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Endocrinology Vol. 147, No. 4 1838-1846
Copyright © 2006 by The Endocrine Society

Cloning of the Chicken Pituitary Receptor for Growth Hormone-Releasing Hormone

Andrew A. Toogood, Stephen Harvey, Michael O. Thorner and Bruce D. Gaylinn

Division of Endocrinology and Metabolism (A.A.T., M.O.T., B.D.G.), University of Virginia Health System, Charlottesville, Virginia 22908; and Department of Physiology (S.H.), University of Alberta, Edmonton, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. Bruce Gaylinn, Ph.D., Division of Endocrinology, University of Virginia Health System, Box 801411, Charlottesville, Virginia 22903. E-mail: bg2g{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Details of the regulation of GH in birds are unclear. In this report, a receptor was cloned from chicken pituitary cDNA with 61% amino acid sequence identity to the human pituitary GHRH receptor. Phylogenies inferred from sequence alignments support that this is the chicken counterpart of the GHRH receptor known in mammals. Northern blotting shows that this receptor message is expressed in chicken pituitary, with lesser amounts seen in hypothalamus and brain but not in liver. The recombinant chicken receptor binds human GHRH with high affinity and specificity and signals cAMP accumulation. Surprisingly, available peptides synthesized to the published sequence for chicken GHRH-like peptide (cGHRH-LP) were inactive at this receptor. To address this we recloned the cDNA for this cGHRH-LP from chicken hypothalami. The revised sequence encodes lysine at position 21, which is consistent with all reported GHRH sequences from other species but different from the originally published chicken sequence. When this revised cGHRH-LP sequence was synthesized, it had improved but still weak potency at the cloned receptor. Consistent with the activity at the cloned receptor, human GHRH was potent when assayed in live chickens or on chicken pituitary membranes, but cGHRH-LP was not. We conclude that we have cloned a putative GHRH receptor that is homologous to mammalian GHRH receptors and functionally expressed in chicken pituitary, but that the identity of the endogenous ligand remains unclear. The chicken GHRH receptor cloned in this study can serve as a tool to identify its ligand and to clarify the evolutionary development of the regulation of GH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH RELEASE FROM the anterior pituitary in mammals is under the regulation of two hypothalamic peptide hormones, GHRH and somatostatin, with a further contribution from the endogenous GH-releasing peptide, ghrelin (1). GHRH acts via a specific receptor on the pituitary somatotroph stimulating GH release and GH synthesis. The GHRH receptor belongs to a family of seven transmembrane G protein-coupled receptors (family B) that includes the secretin, parathyroid hormone, calcitonin, vasoactive intestinal peptide (VIP), glucagon, corticotrophin-releasing hormone, and pituitary adenylate cyclase activating polypeptide (PACAP) receptors (2). In man and other mammals, the GHRH receptor binds its ligand specifically but has slight cross reactivity with other structurally related peptides such as PACAP and VIP (3, 4, 5). Although the GHRH receptor has been cloned and characterized in several mammals (5, 6, 7, 8, 9) and two fish (10, 11), no GHRH receptor from birds has been characterized, and the physiological role of GHRH in birds remains unclear.

In the chicken, GH release appears to be under hormonal regulation similar to that in mammals. Administration of human, pig, or rat GHRH to chickens produced a rapid, dose-dependent rise in GH release after iv injection, achieving maximal response within 5–10 min of administration, mirroring the response to GHRH observed in humans (12). Intravenous somatostatin administered simultaneously with mammalian GHRH inhibits the GH response to GHRH. In the chicken, surgical lesions sited in the arcuate nucleus of the hypothalamus, the site of GHRH-containing neurones in mammals, significantly reduced GH secretion, suggesting that a GHRH-like substance is released from this area (12).

Despite the evidence of a stimulatory GH regulatory system analogous to that observed in mammals being present in the chicken, the nature of the endogenous GHRH and the receptor that binds it, and through which it exerts its effect, remains unclear in chickens. The gene for a putative avian GHRH [chicken GHRH-like peptide (cGHRH-LP)] has been cloned and characterized (13) but the peptide had poor activity in chickens failing to stimulate GH release from somatotrophs in vitro and having only a slight effect in vivo at very high doses (14). One possible reason for the failure of this cGHRH-LP to stimulate GH release is an error in the cloned sequence. In all the GHRH proteins sequenced to date, the lysine at residue 21 is conserved and is vital for activity, but in published sequences for chicken GHRH (cGHRH) this residue was replaced by an asparagine (13).

