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Endocrinology Vol. 139, No. 8 3390-3398
Copyright © 1998 by The Endocrine Society

ARTICLES

Cloning and Characterization of the Chicken Thyrotropin-Releasing Hormone Receptor1

Yuh-Man Sun, Robert P. Millar, Hao Ho, Marvin C. Gershengorn and Nicola Illing

Medical Research Council/University of Cape Town Research Unit for Molecular Reproductive Endocrinology (Y.-M.S., R.P.M.), University of Cape Town, Observatory 7925, South Africa; the Division of Molecular Medicine, Department of Medicine, Cornell University Medical College (H.H., M.C.G.), New York, New York 10021; and the Department of Biochemistry, University of Cape Town (N.I.), Rondebosch 7700, South Africa

Address all correspondence and requests for reprints to: Dr. Nicola Illing, Department of Biochemistry, University of Cape Town, Rondebosch 7700, South Africa. E-mail: illing{at}molbiol.uct.ac.za


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helixes of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH (pGlu-His-ProNH2) is synthesized in the hypothalamus and is transported via the hypophysial portal circulation into the pituitary gland to regulate the biosynthesis and release of TSH. It also functions as a paracrine regulatory factor and a neurotransmitter/neuromodulator in central and peripheral nervous systems (1). Varying from these physiological roles, TRH is well documented as a potent GH-releasing factor in vertebrates (2). In the chicken, TRH stimulates GH release in vivo and in vitro, and the effect is even more potent than that of mammalian GRF (3, 4, 5). TRH exerts these physiological roles by binding to its specific receptor (TRH receptor) on the membranes of somatotrophs and thyrotrophs (6, 7, 8).

To date, five types of TRH receptor complementary DNAs (cDNAs) have been cloned from mammals, two isoforms from the mouse (9, 10) and two isoforms from the rat (11, 12, 13) and human (14, 15, 16), but not in any other vertebrate classes. The structure of the TRH receptors reveals that the receptor is a member of the G protein-coupled receptor (GPCR) family. GPCRs are characterized by having seven transmembrane (TM) helixes, which are connected by alternating hydrophilic extracellular loops (EL) and intracellular loops (IL). The N-terminus is extracellular, whereas the C-terminus is on the intracellular side of the membrane. The TRH receptor has been shown couple to several G proteins (e.g. Gq, G11, Gi-2, Gi-3, and a Gs-like protein that does not activate adenylyl cyclase), which, in turn, promote the secondary messengers [diacylglycerol, inositol trisphosphate, and Ca2+] signaling cascade after being bound by its ligand (see Ref. 17 for a review). In addition, some findings have suggested that TRH receptor also couples to Gs and stimulates adenylyl cyclase in rat GH3 cells (18, 19). In chickens, cAMP may participate in the effect of TRH-induced GH release (20).

This study has been undertaken to clone the TRH receptor gene from the chicken and pharmacologically characterize the receptor by measuring inositol phosphate and cAMP formation. We found that the chicken TRH receptor has been highly conserved in millions of years of evolution, as it retains a large degree of primary sequence homology with the mammalian TRH receptors and shows a similar pharmacological response to a range of TRH analogs. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Desaza1TRH was a gift from the late Dr. L. A. Cohen (Bethesda, MD), and Pyr3TRH was a gift from Dr. T. K. Sawyer (Parke-Davis Pharmaceutical Research, Detroit, MI). Val2TRH and Phe2TRH were obtained from Peninsula Laboratories (Belmont, CA). TRH and [N{tau}-methyl-His] TRH (MeTRH) were purchased from Sigma Chemical Co. (St. Louis, MO), and [3H]methyl-TRH was obtained from DuPont-New England Nuclear (Boston, MA). All other chemicals were obtained from Sigma Chemical Co.

Constructing the cDNA library of the chicken pituitary gland
Total RNA was isolated by a guanidinium thiocyanate-phenol-chloroform extraction method (21) from 70 chicken pituitary glands (Golden Grove Poultry Co., Cape Town, South Africa). Polyadenylated [poly(A)+] RNA (6.6 µg) was purified by affinity chromatography using an oligo(deoxythymidine)-cellulose column. The poly(A)+ RNA was used to make a cDNA library in the Uni-ZAP XR vectors digested with EcoRI/XhoI (Stratagene, La Jolla, CA). Before amplification, the titer of the cDNA library was 2 x 106 plaque-forming units/ml.

