: GSP-1, 5'-CAA GGT ATC GTA GGT TTT GTT CTG GCT-3' (nucleotide position 355–381), and GSP-2, 5'-CGT CAA GTC ATT CTT CTT CCA GTA CTG-3' (nucleotide position 217–243). First PCR was done using GSP-1 and universal primer mix A (UPM). To obtain more of the upstream coding sequence, GSP-2 and UPM were used in a second PCR. The primers for 3'-RACE were as follows: GSP-3, 5'-CTG GAG GTC CCA TGC CCC GGG CTC CAG-3' (nucleotide position 1282–1308), and GSP-4, 5'-CGC CTT CTG CCG AGA GCA AGC CCC ACC AG-3' (nucleotide position 1682–1710). The first PCR of 3'-RACE was done using GSP-3 and UPM. To obtain more of the downstream coding sequence, GSP-4 and UPM were used in PCR. Finally, the open reading frame was amplified by PCR using SLR 5' untranslated region 5'-TGA CAT CTT TGT GTT AGC AAA GGA AAG C-3' (nucleotide position –38 to –10) and SLR 3' untranslated region 5'-ATG ACA AAT GGC TTC CAA CCT CTA CG-3' (nucleotide position 2006–2031) with High Fidelity Platinum Taq polymerase (Invitrogen, Carlsbad, CA). All PCR products were subcloned into the pGEM Easy T-vector and sequenced by the dideoxy chain termination method (ABI 310).
Cloning of the partial cDNA encoding the coho salmon SLR was done to provide sequence design of primers and probes for real-time quantitative RT-PCR. Total RNA was isolated from the liver using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) followed by the Molecular Research Center protocol, and the cDNA was synthesized as described above. The PCR was done using the following primers: 5'-CAC TGT GCA ATG AGG GCC TTC AA-3' (forward; nucleotide position 655–677) and 5'-GAC CCA GCA GTC TCT GTA AGT CTG AAC-3' (reverse; nucleotide position 1052–1078). The PCR products were sequenced by the dideoxy chain termination method (ABI 3100).
Structural and phylogenetic tree analyses
Predictions of a signal peptide, transmembrane region, and potential N-glycosylation sites were performed using prediction server of the Center for Biological Sequence Analysis (www.cbs.dtu.dk) based on the deduced amino acid sequences. Conserved peptide domains (fibronectin type III domain and cytokine receptor domain) were predicted by prsBLAST from the National Center for Biotechnology Information database (NCBI) (www.ncbi.nlm.nih.go) and by InterProscan from European Molecular Biology Laboratory-European Bioinformatics Institute (www.ebi.ac.uk), respectively. Multiple amino acid sequence alignments were constructed using the ClustalW from the DNA Data Bank of Japan website (www.ddbj.nig.ac.jp) (18). After manual correction of the alignments, the amino acid sequences were subjected to the ClustalW analysis to construct a phylogenetic tree using a neighbor-joining method (19). The ClustalW analysis was performed using default settings except for gaps that were treated as missing characters. Relative branch support was evaluated by bootstrap analysis. NJ plot software (http://pbil.univ-lyon1.fr/software/njplot.html) was used to prepare a graphical view of the phylogenetic tree (20).
Expression, refolding, and purification of the recombinant ECD of SLR (SLR-ECD)
The fragment encoding the ECD of SLR was amplified by PCR using the full-length SLR cDNA as the template. Primers for SLR-ECD were as follows: SLR-ECD (F; nucleotide position 61–83), 5'-TCC TCG CTG ATG GAC CCT GGC TC-3', and SLR-ECD (R; nucleotide position 721–745), 5'-CGT TGA CTC TTT ATT GGG AAT CTC-3'. PCR was done using proofreading Taq polymerase (Advantage 2 polymerase; Clontech). PCR products were cloned to the pCR-T7 vector (Invitrogen) and transfected to Top-10F' E. coli cells. Vectors from positive clones were purified and transformed into the BL21(DE3) pLysS E. coli strain for protein expression.