At the start of this work a specific cGHRH receptor (cGHRH-R) had not been identified; however, the observation that mammalian GHRHs stimulate GH release in the chicken suggests that such a receptor is present on the somatotroph. We now report the cloning and characterization of a GHRH receptor from the anterior pituitary of the chicken.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care
Chicken pituitary glands and hypothalami were collected at a slaughterhouse (Lillydale, Edmonton, Canada). Tissues used for Northern blotting were obtained from birds reared from hatch at the University of Alberta Poultry Station and killed by cervical dislocation using an Animal Use Protocol approved by the University of Alberta Animal Care Committee.

Tissue collection, RNA extraction, and library creation
The anterior pituitary glands were removed from 100 Hubbard/Ross cross-strain broilers (42 d old) within 2 min of death and snap frozen. The hypothalami were dissected from five birds and snap frozen. The tissue was stored at –70 C until required. RNA was extracted from these tissues using TriReagent (MRC, Cincinnati, OH). The bulk of the poly-A mRNA extracted from the pituitaries was used to create a size-selected cDNA library in Lamda Zap II phage using a combination of random and oligo-deoxythymidine priming (Stratagene, La Jolla, CA). The complexity of the library was 106 independent clones.

PCR cloning of the cGHRH receptor
Initially, RT-PCR was performed using a set of degenerate primers designed to detect all known GHRH receptors (Table 1Go). The first strand cDNA synthesis was performed in a 20-µl volume using 1 µg pituitary mRNA, 100 U Superscript II reverse transcriptase (Life Technologies, Inc., Rockville, MD), 5 mM MgCl2, 1 mM deoxynucleotide triphosphate (Promega, Madison, WI), 2.5 µM oligodeoxythymidine, 20 U ribonuclease inhibitor (Roche Molecular Biochemicals, Basel, Switzerland) and 5 mM dithiothreitol. The reaction was incubated at 42 C for 45 min before being terminated by heating to 99 C for 10 min. The PCR was performed by adding 2.5 U of Taq DNA polymerase (Promega) and 0.17 mM of each primer to the RT reaction, making a total reaction volume of 30 µl. The reactions were performed using a PerkinElmer DNA Thermal Cycler 480 (PerkinElmer, Wellesley, MA). The reaction conditions were a 94 C denaturation phase (30 sec), a 55 C annealing phase (45 sec), and a 72 C extension phase (30 sec) for 35 cycles followed by 72 C for 7 min. The PCR products were electrophoresed through 1% agarose gels. Each permutation of forward and reverse degenerate primer pairs (Table 1Go) produced multiple product bands from chicken pituitary mRNA, but each set of bands included one that matched the expected size seen with these primers when used on the human GHRH receptor (hGHRH-R). Nested PCR of the appropriate sized bands with internal degenerate primers gave the expected smaller sized bands, suggesting that the products could be from the chicken receptor. These bands (before nesting) were cut out of the gel and purified using a QIAquick gel extraction kit (QIAGEN, Valencia, CA) and sequenced using an ABI Prism dye sequencer (PerkinElmer, Cetus). The overlapping snippets of sequence were compared with the sequences stored on GenBank using Blastn, which confirmed that they were from a novel gene closely related to other GHRH receptors. These partial sequences were used to design primers that were specific to the cGHRH receptor that was used in PCR using the chicken pituitary cDNA library as the template and plasmid-specific primers to determine the 5' and 3' ends of the receptor cDNA.


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  		TABLE 1. Degenerate primers to conserved regions of GHRH-Rs

 
High-fidelity PCR using Pfu polymerase (Promega) (a proof-reading enzyme) was used to synthesize the coding region of the cGHRH-R cDNA by screening amplified subpools of the phage library for those enriched in receptor message (primers given in Table 1Go).