Screening of the cDNA library of the chicken pituitary gland
Approximately 1 x 106 recombinants were screened in 20 (150-mm) petri plates according to the protocol of Stratagene (1994). Duplicated nylon filters were lifted sequentially (1-min absorption for the first filter; 2-min absorption for the second), denatured, neutralized, and baked. The filters were hybridized with an {alpha}-32P-labeled 2.3-kb fragment of the mouse TRH receptor (mTRH-R) cDNA (9) by random hexamer priming using the Megaprime labeling kit (Amersham, Arlington Heights, IL). The filters were prehybridized and hybridized at 42 C in 50% formamide solution (containing 0.8 M NaCl, 0.02 M piperazine-N,N'-bis(2-ethane sulfonic acid), 50% deionized formamide, and 0.5% SDS), then were washed with 1 x SSC (standard saline citrate) and 0.1% SDS at room temperature for 15 min, with 0.5 x SSC and 0.1% SDS at 50 C for 30 min, and with 0.2 x SSC and 0.1% SDS at 50 C for 20 min. Three positive clones were isolated after tertiary screening and identified by sequencing. Three clones (Trun-1, Trun-2, and Trun-3), have shown two types of nucleotide sequences, and all contained a premature stop codon at IL3. The downstream region, from a point 20 amino acid sequences before the stop codon, has no homology with the mammalian TRH receptor and was termed the divergent region (Fig. 1AGo).



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  		Figure 1. Diagram of the isolated chicken TRH receptor cDNAs. A, Three truncated clones contain a premature stop codon at nucleotide 1006 that corresponds to a codon in intracellular loop 3. A consensus 5'-end sequence of the exon-intron junction, AGgtag, is located 60 nucleotide sequences upstream of the stop codon, which results in a divergent region compared with those of mammalian counterparts. The Trun-1 clone has a 72-bp deletion at the 5'-untranslated region. B, Nine truncat-ed clones, which possess a large 3'-untranslated region (>3 kb), exhibit two different lengths of the coding region, that is from TM4 or from intracellular loop 3 to the poly(A)+ tails. C, A full-length clone was constructed by ligating two fragments, the HindIII/SspI digest of the Trun-3 clone and the SspI/BamHI digest of the Trun-4 clone, together. The HindIII restriction site is in the pSK+ vector. ORF, Open reading frame; *, stop codon; - and +, up- and downstream of the open reading frame (+ 1); {square}, putative TMs in the coding sequence. Numbers indicate the positional number of the nucleotide sequence.

 
Constructing the full-length chicken TRH receptor (cTRH-R) cDNA
The cDNA fragment of cTRH-R, which encodes a region from EL2 to TM6, was amplified by PCR with a pair of primers, i.e. a chicken-specific primer CH1 (sense) and the mouse TRH-R primer EB1 (antisense) from pituitary cDNA. The nucleotide sequences of the primers were: 5'-ACAAGGTGTCCAGGAGCT-3' and 5'-CACAAGAGT-CAGGAGGGCATC-3', respectively. Using this cloned fragment as a probe to screen the chicken cDNA library, 25 positive clones were isolated. Of the 25 clones, 9 clones (Trun-4 to Trun-12) were identified by partial sequencing and restriction enzyme mapping as incomplete receptor cDNAs, encoding from either the putative fourth transmembrane helix or intracellular loop 3 to the poly(A)+ tails. To construct the full-length cTRH-R cDNA for subcloning into the pcDNA I/Amp vector (Invitrogen Corp., San Diego, CA), the HindIII/SspI fragment from Trun-3 was ligated to the SspI/BamHI fragment from Trun-4 and cloned into the pcDNA I/Amp vector digested with HindIII/BamHI (Fig. 1CGo). The nucleotide sequences of the constructed full-length chicken TRH-R were confirmed by sequencing and are shown in Fig. 2Go. The truncated cTRH-R and mTRH-R cDNA clones were also subcloned into the pcDNA I/Amp vector to express the receptor for pharmacological characterization.