Transfected cells were grown in 100 ml of LB-broth medium containing 20 μg/ml ampicillin at 37 C in a 250-ml flask. When the absorbance at 600 nm reached 0.4, isopropylthiol-?-D-galactoside was added to the tube at a final concentration of 0.4 mM. Cells were grown for an additional 4 h and then collected by centrifugation at 5000 x g for 15 min and frozen at –30 C. Inclusion bodies were purified using BugBuster protein extraction reagent (Novagen). Refolding and purification of histidine-tagged SLR-ECD (His-SLR-ECD) were done as described in our previous report (17). Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL) using BSA as a standard.
Electrophoresis
SDS-PAGE was carried out according to Laemmli using a 15% acrylamide gel. The gel was stained with Bio-Safe colloidal Coomassie brilliant blue G-250 (Bio-Rad, Hercules, CA). Sizes of recombinant proteins were estimated using the following standards (Amersham Bioscience Corp, Piscataway, NJ): -lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), albumin (67 kDa), and phosphorylase b (94 kDa).
Hormones
Native GH and PRL were purified from pituitaries of coho salmon according to the methods of Jackson et al. (21). Purification of native SL was performed according to the method of Rand-Weaver et al. (1). The purity of the proteins obtained by this method was estimated to be more than 95% by SDS-PAGE. This is similar to that obtained using the same method for purification of GH, PRL, and SL from many species of fish (see Ref. 22 for example).
Iodination of SL
Iodination of SL was performed by the chloramine T method according to the method of Rand-Weaver et al. (5). The specific activity of labeled hormone was determined by a self-displacement method (23) using recombinant His-SLR-ECD (specific activity: 10.2 μCi/μg).
Binding assay
Liver membranes from coho salmon were prepared according to Yao and Le Bail (24). Purified recombinant His-SLR-ECD or liver membranes and 125I-labeled SL were used for the binding tests. Recombinant His-SLR-ECD (25 pmol) in assay buffer (10 mM Tris-HCl, pH 8.0, containing 0.1% BSA, 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride) was incubated with 125I-labeled SL (40,000 cpm) in duplicate in the absence (total binding) or presence (nonspecific binding) of 2 μg of unlabeled SL in a final volume of 250 μl. After incubation at room temperature for 20 h, free 125I-labeled SL was separated according to the method of Sandowski et al. (25). Radioactivity of the pellets was measured in a -counter (3 min/tube). Specific binding was calculated by subtracting the nonspecific binding from the total binding. Affinity constants (Ka) were estimated using GraphPad Prism software version 4.0 (GraphPad Software Inc., San Diego, CA).
Real-time quantitative RT-PCR for SLR
Total RNA was extracted from tissues using TriReagent described as above. Integrity of the RNA was verified by an OD absorption ratio of OD 260 nm/OD 280 nm greater than 1.9, and 300 ng of total RNA was used for the synthesis of the first-strand cDNA. RT was performed in a reaction mixture containing 0.5 mM dNTPs, 112.5 μg random hexamers (Promega), 6 U RNase inhibitor (ABI), 10 mM dithiothreitol, 37.5 U Maloney murine leukemia virus reverse transcriptase (SuperScript II; Invitrogen), and 1x RT buffer for SuperScript II in a final volume of 15 μl. RT was performed for 10 min at 25 C, followed by 60 min at 48 C, and finally for 5 min at 95 C.
For design of specific primers and probes for the assay of coho salmon SLR, SLR cDNA was partially cloned from liver of coho salmon. Primers and probe for real-time quantitative PCR were designed by primer express program (ABI) as follows: forward primer, 5'-CAG CAT TGC TTA AGA AGG GAA AG-3'; reverse primer, 5'-TGG AGA GCC CGC ATA CCA-3'; and probe, 5'-FAM-CCA CTC AGG CTG AAG TTC AGC TCG TCC A-TAMRA-3'. The primers and probe were designed to a region in the intracellular domain. The predicted length of the amplicon is 73 bp. The forward primer spans a predicted intron/exon boundary to avoid amplification of genomic DNA. For normalization of data, an 18S ribosomal gene was used. Primers and probes for 18S were purchased from ABI. PCR (25 μl) contained 12.5 μl TaqMan Universal PCR Master Mix (ABI), 3 μl of the first-strand cDNA for SLR assay, or 2 μl of a 1/100 dilution for the first-strand cDNA for the 18S assay, 0.9 μM forward and reverse primers, and 0.2 μM probe. Amplification and detection of samples were performed with the ABI 7700 system using the following thermal cycling conditions: 50 C for 2 min, 95 C for 15 sec, and 60 C for 1 min (40 cycles). Four serial dilutions of cDNA were run to determine PCR efficiency. The efficiencies (E) were calculated from the slope of the relationship log input cDNA vs. the threshold cycle (Ct) for the serial dilution of a sample: E = 10–1/slope. Steady-state SLR mRNA levels were calculated relative to the 18S gene as in the method of Pfaffl (26). Relative expression was calculated from the Ct for SLR and 18S from a given sample and PCR efficiencies of the SLR (ESLR) and 18S (E18S) amplifications: relative expression = [(E18S + 1) x Ct18S]/[(ESLR + 1) x CtSLR].
