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Department of Biochemistry and Molecular Biology, University of New Hampshire, Room 310, 46 College Road, Durham, New Hampshire 03824, USA
1 Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan
2 Sado Marine Station, Niigata University, Sado, Niigata 952-2135, Japan
(Requests for offprints should be addressed to S A Sower; Email: sasower{at}cisunix.unh.edu)
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Introduction |
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-helices, connected by intra- and extracellular loops and an intracellular C-terminal domain (Grossmann et al. 1997, Dufau 1998, Ascoli et al. 2002, Moyle et al. 2005). To date, approximately 79 GpHRs have been identified and described in 36 different species, mostly in mammals and also in three species of birds, two species of reptiles, one amphibian, and ten species of fish (Hovergen Database, http://pbil.univ-lyon1.fr). There have been no GpH-Rs described in any species of agnathans.
The glycoprotein hormone family consists of two gonadotropin hormones (GTHs), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and one thyroid-stimulating hormone (TSH). They are part of the cystine-knot family of growth factors with two non-covalently bound subunits, and ß. The subunit is common within a single species (Kawauchi et al. 1989). The ß subunits are homologous and convey hormone specificity (Themmen & Huhtaniemi 2000, Szkudlinski et al. 2002, Swanson et al. 2003). Therefore, these glycoprotein hormones are believed to have evolved from a common ancestral molecule through duplication of ß-subunit genes and subsequent divergence (Moyle et al. 1994, Li & Ford 1998). Two GTHs have been identified in all taxonomic groups of gnathostomes, including actinopterygians (Suzuki et al. 1988, Kawauchi et al. 1989, Querat et al. 2000), sarcopterygians (Querat et al. 2004), and chondrichthyans (Querat et al. 2000), and only recently in jawless vertebrates (agnathans) (Sower et al. 2006).
Lampreys (Petromyzontidae), along with hagfish (Myxinae), are the only living representatives of the agnathans, the most ancient class of vertebrates, whose lineage dates back over 530 million years (Kuratani et al. 2002). Although the problem of the evolutionary relationship between these two surviving groups of agnathans has not yet come to a definitive conclusion, it is well accepted that they form a monophyleticgroup to therest of vertebrates –the gnathostomes. Lamprey and gnathostomes share the same jawless ancestors and this is of foremost importance for the contribution of the study of lamprey in understanding the evolutionary mechanisms which led to the definition of the functional architecture of endocrine control in vertebrates. This is particularly true for the pituitary GpH/GpH-R pairs, since it is estimated that the structural and functional divergence of GpH-Rs coincide with the geological time of divergence of the gnathostomes from their jawless ancestors.
Lampreys, which express two forms of gonadotropin releasing hormone (GnRH), lamprey GnRH-I and -III (Gorbman & Sower 2003), are important to our understanding of the reproductive success of the first vertebrates and are likely to have retained key characteristics of the ancestral GTH and GTH-R(s) from which modern GTH and GTH-Rs arose. Although the lamprey GnRH-I and -III with a known role in the hypothalamic–pituitary–gonadal axis of this animal have been well demonstrated (Sower 2003), the lamprey homologs of gnathostome GTHs, TSHs, and their receptors have eluded numerous attempts in identification. Here, we report the identification of a functional lamprey GpH-R I (lGpH-R I) similar to gnathostome GTH-Rs, exhibiting its highest level of expression in the testis of the lamprey.
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Materials and methods |
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Fifty reproductive mature sea lampreys were collected from CochecoRiver fish ladder inDover, NH, USA during their upstream migration in May. They were transported to the Anadromous Fish and Aquatic Invertebrate Research (AFAIR) Laboratory of the University of New Hampshire and maintained under continuous flow of river water and aeration at ambient temperature until sampled. Sea lamprey tissue samples were collected at the UNH AFAIR laboratory. Approximately 100 mg tissue fragments were flash frozen in liquid nitrogen and then kept on dry ice until stored at –80 °C.