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  		Figure 2. Alignment of the deduced amino acid sequences of the cTRH-R (ch) and the mTRH-R (m). Putative TM helixes for the cTRH-R were assigned based on those of the mouse receptor (17 ). Identical residues between both species are shown in bold letters. · · ·, space; *, stop codon. Numbers indicate the positions of amino acids (top number for the chicken receptor; bottom number for the mouse receptor).

 
Sequencing
The 12 positive clones were rescued from the Uni-ZAP XR arms and subcloned into the pBluescript S/K+ vectors (Stratagene). Plasmid DNA of 12 clones and the full-length cTRH-R cDNA were purified by Wizard Maxi Prep column (Promega, Madison, WI) and sequenced manually, using a Sequenase kit (U.S. Biochemical Corp., Cleveland, OH). To confirm the nucleotide sequences of the full-length cTRH-R cDNA, the plasmid DNA was sequenced in both directions.

Northern blot analysis
Total RNA (30 µg) extracted from chicken and sheep pituitaries, respectively, was run on a formaldehyde denaturing gel and transferred onto the Hybond N membrane (Amersham). The samples was fixed in the membrane using a UV cross-linker (Amersham). The membrane was hybridized with the full-length chicken TRH-R cDNA, which was labeled with [{alpha}-32P]deoxy-CTP (Amersham) using the Megaprime labeling kit (Amersham). The autoradiograph of the membrane was exposed for 5 days.

Analysis of the structure of the divergent region in the cTRH-R gene
To analyze the divergent region of the Trun-1, -2, and -3 clones (Fig. 3Go, top panel), the cTRH-R gene was isolated from a chicken genomic library constructed in bacteriophage {lambda} charon 4A (22) by hybridization with {alpha}-32P-labeled Trun-3 cDNA. Two clones were isolated after tertiary screening (YS1 and YS2). Restriction enzyme digests of the YS1 clone were subjected to Southern blot analysis by hybridizing with probe A, which contained the coding region of TM1 to most of intracellular loop 3, then were stripped of the probe and rehybridized with probe B containing the entire divergent region (Fig. 3Go, top panel). PCR was applied to reconfirm the location of the divergent region using two pairs of gene-specific primers, CH1/CH4 and CH2/CH3 (Fig. 3Go, top panel).



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  		Figure 3. Schematic representation of the cTRH-R genomic and truncated cDNA clones. The divergent region of the truncated cDNA was verified as an intron by Southern blot analysis and PCR (shown in Fig. 4Go). The intron follows on from exon 1 where the consensus 5'-end sequence of the exon-intron junction (AGgtag) is shown. Exon 1 encodes a 5'-untranslated region through most of intracellular loop 3. The probes A and B and four primers, which are indicated in Fig. 4Go, are shown at the corresponding regions of the cDNA clone. ORF, Open reading frame; - or +, up- or downstream of the open reading frame (+1); //, region not shown. Numbers indicate the positional number of the nucleotide sequence.

 
Cell culture and transfection
COS-1 cells were grown at 37 C in an humidified atmosphere containing 5% CO2. One to 2 days before transfection, COS-1 cells were harvested with trypsin and plated out at 0.6–0.8 x 105 cells/25-mm well for inositol phosphate (IP) or cAMP formation assays and at 0.7–1.0 x 106 cells/100-mm dish for receptor binding experiments. The cells were transfected for 3.5 h with the cTRH-R/pcDNA amp subclone (cTRH-R subclone), the truncated cTRH-R/pcDNA amp subclones, or the mouse TRH-R/pcDNA amp subclone (mTRH-R subclone), using a modification of the diethylaminoethyl-dextran method (23). For IP formation assay, the cells were labeled with myo-[3H]inositol (1 µCi/ml; Amersham) in DMEM (supplemented 2% FCS and penicillin/streptomycin) for 16–22 h in a 37 C incubator (5% CO2), 48 h after transfection.