Northern blots
Total cellular RNA was extracted from pituitary, hypothalamus, whole brain, retina, and liver tissue pooled from three adult birds and subjected to Northern blot analysis using a digoxigenin (DIG)-labeled 1300-bp cGHRH-R cDNA. Briefly, the RNA was electrophoresed in 1% (wt/vol) agarose, 3.1% (wt/vol) formaldehyde gels (3 µg total RNA per lane for each tissue) and transferred to nylon membranes by capillarity blotting. After transfer, the RNA was immobilized in 60% (wt/vol) formamide [containing 0.75 mol NaCl, 1.0 mmol PIPES, 25 mmol EDTA, 0.2% (wt/vol) SDS, and 1x Denhart’s reagent (0.1% (wt/vol) Ficoll, 0.1% (wt/vol) BSA, 0.1% (wt/vol) polyvinylpyrrolidine, and 100 µg salmon sperm DNA per liter, pH 6.8)] in the presence of the probe (10 pmol/ml hybridization buffer) for 12 h at 55 C. After a brief rinse in 2x standard saline citrate (SSC), the nylon membranes were washed at room temperature in 0.1% (wt/vol) SDS containing 2x SSC and subsequently at 75 C in 1% (wt/vol) SDS containing 0.1x SSC. The blot was then incubated in blocking reagent (Roche Applied Science) for 1 h with gentle shaking and then with alkaline phosphatase-conjugated anti-DIG antibody (Roche Applied Science), diluted 1:5000 in 1x Blocking Reagent. Colorimetric detection of the DIG-labeled probe was carried out using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (Roche Applied Science).

Cloning cGHRH-LP
CGHRH-LP was cloned from hypothalamic RNA using oligonucleotide primers based on the published sequence (13), in a high-fidelity PCR using pfu polymerase (Promega) to obtain a full-length sequence. Forward primers were 5'-TGG GGC AGG TGT ACG GCG CTG TAC TAC C-3' and 5'-CGG GAA TAC CCT ACA GGA CTT CGC ACT ACG-3'. Reverse primers were 5'-TAG CGG CTG TAG CTG TCC GTG AAG ATG C-3' and 5'-CAG GAC GGC CGC TAA GTA TTT CTT GAC AGC-3'.

Peptide sources
K21GHRH-LP (1–33) and K21cGHRH-LP (1–44) were synthesized independently at the University of Virginia Biomolecular Research Facility using solid-phase fmoc chemistry. The products were prepared to greater than 95% purity by reverse-phase HPLC, and the molecular structure was verified by mass spectrometry. All other peptides were from Bachem/Peninsula (Peninsula Labs, San Carlos, CA) except for N21cGHRH-LP (1–46) and carp GHRH-LP (gifts from J. E. Rivier and W. W. Vale, The Salk Institute for Biological Studies, La Jolla, CA) and chicken VIP and turkey peptide histidine-isoleucine (PHI) (gifts from M. E. El-Halawani, Department of Animal Science, University of Minnesota, Saint Paul, MN). Because the N21cGHRH-LP (1–46) and carp GHRH-LP were less active than anticipated, mass spectrometry at the University of Virginia Biomolecular Research Facility was used to confirm that these peptides had the expected mass, had not become oxidized or cleaved, and were present at the expected concentration. No problems with the peptides were found. HPLC purified iodinated His1,Nle27,hGHRH(1–32)NH2, N21cGHRH-LP (1–46), K21GHRH-LP (1–33), and carp GHRH-LP were prepared in our lab as previously described (15).