Degenerate primers design
Degenerate primers were designed using the PRINTS database (http://umber.sbs.man.ac.uk/dbbrowser/PRINTS/) GLYCHORMONER protein fingerprint alignment blocks as input. The PRINTS database contains protein fingerprints derived by extensive statistical analysis of protein alignments for identification of the alignment blocks (fingerprint elements), which uniquely identify the corresponding protein family (Attwood et al. 1998, 2003). Selection of primers was based on a simple optimality score calculated using the following formula: S=sum(si)/D, where S is the final optimality score for a given degenerate primer candidate, si are the scores assigned to each of the properties of the individual oligos corresponding to the degenerate primer candidate and D is the degeneracy of the primer.
RNA isolation and degenerate PCR amplification
Total RNA of tissues was extracted using the TRI-reagent (MRC, Cincinnati, OH, USA) method following the manufacturer’s protocol. Single-strand cDNA was synthesized from ca 1 µg testis total RNA using the avian myeloblastosis virus reverse transcriptase and the Not-I poly-A anchored reverse primer from Amersham. Amplification with degenerate primers (IDT, Coralville, IA, USA) was performed with 1.25 U Taq Polymerase (Promega Corp.) in 25 µl reaction volume containing 200 nM dNTP, 3 mM MgCl2, 0.5 µl GC-Melt (Clontech) and 0.4 µM of each primer under the following cycling conditions: (1) 30 min initial incubation at 65 °C, (2) initial denaturation 5 min/95 °C, and (3) 40 cycles with 1 min/94 °C denaturation, 30 s/65 °C annealing, 30 s/72 °C extension. The PCR products were separated on NuSieve low-melting temperature agarose gel (FMC BioProducts, Rockland, ME, USA) and excision of DNA bands was followed by in-gel ligation into the pGEM-T Easy vector (Clontech). The ligation product was then used for transformation of JM109 highly competent cells (Clontech), which were plated on Luria-Bertani (LB) plates treated with IPTG/X-Gal for blue/white colony screening. Positive colonies were grown overnight in LB medium with ampicillin. Plasmid DNA was isolated using the Wizard(r) DNA Purification System (Promega) and sequenced at the Sequencing Service of the University of Utah. At least two samples resulted from different amplification experiments were sequenced for each amplicon.
RT-PCR assay
Gene-specific primers derived from the transmembrane region of the putative lGpH-R identified by degenerate PCR were used for a semiquantitative estimation of the expression of the corresponding genes in various lamprey tissues (brain, heart, intestine, kidney, liver, muscle, testes, and thyroid) by RT-PCR. Total RNA was extracted using the TRI-reagent (MRC) method as previously described. The amount and quality of RNA were estimated by optical density measurements at 260 and 280 nm in 10 mM Tris buffer (pH 8.0) and by non-denaturing agarose gel electrophoresis. Ten micrograms total RNA samples were incubated with 1 IU RQ1 DNase (Promega) in PCR buffer for 2 h at 37 °C to remove any contaminating genomic DNA. Aliquots of the DNase-treated total RNA were run on a non-denaturing agarose gel and the concentration of RNA for PCR was normalized based on the relative intensity of the 28S rRNA bands. An alternate method for normalization of the total RNA amount has been employed by spotting equal volumes (1 µl) of the DNase-treated total RNA on an agarose gel. The integrated optical density of each spot was calculated after background subtraction (NIH/ImageJ application) and the obtained values were then used for correction of the concentrations of the total RNA samples used as templates in reverse transcriptase (RT)-PCR. The one-tube AccessQuick RT-PCR system (Promega) was used for amplification of a 377 bp fragment from the TM region of lGpH-R I (primers C2P6f/C2P2r). A no-(RT) control reaction was run for every sample. The PCR program was: initial denaturation 5 min/95 °C, denaturation 40 s/94 °C, annealing 30 s/63 °C, extension 45 s/72 °C for 40 cycles, and final extension step 5 min at 72 °C.
Cloning of full-length lGpH-R I cDNA
Ten micrograms lamprey testes total RNA was reverse transcribed at 55 °C for 1 h with SuperScript III RT (Invitrogen) in the presence of the CDS III primer and SMART IV template-switch oligo (Clontech) followed by 15-min incubation at the same temperature in the presence of 5 mM MnCl2 to enhance the template-switch extension of cDNA. The reaction product was then treated with RNase H to release the single-strand cDNA from the cDNA–RNA heteroduplexes. The 5' end of the lGpH-R I transcript was cloned by step-out RACE (SO-RACE), while the 3' end was amplified using a gene-specific forward primer and the CDS III primer (Clontech). The SO-RACE used a modified version of the two amplification stages protocol described in Matz et al.(2003). The PCR products whose agarose gel migration pattern matched the one predicted from the positioning of the gene-specific primers were isolated, cloned, and sequenced as described previously. The 5' and 3' fragments were assembled using the pregap4 and gap4 programs from the Staden Package (http://staden.sourceforge.net/; Staden 1996).