IP formation assay
IP formation was measured as previously described (24). Briefly, cells were washed twice with buffer A [140 mM NaCl, 4 mM KCl, 20 mM HEPES, 0.1% BSA, 8 mM D-glucose (pH 7.4), 1 mM CaCl2, and 1 mM MgCl2] at 37 C for 5 min. Then, the cells were incubated with buffer A (containing 10 mM LiCl) in the presence or absence of TRH analogs for 1 h at 37 C. All experiments were performed in duplicate and were repeated at least twice.

cAMP formation assay
The cells were washed twice with buffer A and incubated with the buffer (containing 10 mM LiCl) in the presence or absence of TRH (1 nM to ~10 µM) for 60 min with 0.25 mM isobutylmethylxanthine (IBMX; a cyclic nucleotide phosphodiesterase inhibitor) at 37 C, 48 h after transfection. COS-1 cells, transfected with human ß2-adrenergic receptor cDNA, were incubated with 1 µM epinephrine (in the presence of 0.25 mM IBMX) as a positive control. cAMP was extracted using 4 mM EDTA and was measured by RIA (Amersham kit, TKR 342). Individual experiments were repeated twice in duplicate.

Receptor binding assay
One day after transfection, the cells were harvested and seeded into 12-well plates at 100,000 cells/well in DMEM with cells in monolayer for 1 h at 37 C. Receptor binding was performed using [3H]MeTRH as described previously (25).

Data analysis
The values of Ki (receptor affinity) and EC50 (IP formation) were estimated by nonlinear regression analysis using the PRISM program (GraphPad, San Diego, CA). Dose-response curves (each data point is the mean of all experiments) of IP and cAMP formation assays and competition binding assay were drawn using the same program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Twelve truncated cTRH-R cDNAs were isolated by screening a cDNA library that was constructed from RNA extracted from chicken pituitary glands (Fig. 1Go). Trun-1, -2, and -3 were identified by sequencing in both directions and were about 1.4 kb in length (Fig. 1AGo). They all had a stop codon at nucleotide +1006 (downstream), which results in a receptor that prematurely terminates in IL3. There was no homology with mammalian TRH receptors from the 20 amino acids that precede the stop codon onward (called the divergent region). The consensus 5'-end sequence of the exon-intron junction, AGgtag, was found at the point of homology divergence. Trun-2 and -3 were identical. However, Trun-1 showed slightly different nucleotide sequences from the other 2 clones in its lack of 72 bp at 13 nucleotides (-13) upstream from the start of the open reading frame (+1). The other 9 truncated receptor cDNA clones (~3.8–4.1 kb) were analyzed by restriction enzyme mapping and partial sequencing (Fig. 1BGo). They appeared to be two types of incomplete cDNA clones, encoding either from half of the TM4 or from half of the IL3 to the poly(A)+ tails. These clones were probably an artifact of the reverse transcriptase reaction prematurely stopping during the synthesis of first strand cDNA in the library construction. All of these clones had a large 3'-untranslated region. As we were unable to isolate a full-length cDNA from the library, a full-length chicken TRH receptor cDNA was constructed by ligating two fragments (HindIII/SspI and SspI/BamHI) together from two truncated cDNA clones (i.e. Trun-3 and -4; Fig. 1CGo). Although a full-length cDNA product was amplified by PCR, the PCR product had a number of nucleotide substitutions compared with the cDNA library clones and was not used for further study.

The constructed full-length cTRH-R cDNA showed 76% and 84% identity to the nucleotide and amino acid sequences of the mouse TRH-R, respectively (Fig. 2Go). Completely conserved regions in both receptors are the IL1, IL2, EL3, TM2, and TM7. The most divergent parts are the extracellular N-terminus, the middle part, and the C-terminal part of the IL3 (if the loop is divided into three parts), as these parts only share 55%, 53%, and 60% homology, respectively. The cTRH-R contains two potential sites for N-linked glycosylation (N-X-S/T) in the N-terminus, which are also conserved in the mouse counterpart. However, there is an additional glycosylation site in EL2 of the mammalian receptor that is absent in the chicken receptor. The putative binding sites of the mammalian TRH-R, Tyr106, Asn110, Tyr282, and Arg306, are all conserved in the chicken receptor.