Expression, binding studies, and cAMP measurements
The full-length, receptor-encoding Pfu PCR product was cloned into the pTargeT mammalian expression vector. Human embryonic kidney (HEK) 293 cells were transfected with cGHRH-R/pTargeT and selected in 400 µg/ml G418. The GHRH binding assay used in this study has been described previously (15) and is presented in this study briefly. Cell monolayers were rinsed three times with 5 ml PBS. The cells were scraped into 5 ml PBS and centrifuged. They were then resuspended and homogenized in a HEPES buffered saline with 10 mM EDTA and protease inhibitors. After centrifugation at 10,000 x g, the upper, membrane layer of the pellets was resuspended in binding buffer (Tris buffered saline with 5 mM MgCl2), incubated with [His1,125I-labeled Tyr10, Nle27]hGHRH-(1–32)-NH2 [125I-labeled hGHRH (1–32)], 100,000 cpm/0.5 ml incubation volume (peptide from Peninsula Labs, binding assay and in-house iodination detailed in Ref.15) with or without increasing amounts of hGHRH or other test peptides at room temperature for 1 h. The membranes were then pelleted, and the pellets were counted in a {gamma}-counter. The data were adjusted for the GHRH probe concentration and analyzed by nonlinear least squares fitting to mathematical binding competition models using the computer program GraphPad Prism (GraphPad Software, Inc., Oberlin, OH). In stably transfected cell lines, 75–85% of the total bound counts can be displaced with cold GHRH. Calculations give receptor densities of 90–200 fmol/mg crude membrane protein.

For cAMP assays, cells were removed from 150-mm culture dishes and replated into 24-well cluster plates (Costar, Cambridge, MA) at 200,000 cells per well for 24 h before testing. Cells were incubated at 37 C with 1 mM isobutylmethylxanthine for 20 min without serum. Test peptides were added in fresh warmed media, and plates were incubated for an additional 15 min at 37 C. The medium was then aspirated, plates were frozen in a –70 C freezer, and cells were extracted with 100 mM HCl. cAMP levels in cell lysates were measured by previously described methods (5, 16).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and analysis of the cGHRH receptor cDNA clone
The sequences of four, full-length cDNAs obtained from separate high-fidelity PCR amplifications from subpools of the chicken pituitary mRNA library were determined in both the forward and reverse directions. The coding region of the cGHRH receptor cDNA was 1260 bases in length (submitted to GenBank, accession no. DQ114791), encoding a 419-amino-acid protein highly similar to other known GHRH receptors (Fig. 1Go). The chicken receptor sequence predicts a mature protein of the identical amino acid length seen in most mammals, but has a signal peptide that is four amino acids shorter. There is an additional alanine inserted in the extracellular domain at position 86, and a threonine is deleted in the intracellular C-terminal tail at position 410. Alignment of the amino acid sequence with related receptors shows that it is similar to other GHRH receptors, being 56–61% identical to the known mammalian GHRH receptors and approximately 40% identical to those of fish (Table 2Go). Phylogenetic analysis of sequence alignments (17, 18, 19) also supports that this is a GHRH receptor from chicken placing it between homologous receptors from mammals and fish and distinct from receptors for related peptides (Fig. 2Go). Note that GHRH receptors from different species are less well conserved than the corresponding receptors for PACAP, VIP, and secretin. This is consistent with the fact that GHRH peptide sequences are not well conserved (only 62% identity between mouse and man), whereas PACAP and VIP sequences are 100% conserved in most mammals and better than 90% identical in more distant species.


Figure 1
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  		FIG. 1. Alignment of known GHRH receptor amino acid sequences. Bold italics, Predicted signal peptide (32 ). Underlined, Potential sites for N-linked glycosylation. Dots, Gaps introduced for alignment. Shaded boxes, Predicted transmembrane helices (33 ). Refs for sequences: human (5 6 ), pig (8 ), sheep (7 ), cow (7 ), mouse (9 ), rat (6 ), goldfish (10 ), and puffer fish (11 ).

 

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  		TABLE 2. Percent amino acid identity for aligned GHRH-Rs

 

Figure 2
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  		FIG. 2. Phylogenetic distance tree derived from computer alignment of amino acid sequences of similar receptors. Branch points represent statistically chosen hypothetical common ancestral sequences. Branch lengths are proportional to the number of amino acid changes from that hypothetical sequence. Scale bar indicates 0.1 changes per amino acid. Alignment was generated with the program ClustalW (17 ), unrooted tree by the Fitch-Margoliash distance matrix method using Phylip (19 ), and the image was produced by Treeview (18 ). Secretin-R, secretin receptor; PAC1-R, PACAP-specific receptor; VPAC1-R, PACAP/VIP receptor type 1; VPAC2-R, PACAP/VIP receptor type 2.