COS-7 expression construct
Gene-specific primers (lGpH-R I_TOPOf and lGpH-R I_TOPOr respectively) containing the START and STOP codons were designed and used for cloning of the full-coding sequence of lGpH-R I. Briefly, lamprey testes total RNA were reverse transcribed with SuperScript III as previously described and the cDNA used for PCR amplification of lGpH-R I full-coding sequence with Phusion High-Fidelity DNA Polymerase (Finnzymes/NEB, Ipswich, MA, USA). The PCR product was ligated into the pcDNA 3.1/V5-His-TOPO vector (Invitrogen), cloned in TOP10 competent cells and checked by sequencing using vector-specific sequencing primers as well as gene-specific primers to ensure full coverage of the lGpH-R I coding domain sequence (cds). The EndoFree Plasmid Maxi Kit (Qiagen) was used for preparation ofthe DNAfor mammaliancelltransfections.
Transient expression of lGpH-R I in COS-7 cells and cAMP accumulation assay
The COS-7 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cultures were transiently transfected with lGpH-R I construct and with blank pcDNA3.1 vector (negative control) using lipofectamine or lipofectamine 2000 methods (Invitrogen) in 24-well plates according to the manufacturer’s instructions. Efficiency of transfection was estimated by control transfections with pcDNA3.1/V5-His-LacZ vector followed by B-Gal staining (Invitrogen). The putative lamprey native ligand for this receptor was not available at the time of these experiments, so human chorionic gonadotropin (hCG, Sigma-Aldrich) was used instead for stimulation of transfected COS-7 cells. Intracellular accumulation of cAMP was estimated after 3-h incubation with various concentrations of hCG in Krebs–Ringer buffer containing 0.1% pyrogen free BSA (Calbiochem) and 0.1 mM IBMX (Sigma). The buffer was aspirated, cells lysed in 0.1 M HCl or in an Amersham proprietary lysis reagent and concentration of cAMP measured by radioimmunoassay (RIA). The 125I cAMP tracer and cAMP antiserum were a generous gift from Dr William Moyle (University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA). Results are expressed as fold increase over the basal level of the intracellular cAMP±S.E.M. versus the concentration of hCG in the stimulation medium.
Effect of lGpH-R on basal levels of intracellular cAMP
To estimate the effect of lGpH-R I on the basal production of cAMP in transiently transfected COS-7 cells a secreted alkaline phosphatase (SEAP)-based reporter system was used. It showed a higher sensitivity compared with the RIA procedure in experiments, where the rat LH-R was stimulated with hCG or ovine LH (data not shown). The reporter plasmid cAMP responsive elements (pCRE)-SEAP contains three CRE upstream from the gene encoding a modified human placental alkaline phosphatase, which is secreted into the culture medium upon stimulation by intracellular cAMP. COS-7 cells were co-transfected with various amounts of pcDNA3.1/lGpH-R I or empty pcDNA3.1 (0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 µg) and 0.8 µg pCRE-SEAP or pTAL-SEAP (negative control) in 24-well plates with 2 µl lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s protocol. After 24-h incubation, the culture medium was aspirated and replaced with low serum Opti-MEM medium and incubated overnight. The next day the medium was replaced with serum and phenol red free Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) containing 0.1% BSA (Calbiochem) with or without 0.1 mM IBMX. A third treatment consisted of 0.1% BSA, 0.1 mM IBMX, and 5 µM forskolin DMEM for each lGpH-R I vector amount for normalization of the transfection efficiencies. After 18-h incubation, the medium was collected, centrifuged for 10 min at 10 000 g at 4 °C then heated at 65 °C for 30 min to eliminate the intrinsic alkaline phosphatase activity. A 20 µl aliquot of each sample was then mixed with reaction buffer (50 mM Tris, pH 8.2) containing 28 mM pNPP (para-nitrophenyl phosphate) alkaline phosphatase chromogenic substrate and then incubated at 37 °C. The optical density was read at 405 nm on a microplate autoreader (BioTek, Instruments, Winooski, VT, USA) at approximately 3-h time intervals. The results reported here are derived from end-point optical density measurement data obtained after approximately 24-h incubation. Absorbance in each well was corrected by subtraction of the pTAL-SEAP corresponding measurement and normalized with respect to the corresponding response to forskolin treatment. The same procedural outline was applied for analysis of the effect of different glycoprotein hormone treatments(hCG,ovine LH,and ovine TSH)onactivation of cAMP-mediated signal transduction by lGpH-R I.