The position of the divergent region of the Trun-1, Trun-2, and Trun-3 clones in the TRH receptor gene was analyzed by comparing the cDNA clones to a genomic clone of the TRH receptor gene. Two TRH receptor genomic clones (YS1 and YS2) were isolated by screening a chicken genomic library (22) with the chicken TRH receptor cDNA probe. Two probes were prepared from the Trun-3 cDNA (probe A, which contained the conserved region of the cTRH-R, and probe B, which contained the divergent region) and were used in Southern blot analysis of YS2 digested with various restriction enzymes (Fig. 4Go). Probe A hybridized to an expected 1.2-kb EcoRI fragment, whereas probe B hybridized to a 4.8-kb band (Fig. 4Go), confirming that the YS1 genomic clone contained both the conserved coding region of Trun-3 as well as the divergent region. Probes A and B both hybridized to 8-kb BamHI and HindIII fragments from YS1. This information was used to construct a restriction enzyme map of YS1 (Fig. 3Go). The divergent region was adjacent to exon 1, as the PCR products amplified from YS1 and the Trun-3 cDNA clone, were the same size (Fig. 4BGo). In addition, there was no PCR product amplified from the Trun-4 cDNA, in which the divergent region intron had been spliced out. This finding suggests that the divergent region of the Trun-3 cDNA is a part of a intron (Fig. 3Go).



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  		Figure 4. Identification of the divergent region of the Trun-3 clone. A, Southern blot analysis of the TRH receptor genomic clone (YS1 clone in the {lambda} charon 4A vector digested with EcoRI) digested with EcoRI (E), XbaI (X), HindIII (H), and BamHI (B) or combinations thereof and hybridized with {alpha}-32P-labeled probes (A and B; shown in Fig. 3Go). The 1.2-kb EcoRI and 1.5-kb EcoRI/HindIII (or BamHI) fragments were hybridized with probes A and B, respectively. The two probes hybridized to a 2.7-kb HindIII fragment of the chicken receptor gene (excluding a 5.3-kb {lambda} charon 4A vector). B, PCR was applied to identify the divergent region using its specific primers (shown in Fig. 3Go). The 0.5- and 0.25-kb PCR products were amplified with two pairs of primers (CH1 and CH4, and CH2 and CH3, respectively) from two templates [Trun-3 clone (lane 1) and genomic clone (lane 2)], whereas the negative controls [Trun-4 clone (lane 3) and water (lane 4)] are blank. Marker is present in lane 5.

 
The full-length TRH receptor cDNA was found to hybridize to three messenger RNA (mRNA) transcripts in Northern blot analysis of RNA extracted from chicken pituitaries(Fig. 5Go). The major transcripts were 5 and 1.4 kb in size. The latter transcript was similar in size to the truncated cDNAs, Trun-1, Trun-2, and Trun-3. A minor transcript of 3.2 kb was also detected. These hybridization signals were specific for the RNA extracted from chicken pituitaries, as no signal was detected in the RNA extracted from sheep pituitaries (Fig. 5Go).



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  		Figure 5. Northern blot analysis of total RNA extracted from chicken and sheep pituitary glands. Thirty micrograms of total RNA isolated from chicken and sheep pituitaries were transferred onto Hybond N membrane, and the membrane was hybridized with {alpha}-32P-labeled cTRH-R cDNA. Three mRNA transcripts of the chicken receptor (5, 3.2, and 1.4 kb) were detected in the membrane by autoradiograph after 5-day exposure.

 
The isoforms of the chicken receptor were expressed alone or coexpressed in COS-1 cells and were characterized by measuring IP formation in the presence of various doses of TRH. None of the truncated isoforms (either individual or combined) stimulated IP formation (Fig. 6Go). Additionally, there was no significant difference in EC50 values in the COS-1 cells expressing the wild-type receptor alone or coexpressed with the Trun-3 cDNA clone. These findings indicated that the truncated receptors were nonfunctional and, furthermore, did not exhibit a negative effect on the ability of the wild-type cTRH-R to stimulate IP production in response to ligands.