Statistics
Comparison of SLR gene expression in various tissues from male and female coho salmon was done using a two-way ANOVA followed by Bonferroni post hoc tests with Prism version 4.0. Differences between groups were considered significant at P < 0.05.
Results
Cloning and phylogenetic analysis of SLR
The SLR cDNA was cloned and sequenced to reveal that it consists of 2589 bp encoding 657 amino acid residues (Fig. 1). The deduced amino acid sequence of SLR is composed of a signal peptide (20 amino acids), an extracellular domain (228 amino acids), a single transmembrane region (23 amino acids), and an intracellular domain (386 amino acids). The amino acid sequence shows relatively high identity to GHRs (37–60% to mature GHR) and low identity to PRLRs across all species (28–33% to mature PRLR) (Table 1). In general, the deduced SLR protein has higher identity to nonsalmonid GHRs than it does to salmonid GHRs. The partial cDNA for the coho salmon SLR (371 bp) was 98% identical to the masu salmon SLR (data not shown). SLR has common features of a GHR including a FGEFS motif, three pairs of cysteine residues in the ECD, a single transmembrane region, and Box 1 and Box 2 regions in the intracellular domain (Fig. 1 and Table 2). A cytokine receptor domain (amino acid position 38–139) and fibronectin type III domain (amino acid position 137–230) were found in the ECD.
FIG. 1. Nucleotide and deduced amino acid sequences of masu salmon SLR. Nucleotides and amino acids are positively numbered beginning with the initiation methionine. The putative regions are as follows: signal peptide (underlined), conserved cysteine residues in the extracellular domain (circled), potential N-glycosylation sites (boxed in open squares), site B (bold), FGEFS motif (underlined with a dotted line), single transmembrane domain (underlined with two solid lines), Box 1 and Box 2 (boxed in shaded rectangles), tyrosine residues in the intracellular domain (triangles), and stop codon (*).
TABLE 1. Amino acid sequence identities of masu salmon SLR with GHRs and PRLRs
TABLE 2. Comparison of conserved regions among SLR, GHRs, and PRLR in teleosts
The deduced amino acid sequences of the SLR cDNA, and representative vertebrate and available teleost GHR and PRL sequences were used to infer a phylogeny of SLR (Fig. 2). This analysis clearly divided the sequences into GHR and PRL groups and also separated teleost GHR and PRLR groups from those of tetrapod vertebrates. SLR was included in the GHR group, and it was especially close to marine fish species and relatively distant from salmon and eel GHRs based on high bootstrap values.
FIG. 2. Phylogenetic analysis of masu salmon SLR sequences including other major vertebrate and all available teleost GHRs and PRLRs. The analysis used the mature proteins excluding the signal peptide. Numbers shown at each branch are bootstrap values. Branch lengths indicate proportionality to amino acid changes on the branch. Accession numbers of those proteins in National Center for Biotechnology Information database are shown in Table 1. Scale bar shows substitutions per site.
Expression of His-SLR-ECD in E. coli
The recombinant His-SLR-ECD protein was expressed in BL21 (DE3) pLysS E. coli cells, refolded, and purified. Most of His-SLR-ECD protein eluted from the diethylaminoethyl-52 column with 0.2 M NaCl. Purified His-SLR-ECD subjected to SDS-PAGE with or without ?-mercaptoethanol revealed a single band in reducing conditions (31.5 kDa) and two bands in nonreducing conditions (65 and 31 kDa) (Fig. 3).