 
Northern blotting found that the mRNA matching this sequence was expressed in chicken pituitary with lesser amounts found in hypothalamus, whole brain, and retina, but not in liver (Fig. 3Go). mRNA transcripts of approximately 1.4, 2.0, and 3.2 kb were detected in the chicken. By comparison, with a mammalian GHRH receptor probe, bands of 2.0 and 3.1 kb are seen in rat pituitary and 2.0 and 3.4 kb are seen in ovine pituitary (5). The most abundant transcript is 2 kb in all species. The significance of multiple pituitary GHRH receptor transcripts is unknown.


Figure 3
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  		FIG. 3. Northern blot showing relative expression of cGHRH-R mRNA in chicken tissues. An equal loading of 3 µg total RNA per lane was used for each tissue. 1, Pituitary; 2, hypothalamus; 3, whole brain; 4, retina; and 5, liver.

 
RT-PCR cloning of cGHRH-LP
RT-PCR cloning of cGHRH-LP revealed that the mRNA sequence was similar to that previously published with the exception that the nucleotide at position 63 was adenosine, not thymidine. This new sequence encoded a peptide with lysine at position 21 instead of asparagine (Lys21-cGHRH-LP). A total of nine clones, forward and reverse sequences, derived from four independent PCR were sequenced, and all encode the lysine at position 21. All GHRH peptides reported to date from other species include this lysine at position 21 (see Fig. 4Go), and recent sequencing of this chicken mRNA by other groups also finds this residue to be lysine (GenBank AY95323, BX932280, BX932926).


Figure 4
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  		FIG. 4. Alignment of GHRH, GHRH-like, and PRP sequences from different species. Areas of N- and C-terminal homology are shaded, conserved lysine is bold and underlined. ?, Unknown residue. The conserved N-terminal domain is known to be required for interaction with GHRH receptors (31 ), whereas the conserved C-terminal domain has no known function. Long forms (lf) of some of these peptides are listed to demonstrate homologies and because endogenous cleavage sites can vary with species and tissue. Species abbreviations and GenBank accession numbers: hGHRH1–44, human NP_066567; porGHRH, porcine P01287; bovGHRH, bovine AF242855; ovGHRH, ovine P07217; ratGHRH, rat P09916; mousGHRH, mouse M31658; cGHRH-L2, chicken BX929984; cGHRH-LP chicken, sequence presented here; DuGHRH-L, duck AF343119; MfrGHRH-L, marsh frog AF221632; XfrGHRH-L, clawed frog AF187877; carGHRH-L, carp P42692; salGHRH-L, salmon x 73233; cfiGHRH-L, catfish x 79078; TuGHRH-L1, tunicate (21 ); TuGHRH-L2, tunicate (21 ); hPRPlf, human PRP long form S83513; ovPRP, ovine NM_001009776; RatPRP, rat XM_579383; PigPRP, porcine NM_001001544.

 
Membrane binding studies
Figures 5Go and 6Go show competition binding studies using the hGHRH analog His1,125Tyr10,Nle27hGHRH(1–32)NH2 as probe. Figure 5Go examined binding by two independent cGHRH-R clones, P1 and P4, each expressed in HEK293 cells. The two clones were indistinguishable. Unlabeled His1,Nle27hGHRH(1–32)NH2 displaced the probe at both expressed receptor clones in a pattern suggesting a single high affinity binding site with KD of 1.04 nM. No specific GHRH binding was detected on nontransfected cells. When tested at the human receptor by this method, hGHRH gives a KD of 0.136 nM (20), yet this same hGHRH probe gives no detectable specific binding at the mouse GHRH receptor (20). Thus the hGHRH activity at the chicken receptor is fortuitous and better than would be predicted given its very poor activity at the mouse receptor, but is consistent with the known in vivo response to hGHRH in live chickens (12).


Figure 5
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  		FIG. 5. Competitive binding studies using hGHRH and 125I-labeled hGHRH as probe. Two independent clones [clone P1 ({blacktriangleup}) and clone P4 ({blacktriangledown})] were expressed separately in HEK293 cells. KD = 1.04 nM in each clone (four replicates per point).