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  		Figure 6. Dose-response curves for IP formation stimulated by TRH in COS-1 cells expressing the cTRH-R isoforms Trun-3 and Trun-4, and the full-length TRH receptor cDNA (W). The COS-1 cells were either transfected with single cDNAs or were co-transfected as indicated.

 
To further characterize the constructed full-length cTRH-R, receptor binding and IP formation assays were performed in COS-1 cells transfected with either the cTRH-R or the mTRH-R. The assays were determined by employing several TRH analogs that have different substitutions at residues 1, 2, and 3 of TRH, as it has been proposed that the side-chains of all three amino acid residues of TRH are involved in binding to TRH-R. The pharmacological studies on the receptor binding and IP formation revealed that the constructed full-length cDNA clone is a functional receptor and exhibits pharmacological properties similar to those of the mouse homolog. In binding experiments (Fig. 7Go and Table 1Go), the affinity (Ki = 8.1 nM) of TRH to the chicken receptor was 500-fold higher than those of Desaza1TRH and Phe2TRH, and was 1500- and 1850-fold higher than those of Val2TRH and Pyr3TRH, respectively. TRH and MeTRH showed a similar affinity for the chicken receptor. The mouse receptor displayed a similar pattern of decreased affinity for these analogs. There was no significant difference in affinity between chicken and mouse receptors for any analog. The EC50 (10.2 nM) for stimulation of IP formation by TRH in the chicken receptor was about 40-fold lower than those of Desaza1TRH and Phe2TRH, but was 10-fold higher than that of MeTRH (Fig. 8Go and Table 1Go). Additionally, TRH was more potent than Val2TRH (350-fold) and Pyr3TRH (900-fold). The mouse receptor exhibited a similar pattern of potency for stimulating IP formation by these analogs, with the exception of Val2TRH and Pyr3TRH. The peptides showed a higher potency with the mouse receptor than with the chicken receptor.



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  		Figure 7. Binding of TRH analogs to COS-1 cells expressing chicken or mouse TRH receptors. Competition binding of [3H]methyl-TRH in the presence of various concentrations of TRH analogs is shown.

 

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  		Table 1. Receptor binding affinities and potencies of stimulation of inositol phosphate (IP) formation by TRH analogs in COS-1 cells expressing chicken or mouse TRH receptors

 


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  		Figure 8. Dose-response curves for IP formation stimulated by TRH analogs in COS-1 cells expressing chicken or mouse TRH receptors. Data shown are the mean ± SE. Each curve is representative of three experiments performed in duplicate.

 
Figure 9Go shows that there was no cAMP formation in the presence of various doses of TRH (up to 10 µM) in COS-1 cells transfected with chicken or mouse TRH receptor cDNAs. However, the positive control (COS-1 cell transfected with ß2-adrenergic receptor cDNA) showed cAMP formation in the presence of 1 µM epinephrine.



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  		Figure 9. Lack of stimulation of cAMP formation by TRH in COS-1 cells expressing cTRH-R or mTRH-R. The COS-1 cells expressing ß2-adrenergic receptor (ß2-AR) was stimulated with 1 µM epinephrine as a positive control. The levels of cAMP were measured in the presence of 0.25 mM IBMX. Each curve is representative of two experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The deduced amino acid sequence of the cTRH-R exhibits 80% identity to the mammalian counterparts (excluding two isoforms of rodents). It is of interest to note that the related chicken GnRH receptor shares only 40% identity with mammalian GnRH receptors (26). It appears that there has been considerable evolutionary constraint on the TRH receptor structure in its interaction with the smaller ligand. This is consistent with the finding that there is high conservation of primary amino acid sequences between receptors for small ligands among different species in the biogenic amine receptors (27).