Software applications
Assembling of sequencing readings was performed with the pregap4 and gap4 programs from the Staden Package (Staden 1996). Sequence alignments were performed using the ClustalW (Thompson et al. 1994) method. The signal peptide cleavage site was predicted using the SignalP (Nielsen et al. 1997) program (http://www.cbs.dtu.dk/services/SignalP/). Trans-membrane regions were characterized from the thermodynamic profile of the region with programs from the European Molecular Biology Open Source Software (http://emboss.sourceforge.net/) (EMBOSS) package (octanol), the GPCR-specific Prosite (Bucher & Bairoch 1994) signature was also identified using the Prosite motif finder from EMBOSS package (patmatmo-tifs) with a locally installed copy of the Prosite database. For identification of protein motifs and patterns, the Prosite (http://ca.expasy.org/prosite) and InterPro (http://www.ebi.ac.uk/interpro/) search services were used with the lGpH-R I amino acid sequence as query. The maximum parsimony algorithm as implemented in the protpars application of the PHYLogeny Inference Package (PHYLIP; Felsenstein 1989) version 3.6 was applied to 1000 bootstrap replica (seqboot, PHYLIP package) of the initial amino acid sequence, then the consensus tree was derived using the consense program from the same package. Analysis of the RT-PCR gel images was performed with ImageJ application from NIH (Abramoff et al. 2004). Functional data analysis and graphing were performed in Prism (GraphPad, San Diego, CA, USA).
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) amplified DNA fragments with sizes varying between 150 and 800 bp highly similar with gnathostome GpH-Rs. The sequencing readings were assembled in gap4 (Staden package) and the resulting contig approximately 800 bp has been named lGpH-R I. A smaller number of readings were assembled into a second, distinct contig, which we consider a fragment of a second glycoprotein hormone receptor II (lGpH-R II).
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Structural domains of the lGpH-R I were identified based on the detection of specific patterns and motifs and their limits were established relative to the alignment of the protein sequence with other vertebrate GpH-Rs (Fig. 1). lGpH-R I shows the typical structural domain composition of the gnathostome GTH- and TSH-Rs: extracellular domain, rhodopsin-like transmembrane domain, and cytoplasmic segment domain. In the extracellular domain of the lGpH-R I were also identified a Cys-rich N-terminal domain followed by a LRR-containing region and a C-terminal Cys-rich region. Putative N-glycosylation sites in positions 86, 207, and 285 in the extracellular domain and one putative N-glycosylation site have been predicted at position 651 in the cytoplasmic segment of the receptor. Seven hydrophobic helical segments (Fig. 1, TM1–7), ca 20 residues in length are linked by three intracellular and three extracellular (EL) loops. A disulfide bridgebetween Cys residues located in EL1 and EL2 is also important for stabilization of the receptor conformation. The C-terminal end of the third transmembrane helix harbors a protein motif shared by all members of the GPCR superfamily. The Cys residue in position 630 matches the well-conserved Cys residue shown to be palmitoylated and anchored to the cell membrane in the rest of the members of the GpH-R family.
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Forty-nine vertebrate GpH-R protein sequences were used for reconstruction of the molecular phylogenetic relationships within this class of proteins, using the GpH-R isolated from sea anemone (Anthopleura elegantissima) (Nothacker & Grimmelikhujzen 1993) as an outgroup. The resulting tree (Fig. 4) has the usual topology of the phylogenetic trees most often reported for this family of proteins, with two major clades mirroring the functional distinction between the TSH-Rs and GTH-Rs. lGpH-R I and the GTH-Rs form a monophyletic group, the lamprey receptor being the earliest diverged member of this clade.