FIG. 3. Results of 15% SDS-PAGE of purified SLR-ECD under reducing (1 ) and nonreducing conditions (2 ). The gel was stained with Coomassie Brilliant Blue. Numbers show the molecular weight (MW) of standards (M)
Saturation and competition experiments
To examine specific binding, a constant amount of His-SLR-ECD or solubilized liver membrane was incubated with increasing concentrations of 125I-labeled SL. Specific binding increased in a concentration-dependent manner and approached saturation at high concentrations of 125I-labeled SL (Fig. 4, A and B). The Scatchard plot showed a better fit in a single-binding-site model (R2 = 0.962) compared with a two-binding-site model (R2 = 0.693). The Ka of recombinant His-SLR-ECD was 2.60 x 109 M–1, and the binding capacity was 56.70 nM (Fig. 4C). The Ka and binding capacity of native SLR in solubilized liver membrane were similar to the purified recombinant His-SLR-ECD (Ka = 2.01 x 109 M–1 and 26.12 fmol/mg protein) (Fig. 4D). Specific binding was displaced by SL (ED50 = 3.9 ng/tube; 15.6 ng/ml) (Fig. 5) and higher concentrations of GH (ED50 = 31 ng/tube; 124 ng/ml) and PRL (ED50 = 3000 ng/tube; 12,000 ng/ml).
FIG. 4. A representative binding saturation curve using increasing amounts of 125I-labeled coho SL and either SLR-ECD (A) or coho salmon liver membrane (B). Each point represents the mean of duplicate determinations. Scatchard plots (C and D) were obtained from the data in A and B, respectively. Ka, Affinity constant; Bmax, maximum binding capacity.
FIG. 5. Competition of unlabeled coho salmon SL (), GH (), and PRL (). Each point represents the mean of duplicates. Recombinant SLR-ECD was used for this experiment.
Tissue distribution and abundance of SLR
The level of SLR mRNA in various tissues of coho salmon was detected and quantified by real-time RT-PCR (Fig. 6). Data were normalized to 18S mRNA levels; the Ct of 18S was similar among various tissues. No significant differences in the relative abundance of SLR mRNA in various tissues were found between males and females. All tested tissues expressed SLR, with the highest expression levels in liver and visceral fat. SLR mRNA abundance was lowest in kidney and spleen.
FIG. 6. Steady-state levels of SLR transcripts measured by real-time quantitative RT-PCR relative to 18S RNA in various tissues from coho salmon. The black column shows male and the stippled column shows female values. Each column represents mean ± SE (n = 4).
Discussion
In this study, the full-length SLR cDNA from masu salmon was cloned and found to be structurally similar to GHR and PRLR. The SLR shares common features of the class I cytokine receptors including a single transmembrane domain, extracellular N-terminal and intracellular C-terminal domains, a cytokine receptor region, a fibronectin type III domain, and conserved cysteine residues in the ECD (27). The SLR protein shares common features of a GHR including Box 1, Box 2, and FGEFS motif. However, the region of SLR that corresponds to site B of GHR differs from all related teleost GHRs. Site B of GHR has been suggested to be part of the GH-binding site (28). Thus, based on these comparisons coupled with the results of our ligand-binding studies (discussed below), the cloned cDNA appears to encode the previously undescribed SLR of teleosts.
Analysis of the evolution of the SL protein, the cognate ligand for SLR, shows clearly that SL evolved from a common ancestor of GH and PRL (29, 30). Although SL appears equally distinct from GH and PRL, in our analyses the SLR is clearly more closely related to the GHR than the PRLR. The absence of SL in tetrapods coupled with the presence of GH and PRL in all vertebrates indicates that GH and PRL and their receptors, by association, evolved early in the evolution of vertebrates and before the rise of tetrapods. Our analysis of the SLR suggests that it evolved from the GHR rather than the PRLR, although more SLR sequences from other species are needed to reveal these evolutionary relationships.