Using a combination of mutagenesis and computer simulation studies, a putative binding site of mouse TRH receptor has been identified (25) and is summarized in Table 2Go. It has been proposed that the binding of TRH to its receptor involves hydrogen bonding interactions and hydrophobic interactions rather than ionic interactions. All of these candidate residues interacting with the ligand are conserved in the chicken receptor. Other residues have been identified (e.g. Gln105 and Tyr282) that are involved in interhelical H-bonding interactions, but do not directly interact with TRH (29). These residues are all conserved in the chicken receptor. We have recently suggested that TRH binds to the TRH receptor by interacting initially with residues in the ELs and is then guided into the TM binding pocket (30). Asn289 is one of the EL interacting sites, and it is also present at a homologous position in the chicken receptor.


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  		Table 2. The putative binding sites of the mouse TRH-R

 
Apart from the binding pocket, the chicken receptor is likely to share similarities in the mechanism of receptor activation with the mammalian receptors. For example, two residues, Asp71 in TM2 and Arg283 in TM6, which play a role in G protein coupling in the mouse receptor (25, 31), are conserved in the chicken receptor. On the other hand, the chicken receptor displays some features that are different from those of the mammalian homologs. The chicken receptor lacks a potential N-glycosylation site in the EL2, which is present in the mammalian receptors (Asn167). It has been shown that glycosylation may play a role in receptor expression or stability (32). Additionally, a difference in the distribution of putative phosphorylation sites (33) exists in the chicken and mammalian receptors. The chicken receptor contains four additional putative phosphorylation sites in the C-terminal part of IL3, whereas the mammalian receptors contain four extra putative phosphorylation sites in the C-terminal tail. Phosphorylation may play a part in desensitization of receptors. The chicken receptor exhibits a difference in down-regulation from that of the mammalian counterpart (34). Down-regulation of the cTRH-R occurs within 30 min after administration of TRH and is maintained for at least 60 min in vivo and in vitro. On the contrary, the down-regulation of the mammalian receptors occurs after several days of TRH challenge in vivo (35, 36) or after 12–24 h of TRH treatment in vitro (37). It remains to be further investigated whether the putative phosphorylation sites are involved in the regulation of receptor down-regulation.

The cTRH-R, like mammalian TRH receptors, has retained several amino acids that are highly conserved in GPCRs. These include TM2-Asp73 and TM7-Asn318 (Asp71 and Asn316 in the mammalian receptors). The Asp residue has been shown to be involved in receptor activation in the mTRH-R (25) and many other GPCRs (38). The highly conserved Cys residues at 100 and 181 (98 and 179 in mammals) have been shown to form a disulfide bond between EL1 and EL2 to stabilize the structure of functional receptor in GPCRs (38) and the mTRH-R (39). Two other Cys residues, Cys337 and Cys339 (335 and 337 in the mammalian receptors), are present in the C-terminal tail. Homologous Cys residues have been shown to be palmitoylated and may anchor the tail in the plasma membrane, which was proposed to be important for interactions with G proteins (40, 41). In contrast, the Cys residues in the mammalian TRH receptor are not necessary for G protein coupling, but may play a role in restraining the receptor in an inactive conformation that is optimal for rapid internalization of the receptor (42) because a truncation mutation (C335Stop) at this locus of the mTRH-R exhibits constitutive (or agonist-independent) activity (43). The highly conserved Asp-Arg-Tyr (DRY) motif at the C-terminal part of TM3, which is involved in G protein coupling and receptor internalization (44), is present as ERY in the chicken and mammalian receptors as in several other receptors. A substitution of the Asp residue with Glu (E) in a mammalian GnRH receptor results in increasing binding affinity, activation, and rate of internalization (45). Following the DRY motif, the XXI/VXXPL/I motif, in which the Pro and Leu residues have been established to play a role in G protein coupling (45, 46), is also conserved in the chicken and mammalian receptors. Moreover, Asn43 (TM1), Trp150 (TM4), Pro203 (TM5), FXXXWXP motif (TM6), and NPXXY motif (TM7), which are conserved GPCR residues present in the mammalian TRH receptor, are also conserved in the cTRH-R.