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. This figure shows the relative levels of the lGpH-R I signal in different tissues calculated as a percentage of the RT-PCR signal found in the testis samples.
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). Indirect estimation of cAMP synthesis induced by increasing amounts of lGpH-R I transfected into COS-7 cells showed an increase in the basal level of cAMP with the amount of lGpH-R I plasmid (Table 2) suggesting that the lamprey receptor may constitutively induce activation of the cAMP signaling at least in a mammalian cell line. Comparison between the abilities of different glycoprotein hormones to induce the cAMP-dependent secretion of the reporter protein in the medium showed a higher response for mammalian (ovine) LH and TSH compared with chorionic gonadotropin.
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Discussion |
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The total length of lGpH-R I (719aa) is intermediary between the lengths of the GTH- and TSH-Rs from gnathostomes (695 and 750 on average) (Kumar & Trant 2001, Oba et al. 2001). The differences are due in most part to the differences in the number of residues in the ectodomain and cytoplasmic domain compared with the other GpH-Rs. lGpH-R I has the shortest extracellular domain (estimated at 320 residues without the signal peptide) compared with around 350 residues in GTH-Rs and around 400 in TSH-Rs. Its cytoplasmic domain is correspondingly the longest in the family (estimated at 106 residues, compared with around 80 in TSH-Rs, 70 in LH-Rs, and only 60 in FSH-Rs).
The N-terminal half of lGpH-R I has the lowest identity with the vertebrate GpH-Rs. The potential functionally important residues at different sites in the extracellular domain of lGpH-R I are unique to this receptor in the context of the conservation of the global domain organization. This might be of particular significance for understanding the functional divergence of this class of receptors. The region is important for ligand binding and contains the determinants of the specificity of binding of agonists to the GpH-Rs (Combarnous 1992, Bhowmick et al. 1996, Grossman et al. 1997, Ascoli et al. 2002).
The Arg in position 192 in lGpH-R I (Fig. 1(i)
) corresponds to a well-conserved Lys residue in TSH-R and LH-R. It has been shown that mutation of the human TSH-R Lys residue in this position to Arg abolishes the ligand specificity of the receptor leading to increased sensitivity of the human TSH-R to chorionic gonadotropin (hCG) (Dufau 1998, Farid et al. 2000). Two other interesting residues are the Lys and Asp residues in positions 89 and 91 respectively of the lGpH-R I, located downstream a glycosylation site, which lGpH-R I shares with TSH-Rs (Fig. 1(ii)). They correspond to Arg and Tyr residues in the TSH-R consensus (positions 80 and 82 respectively in rat TSH-R). It has been shown (Smits et al. 2003) that concomitant mutations Arg80Lys+Tyr82Glu results in a gain of sensitivity of TSH-R to hCG.
The rat LH-R LeuXxxCysXxxGly motif close to exons 1–2 junction shown to be crucial for LH/hCG hormone binding (Hong et al. 1998) matches the lGpHR I ValXxxCysXxxGly residues and is distinct from the TSH-R consensus in the same position. Ala substitution of any of these residues resulted in no hormone binding, although the receptor was successfully expressed on the cell membrane.
Costagliola et al.(2002) described a specific motif in TSH-R and downstream of the C-terminal Cys-rich domain. The motif with consensus sequence YDY is the site for sulfation at one of the Tyr residues and located upstream at the start of the transmembrane domain. Its presence has been shown to be a requirement for binding and activation of TSH-R and suggested to play a similar role in GTH-Rs. The motif is very well conserved in TSH-Rs, common in mammalian LH-Rs and present only sporadically in FSH-Rs (where the consensus FDY can be found instead). This motif is missing in lGpH-R I, which is another element differentiating the lGpH-R I from vertebrate TSH-Rs in an apparent contradiction with the global similarity results.
The glycosylation of the extracellular domain of GpH-Rs has been shown to be important for the function of these receptors (Dufau 1998). The presence of only three glycosylation sites in the extracellular domain differentiates the lamprey receptor from all other known GpHs, which have six (TSH-R and LH-R) or five (FSH-R) such motifs in this domain. However, the glycosylation motif in position 86 aligns with a similar motif in TSH-Rs, the one in position 207 is common to all GpH-Rs, while the NLT glycosylation motif at the N-terminal end of the C-terminal Cys-rich domain is common in most FSH-Rs.