 

Figure 6
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  		FIG. 6. A, Competition for binding of 125I-labeled hGHRH probe at the cloned cGHRH-R by hGHRH (1–32) (bullet), K21cGHRH-LP (1–33) ({blacksquare}) and N21cGHRH-LP (1–46) ({square}). B, Competition for binding of 125I-labeled hGHRH probe on chicken pituitary membranes by hGHRH (1–32) (bullet) and K21cGHRH-LP (1–33) ({blacksquare}) (four replicates per point).

 
Figure 6AGo compares the binding displacement seen with hGHRH with that of the original published cGHRH-LP, N21cGHRH-LP (1–46), and the peptide we developed, K21cGHRH-LP (1–33), based on our sequence. Although the N21cGHRH-LP had no measurable activity, the K21cGHRH-LP was only weakly active with approximately 400-fold less potency at the cloned cGHRH-R than hGHRH.

When crude membranes were prepared for binding studies from flash-frozen chicken pituitaries (Fig. 6BGo), K21cGHRH-LP (1–33) was significantly less potent than hGHRH in displacing hGHRH probe. N21cGHRH-LP (1–46) was not active in displacing hGHRH on these chicken pituitary membranes even at high dose (data not shown). Thus the endogenous receptor found on native chicken pituitary membranes showed binding properties for these peptides that were similar to those seen for the in vitro-expressed cGHRH clones. GHRH probes made from iodination of N21cGHRH-LP (1–46), K21cGHRH-LP (1–33), or carp GHRH-LP were tested and showed little detectable specific binding to either cGHRH-R or hGHRH-R, and so no displacement studies could be performed (data not shown). Tests with an independent synthesis of the peptide K21cGHRH-LP (1–44) gave the same activities as seen with K21cGHRH-LP (1–33) (data not shown).

cAMP generation
To establish whether the expressed cGHRH-R clones were functional in signaling, we examined cAMP accumulation in response to hGHRH in stably transfected cell lines and in nontransfected HEK293 cells. As shown in Fig. 7Go, basal levels of cAMP were low and unaffected by hGHRH in the nontransfected cells. Cells transfected with either clone P1 or P4 gave a similar strong response to 1 nM hGHRH. Stimulation with 1 µM isoproterenol, which acts at the ß adrenergic receptors endogenously found on these HEK293 cells, was used as a control to demonstrate that similar numbers of live responsive cells were present for each cell line.


Figure 7
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  		FIG. 7. Intracellular accumulation of cAMP in response to hGHRH or isoproterenol at wild-type HEK293, and HEK293 cells expressing either of two independent GHRH-R clones, P1 and P4 (eight replicates per point).

 
The potency of the chicken K21cGHRH-LP (1–33) was then tested at the recombinant cGHRH-R. Figure 8Go shows that the chicken peptide was approximately 100-fold less potent than the human peptide. Studies with N21cGHRH-LP (1–46) detected no significant activity at any dose, whereas K21cGHRH-LP (1–44) gave activities very similar to those seen with K21cGHRH-LP (1–33).


Figure 8
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  		FIG. 8. Accumulation of intracellular cAMP in wild-type (nontransfected) HEK293 cells treated with hGHRH ({square}) and in HEK293 transfected with the cGHRH-R treated with hGHRH (1–32) (bullet) or K21cGHRH-LP (1–33) ({blacksquare}) (four replicates per point).

 
To characterize the specificity of the recombinant cGHRH-R, we examined its responsiveness to other peptides (Fig. 9Go). It is known that the sequence-related peptides from the VIP/secretin family often share some cross-reactivity at each other’s receptors (3). In this study, human and chicken PACAP38 were the most potent related peptides with about 100-fold lower potency (higher EC50) than hGHRH. Next, most potent peptides were human and chicken VIP and chicken PACAP27, with nearly 1000-fold higher EC50. Other tested peptides with even lower but detectable potencies were carp GHRH-LP, turkey PHI, and human peptide histidine-methionine. Peptides with no detectable activity included secretin, glucagon, glucose-dependent insulinotrophic polypeptide (also called gastric inhibitory peptide), and PACAP-related peptide (PRP) (all human), GHRP2, and TRH (not all peptides tested are shown in the figure). Nontransfected HEK293 cells were found to have endogenous receptors that give a slight but detectable response to PACAP and VIP but not to other tested peptides (data not shown). Thus the cloned cGHRH-R, like mammalian GHRH receptors, appears most specific for mammalian GHRH, with reduced responses to some closely related peptides. Surprisingly, available nonmammalian GHRH-LPs (chicken and carp) were no more potent than PACAP.