The truncated isoforms of the cTRH-R cDNA that we isolated are not functional receptors. The Trun-1, -2, and -3 cDNA clones that contain a premature stop codon (TAA) in the IL3 are due to retention of an unspliced intron. Intriguingly, these truncated receptor clones were shown by PCR and sequencing studies to contain a poly(A)49 tail and an AATAAA consensus sequence of the polyadenylation signal site upstream of the tail (Sun, Y.-M., R. P. Millar, and N. Illing, unpublished data). Additionally, the sizes of these truncated cDNA clones (~1.4 kb) matched one of the major mRNA transcripts that hybridized to the TRH receptor cDNA probe on Northern blot analysis of RNA extracted from chicken pituitaries. These results suggest that these truncated receptors are fully processed mRNA transcripts and are not cloning artifacts. It is noteworthy that the human TRH receptor gene also contains a large intron (25 kb) at a homologous position (47). In addition, two variant forms of TRH receptor have each been cloned from mouse (10) and rat (11). These isoforms are characterized by different amino acid sequences in their respective C-terminal tails and are functional receptors. The isoforms of the rat receptor are produced by alternative splicing of a retained intron, whereas those of the mouse receptor may be formed by alternative splicing of two exons.

The pharmacological properties of the chicken receptor were evaluated by applying different TRH (pGlu-His-Pro-NH2) analogs, in which residues 1, 2, and 3 were substituted individually with different amino acids, to measure their activities of receptor binding and/or G protein coupling. These experiments confirmed that the side-chains of all three amino acid residues of TRH are involved in receptor binding. Desaza1TRH, in which the NH ring of pyroGlu was substituted by a CH2 group, exhibits an approximately 500-fold decrease in affinity for the chicken and mouse receptors. Modifications in the histidine residue of TRH can lead to marked effects on the binding affinity that, in turn, affect the potency for IP formation and for secretion of TSH. One of these TRH analogs, Val2TRH, in which the histidine residue of TRH was replaced by valine, showed a more than 500-fold decrease in affinity for the chicken and mouse receptors. A portion of the decrease may be due to the loss of a stacking interaction between the ligand and receptor, because it was postulated that the imidazole of the His residue forms hydrophobic interactions with a pocket formed by Tyr282, Tyr188, Tyr192, Phe196, and Phe199 (17, 48). This may also explain why when histidine was substituted with a strongly hydrophobic phenylalanine, there was less than a 100-fold decrease in the binding affinity (29) and in stimulating IP formation in this study. In addition, a portion of the decrease in the affinity of Val2TRH or Phe2TRH may result from the loss of an H bond interaction, because residue Ser113 of the receptor was suggested to form an H bond with the histidine (17). Furthermore, the binding affinity of the chicken receptor for Pyr3TRH, in which the ProNH2 residue is substituted by a pyrolidine ring, is 1800-fold lower than that for TRH, as with the mouse receptor. It was suggested that the CO group of Pro residue of TRH forms an H bond with the guanidino group of Arg306 residue in the receptor.

It has been suggested that the adenylyl cyclase-cAMP pathway plays a role as a secondary messenger in TRH-induced physiological functions, in addition to the phospholipase C-inositol 1,4,5-trisphosphate-1,2-diacylglycerol-Ca2+ pathway. Evidence has been presented that cAMP is involved in TRH-stimulated PRL secretion in the rat pituitary tumor cell line (GH cells) (49) and may participate in TRH-induced GH release in chicken somatotrophs (20). However, we demonstrate here that TRH does not elevate cAMP levels in the COS-1 cells transiently expressing chicken or mouse TRH receptors. The result is consistent with the finding that TRH did not stimulate cAMP formation in the mouse TRH receptor expressed in five different cell types (50).

In conclusion, we have cloned a cDNA for the cTRH-R and found it to be highly homologous and pharmacologically indistinguishable from the previously cloned receptors from mouse, rat, and human species. This degree of evolutionary conservation is unusual and may be related to the small number of contacts that must be maintained between a tripeptide and its receptor to achieve high affinity binding and optimal activation.


   Footnotes
 
1 This work was supported by the British Council, the Foundation for Research Development (South Africa), the Medical Research Council (South Africa), the University of Cape Town, and USPHS Grant DK-43036. Back

Received December 1, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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