Tissue expression pattern and functional assay
Initially, the receptors for GpHs seemed to be characterized by their high tissue expression specificity (Dufau 1998, Ascoli et al. 2002) in accordance with their tissue-specific physiological roles (Hsu et al. 1998). Later, a couple of examples of ‘ectopic’ expression of these receptors in mammals have been described by various authors (Endo et al. 1995, Hermey et al. 1999, Eblen et al. 2001). It has become accepted that LH-Rs are more prone to show extragonadal expression and are less specific, while the FSH-R and TSH-R genes are under a more stringent control over their tissue expression and binding specificity. Identification and cloning of the GTH- and TSH-R homologs in fish species seemed to confirm this paradigm, although the diversity of tissues where the transcripts of GpH-Rs, in particular, of fish LH-Rs could be detected has increased (Bogerd et al. 2001, Kumar et al. 2001, Vischer & Bogerd 2003, Vischer et al. 2003). The low specificity in tissue expression of lGpH-R I seems to confirm an evolutionary trend of decreased stringency of gene expression control in ancient vertebrate lineages. Nevertheless, its highest level of expression in the testicular tissue of the male lamprey supports the hypothesis of the involvement of lGpH-RI in the endocrine control of reproduction in lamprey in completion of a hypothalamo–pituitary–gonadal axis as known for all jawed vertebrates (Dufau 1998, Ascoli et al. 2002).
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Overall, analysis of the primary structural features of the lGpH-R I reveals characteristics, which support the hypothesis of its homology with gnathostome GTH- and TSH-Rs. On the other hand, it does not offer strong support for its classification in either of these two groups. The vertebrate GpH-Rs have been divided into two subclasses based primarily on the functional criterion of implication in two distinct endocrine control axes in gnathostomes. It has been speculated that the GpH-R evolved from possibly ligandless GpH-Rs, whose function was accomplished by (non-hormonal) regulation of their constitutive activity. The increased specificity of the receptors in later evolved vertebrates seems to be associated with the decrease in their constitutive activity in the context of correlated mutations in receptor and ligand leading to increased thermodynamic stability of the complex (Farid & Szkudlinski 2004). Identification of new leucine–rich repeat–containing G protein–coupled receptors (LGR) in rat and human showing a very low tissue expression specificity and high constitutive activity seems to confirm this scenario (Hsu et al. 1998, 2000).
The lGpH-R I described in this paper is the first receptor for glycoprotein hormones described in agnathans. It is highly similar and the key motifs present in its sequence suggest that it is likely a homolog of the vertebrate gonadotropin and thyrotropin receptors. However, it does not lend itself to a formal classification in one of these two gnathostome GpH-R groups. Its detectable response to a mammalian gonadotropin indicates that it is a functional receptor that has a role in the sea lamprey reproductive physiology. To date, there has been no evidence to support the presence of TSH in this species and only one GTH-like hormone has been identified (Sower et al. 2006). It has previously been shown that treatment with salmon gonadotropin extract induces an increase not only in plasma estradiol concentrations, but also in plasma thyroxine concentrations in male and female adult sea lampreys (Sower et al. 1985). Based on these results, it has been hypothesized (Sower et al. 2006) that the thyroid and the gonads of the lamprey are under a pituitary control mediated by the same glycoprotein hormone. From this perspective, the endocrine physiology of lamprey antecedes the evolution of the pituitary–gonadal and pituitary–thyroid axes in gnathostomes (jawed vertebrates). However, further investigations are required for characterization of the ligand binding and signal transduction activities.
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Acknowledgements |
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Funding |
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This research has been supported by the National Science Foundation (IBN0421923) to S A S and Japan Society for the Promotion of Science and Grant-in-Aid for International Scientific research (No. 11691132) and for Basic Research (Nos 12556033 and 15370029) to H K from the Ministry of Education, Japan. This is Scientific contribution No. 2280 from the New Hampshire Agricultural Experiment Station. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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Received in final form 12 April 2006
Accepted 26 April 2006
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