Recombinant His-SLR-ECD was used to determine ligand specificity, particularly whether it was simply another variant of the GHR or PRLR. The His-SLR-ECD revealed high-affinity, saturable binding to 125I-labeled SL. Although members of this receptor family often have two hormone binding sites, Scatchard analysis of the His-SLR-ECD indicated a best fit with a single-binding-site model. In most cases of interactions between ligand and receptor(s), the 1:2 complex rapidly dissociates into the 1:1 complex as shown in rat, rabbit, cattle, and man (31). Therefore, the Scatchard plot of the His-SLR-ECD might appear as the single-site binding-site model similar to what was found for the rabbit GHR (32) and the trout PRLR (33). The affinity of SL binding to His-SLR-ECD (Ka = 2.6 x 109 M–1) was similar to that observed for SL binding to salmon liver membranes (Ka = 2.01 x 109 M–1), GH binding to trout GHR [0.7 x 109 M–1 (24); 2.4 x 109 M–1 (34)], and PRL binding to tilapia PRLR [1.7 x 109 M–1 (35)]. This similar binding affinity suggests that the recombinant His-SLR-ECD was expressed and refolded correctly. The binding capacity of His-SLR-ECD was 56.70 nM, which accounts for approximately half of the added SL, and indicated that only 57% of the recombinant His-SLR-ECD was active. Analysis of the recombinant His-SLR-ECD by SDS-PAGE revealed a singe band of 31.5 kDa under reducing conditions and bands of 31.5 and 65 kDa under nonreducing conditions. Presumably, the larger band is a dimer formed by a disulfide bond between unpaired cysteine residues, which could not be removed by our purification methods. Therefore, the lower than expected binding capacity is probably because of this dimerization of the His-SLR-ECD.
Competitive binding analysis of the SLR showed that SL was most effective in displacing labeled SL. The 50% displacement of labeled SL by GH was achieved at a 7.9-fold higher concentration of GH compared with SL, whereas PRL required a 769-fold higher concentration for equivalent displacement. In addition to verifying that this putative SLR is not just a variant form of GHR, these results suggest that SLR is relatively less specific for differentiating between SL and GH compared with the GHR, which is highly specific for GH (17). Thus, it is possible that under normal physiological conditions, some GH may bind the SLR and that SL and GH may functionally interact through the SLR. For example, during fasting, plasma GH levels increase to 100 ng/ml, whereas SL levels remain relatively low (around 10 ng/ml) (Ref. 36 ; our unpublished data). After many weeks of fasting, it is possible that GH may have significant interaction with the SLR. The relative differences in specificity between GH and SL for GHR and SLR are reminiscent of the salmon gonadotropins and their receptors. The FSH receptor binds both salmon FSH and LH, whereas the LH receptor is highly specific for LH (37).
To shed light on possible functions of SL, levels of SLR mRNA were measured by real-time quantitative PCR in various tissues. The SLR gene was expressed in all tissues, with the highest levels in liver and visceral fat, which suggests that liver and visceral fat are major SL target organs. Although the proposed functions of SL are quite varied (see Introduction), recent work on Mediterranean fishes suggests that SL may play a role in seasonal variation in lipid metabolism (16). Furthermore, injection of SL into European sea bass (Dicentrarchus labrax) inhibits hepatic acetyl-coenzyme A carboxylase and decreases the respiratory quotient, suggesting activation of lipid catabolism (2). The involvement of SL in lipid metabolism has also been suggested by the study of cobalt rainbow trout, which lacks SL-producing cells in the pituitary and accumulates a large amount of ip fat tissue (38). Combined with the physiological information, the SLR mRNA distribution data support the concept that SL has a major function in regulating lipolysis in the liver and visceral adipose tissue. Furthermore, the presence of SLR transcripts in the kidney, spleen, and gonad is consistent with observed effects of SL on ion transport, immune function, and steroidogenesis.
In summary, a SLR cDNA was cloned for the first time from masu salmon, and structural analyses indicate that SLR is a new member of the class I cytokine-type receptor family. High levels of SLR transcripts in liver and visceral fat support the notion that a major function of SL is regulation of lipid metabolism. The availability of SLR sequence and methods to quantify SLR transcripts should provide opportunities to determine the precise biological actions of SL at the cellular level in fishes, and may lead to the discovery of novel receptors for this hormone family in other vertebrates.
Acknowledgments
We are grateful Dr. M. Rand-Weaver for the gift of SL antiserum. We also thank Dr. Munetaka Shimizu, Mr. J. T. Dickey, and Ms. K. Cooper for their assistance.
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