Figure 9
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  		FIG. 9. Intracellular cAMP accumulation in response to different doses of the indicated peptides on HEK293 cells transfected with cGHRH-R. Peptide species: h, human; c, chicken; t, turkey (six replicates per point).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is a large body of evidence to suggest that the endocrine regulation of GH secretion in the chicken is similar to that observed in mammals (12). However, the constituents of the avian hypothalamic-pituitary-GH axis have been elusive. We have cloned a functioning, seven transmembrane domain G protein-linked receptor from the anterior pituitary gland of the chicken, which is also expressed in the hypothalamus and brain, albeit at lower levels. Comparisons made between the sequence of the present clone and the sequences of the known receptors for GHRH, secretin, PACAP, and VIP confirm that this receptor is most closely related to other GHRH receptors and falls between the known GHRH receptors of mammals and fish. Studies using a variety of peptides have shown that the receptor binds and signals cAMP accumulation in response to mammalian GHRH with greater affinity than other ligands from the same or related families, providing further support that this is a GHRH receptor. Other sequences for the cGHRH-R have recently been released on GenBank (XM_418490, DQ284530, and DQ230840). These encode the identical receptor protein sequence reported in this study except for XM_418490, where the computer predicted mRNA based on chicken genomic DNA sequence contains errors.

In vitro and in vivo studies in the chicken have shown that GH release from the pituitary is regulated by peptides such as TRH, ghrelin, and PACAP (12). In the absence of an active GHRH peptide, it has been suggested that these are the primary regulators of GH secretion (12). In the present study, we have clearly shown that TRH is inactive at the cGHRH-R confirming an alternative independent role for this system in the regulation of GH release in the chicken. PACAP is derived from the same gene as the cGHRH-LP and, in our hands, both of these peptides activate the cloned cGHRH-R with similar potency, but they are >100-fold less potent than hGHRH, which has high potency at either hGHRH-R (KD ~ 0.14 nM) or cGHRH-R (KD ~ 1 nM).

Evolutionarily, GHRH-LPs are first reported in protochordate tunicates where they are found on the same gene as PACAP and that gene is duplicated to produce two variants of each peptide (21). The role of GHRH in these animals is unclear because no endogenous GH-like hormone has been found (22). In fish, GHRH and PACAP are also found on the same gene (23), and PACAP acting through a specific PACAP receptor (PAC1) appears to be the major regulator of GH release (24, 25). Recently, an additional GHRH/PACAP gene has been identified in zebrafish such that messages for two very similar variants of each peptide are present (26). Two GHRH-LPs are also found in goldfish (27). It has been reported that chickens have only one GHRH-LP (cGHRH-LP) (21, 22), but others have suggested another peptide may exist (25), and recent large scale sequencing efforts appear to have identified a second cGHRH-like sequence (GenBank accession no. BX929984, expressed sequence tag from cDNA library from chicken head, and also XM_417357), which we will refer to as cGHRH-LP2. Montero et al. (25) have proposed that PACAP is the physiological regulator of GH release in teleost fish, but that GHRH has progressively taken over that role with the evolution of quadrupeds. In mammals, the two copies of the ancestral GHRH/PACAP gene have diverged such that, on one copy, the GHRH-LP has evolved to encode PRP, and on the other gene, the PACAP-like sequence now encodes GHRH-related peptide (22). This allows GHRH and PACAP to be regulated independently from different genes. PRP and GHRH-related peptide are not active at the known PACAP or GHRH receptors, and their functions are not well understood (28). The chicken cGHRH-LP peptide that has been studied here and in the literature (21, 22) is found on the same gene as PACAP, and as shown in Fig. 4Go, shares a C-terminal sequence motif with the PRP of mammals and the GHRH-LPs of fish and frogs. Compared with cGHRH-LP, the new cGHRH-LP2 sequence is more like mammalian GHRH in that it is encoded on a separate gene from PACAP, does not share this C-terminal motif, and has slightly greater overall sequence homology to mammalian GHRH (10 vs. 12 amino acid changes compared with human in the crucial 1–29 region of the peptide). But the endogenous peptide expression and physiological significance of any of these cGHRH-LPs remains unclear.

In addition to cloning the receptor, we have clarified the cDNA sequence of cGHRH-LP, which encodes a lysine residue at position 21 of the translated peptide, a residue conserved in all known GHRH peptides (Fig. 4Go). Despite this revised sequence, the synthetic peptide has only weak activity at the cloned receptor, similar to that of PACAP, compared with the strong activity of hGHRH. This is not an artifact of the cloned receptor, as hGHRH shows potent activity in binding studies using chicken pituitary membranes, in stimulating GH release from isolated chicken pituitary preparations, or when administered iv to live chickens (12), whereas N21cGHRH-LP had none of these activities (14) and both K21cGHRH-LP (1–33) and K21cGHRH-LP (1–44) had little or no activity in live 4- to 6-wk-old chickens (our unpublished data), an age when chickens are optimally responsive to GHRH (29). This suggests that the cGHRH-LP may not be the correct ligand and an alternative peptide such as cGHRH-LP2 may mediate GH release via this receptor. Consistent with this hypothesis, of the two similar GHRH like peptides found in goldfish, only one was active at the goldfish GHRH receptor (27). Because only the mRNA and not the actual peptides expressed in the chicken have been sequenced, it is also possible that the low potency of cGHRH-LP is because the endogenous peptide is processed or modified in some manner required for full biological activity such as is seen with the octanoylation of ghrelin (30). The mRNA for cGHRH-LP is alternatively spliced near the C terminus (residues 33–35 deleted) to yield peptides of 43 or 46 amino acids (13). Based on our knowledge of the mammalian system, this change in the peptide C terminus would not be expected to affect activity at the GHRH receptor as only the first 29 amino acids are needed (31), but this assumption may not hold true in the chicken, and full-length alternative splice forms were not synthesized and tested. A third alternative as to why we saw low potency from cGHRH-LP even after correcting its sequence is that there may have been an error during the synthetic process or that the naturally occurring ligand has a secondary structure that is not replicated during the synthetic process. Although the new K21cGHRH-LP was synthesized twice independently (once 1–33 and once 1–44) and each sequence was confirmed by mass spectrometry, sequence rearrangements or conformational problems are unlikely but not impossible.

In summary, we have cloned a functional GHRH receptor from the anterior pituitary of the chicken and determined a corrected cDNA sequence of a putative ligand for this receptor. Surprisingly, however, peptides synthesized to different lengths of the original published sequence for cGHRH-LP or to the modified sequences reported in this study had little or no potency at this receptor or in live chickens (14). However, this cGHRH-R provides a new tool to identify the endogenous GH-releasing peptide in chickens and to understand the evolutionary development of the regulation of GH.


   Acknowledgments
 
We acknowledge the gift of peptides from J. E. Rivier, W. W. Vale, and M. E. El-Halawani as well as technical support from the Center for Cellular and Molecular Studies in Reproduction (National Institutes of Health P30-HD28934), the University of Virginia Biomolecular Research Facility, and the Pratt Fund.


   Footnotes
 
Support for this work was provided by National Institutes of Health RO1 DK45350 to B.D.G., Pharmacia Ltd. to A.A.T. and Natural Sciences and Engineering Research Council (Canada) (to S.H.).

The cDNA coding sequence for the cGHRH-R reported in this study has been submitted to the DNA Data Base of Japan/European Molecular Biology Laboratory/GenBank databases under accession no. DQ114791.

First Published Online January 5, 2006

Abbreviations: cGHRH, chicken GHRH; cGHRH-LP, chicken GHRH-like peptide; cGHRH-R, cGHRH receptor; DIG, digoxigenin; GHRH-LP, GHRH-like peptide; HEK, human embryonic kidney; hGHRH, human GHRH; PACAP, pituitary adenylate cyclase activating polypeptide; PHI, peptide histidine-isoleucine; PRP, PACAP-related peptide; SSC, standard saline citrate; VIP, vasoactive intestinal peptide.

Received July 22, 2005.

Accepted for publication December 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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