| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Biochemistry and Physiology (K.K.M., O.C.P., S.D.F.,
D.L.H., C.P.T., R.G.S., L.H.T.VdP., A.D.H.) Merck Research
Laboratories Rahway, New Jersey 07065
Department of Human
Genetics (M.S.P.) Merck Research Laboratories West Point,
Pennsylvania 19486
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
2 kb divides the open
reading frame into two exons encoding TM 1–5 and TM 6–7, thus placing
the GHS-R into the intron-containing class of GPC-Rs. The intron maps
to the site of sequence divergence between the human and swine type 1a
and 1b GHS-R mRNAs. In addition, determination of the nucleotide
sequence for the human GHS-R gene confirmed the position of an intron
in the human GHS-R gene at this position. A full-length contiguous cDNA
from rat hypothalamus was isolated and shown to be identical in its
nucleotide and deduced amino acid sequence to the rat pituitary GHS-R.
The cloned rat GHS-R binds [35S]MK-0677 with
high affinity [dissociation constant (KD) =
0.7 nM] and is functionally active when
expressed in HEK-293 cells. Expression of the rat GHS-R was observed
specifically in the pituitary and hypothalamus when compared with
control tissues. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
11 (Ref. 20 and S. D. Feighner, D. L. Hreniuk, O.
C. Palyha, L. H. T. Van Der Ploeg, and A. D. Howard, unpublished
observations). From both human and swine pituitary, two types of cDNA
were isolated: type 1a, encoding a functional protein containing 7-TM
domains, and type 1b, encoding a protein containing TM-1 through 5 with
no measurable functional activity in cell-based assays. In
situ hybridization studies in rhesus brain and RNase protection
with RNA from human tissues demonstrated that GHS-R expression could be
detected in discrete nuclei of the hypothalamus. In this report, we
describe the cloning and characterization of the rat GHS-R type 1a gene
from the pituitary. An intron in the pre-mRNA locates to the same
predicted amino acid position at which the human and swine type Ia and
Ib GHS-R cDNAs diverged. Nucleotide sequence analysis of the human gene
shows that the human and swine Ib mRNA also contain an intron with a
short conserved intron-derived coding sequence. A full-length GHS-R
cDNA from the rat hypothalamus is identical to the rat type Ia GHS-R
from the pituitary gland. The KD for MK-0677 binding to the
GHS-R measured 0.7 nM, and the receptor is functionally
active when expressed in HEK-293 cells. |
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
106 plaques. The
cDNA encoding the GHS-R was excised as a 6.5-kb BsiW1 fragment, and
its nucleotide sequence was determined. A noncontiguous ORF of1.1
kb encoding seven transmembrane domains (TM) that exhibits a high
degree of sequence identity to both human and swine type 1a GHS-Rs was
identified (Fig. 1
; the location of the first ATG is
conserved compared with the human and swine GHS-Rs and is preceded by
several in-frame stop codons, assuring that the correct
translation initiation codon was identified). Approximately 2 kb of
noncoding intronic sequence at nt 790 (corresponds to a splice donor
site [(A/C)AG/GTAAGT)]) was found at the second amino acid of the
predicted TM-6. This intron divides the ORF into an amino-terminal
segment ending at leucine-263 (encompassing the extracellular domain,
TM-1 through TM-5, and the three intra- and first two extracellular
loops) and a carboxyl-terminal segment encoding TM-6, the third
extracellular loop, TM-7, and the C-terminal intracellular domain. The
position of the intron is highly conserved among rat, human, and swine
GHS-R genes (20), and, as previously observed for several other GPC-Rs,
the intron is located between the TM domains (21, 22). The cDNA clone
described here encodes a protein with significant homology to the type
1a GHS-R and presumably represents unspliced GHS-R pre-mRNA. Two types
of human and swine GHS-R cDNA (type 1a and 1b) that most likely arise
from a single gene by alternative mRNA processing were described
previously (20). This assertion was confirmed by determination of the
nucleotide sequence for the proposed human exon-intron boundaries and
the complete intron of the human gene. Type 1a cDNA encodes the
complete 7-TM GHS-R and results from a splicing event that removes the
intron. In type 1b cDNA, the intron is not removed, and an alternative
polyadenylation signal is presumably used in the intron. As a result,
the human and swine type 1b cDNA contain a short, 24-amino acid ORF
fused to leucine-263. Comparison of the rat sequence 3' to the proposed
splice acceptor site with the corresponding region of the human and
swine type 1b cDNA demonstrates significant sequence divergence,
suggesting that the intronic sequences differ among rat, human, and
swine. This genomic organization is also found in the murine GHS-R
gene, which shares 95% nucleotide sequence identity to the rat exon
and intron nucleotide sequence near the splice acceptor site (M.-H.
Jiang, A. D. Howard, L. H. T. Van Der Ploeg, and H. Zheng,
unpublished observations). Interestingly, the rat intron encodes an
80-amino acid extension to the ORF that bears no identity to any known
GHS-R protein sequence.
|
). Compared with the human and swine GHS-R, the rat
GHS-R is two amino acids shorter, with a loss of one residue each in
the amino-terminal extracellular domain and the third intracellular
loop. Six amino acid substitutions were also noted: three being
localized to the N-terminal extracellular region, one in TM-1, and two
in the C-terminal intracellular domain. Several conserved features
ascribed to GPC-Rs were also identified in the rat GHS-R: the signature
aromatic triplet sequence (Glu-Arg-Tyr) adjacent to TM-3, Cys-115, and
Cys-197 in the first two extracellular loops capable of disulfide
bonding, putative amino-terminal N-glycosylation sites
(Asn-Xaa-Ser/Thr), phosphorylation sites in the carboxyl-terminus and
the third cytoplasmic loop, and conserved proline residues in TM-4, 5,
6, and 7 (23).
|
).
Related genes could not be identified under reduced stringency
conditions (lane 1). A similar hybridization profile indicative of a
single copy gene was also observed in other species such as rhesus
monkey, dog, mouse, and rabbit. Hybridizing sequences could not be
observed in yeast (A. D. Howard, C. P. Tan, and K. K. McKee,
unpublished observations). Interestingly, in humans the GHS-R gene is
found at a chromosomal location (3q26.2) in the vicinity of the
possible map position for the Brachmann-de Lange syndrome, a prenatal
and postnatal growth deficiency with developmental delay and dysmorphic
features (24). Determination of the putative involvement of the GHS-R
with the deficiency identified in the Brachman-de Lange syndrome is in
progress.
|
6 fmol/mg pi-tuitary
membrane protein), PCR amplification of swine GHS-R sequences from
pools of an unamplified swine cDNA library (prevalence of 1:300,000),
Northern blot analysis (no signal detected with 10 µg poly
(A)+ mRNA), and in situ hybridization signals
(weak specific signals observed in the central nervous system when
compared with other control GPC-Rs; Refs. 20 and 25). Accordingly,
RNase protection methodology was used to perform an initial analysis of
the tissue-specific expression pattern of the rat GHS-R. The probe used
was designed against the human GHS-R type 1a and 1b sequences (89%
overall nucleic acid sequence identity between rat and human cDNAs) and
therefore contains mismatched residues when compared with the rat
nucleotide sequence. This probe revealed three protected fragments with
human pituitary poly A+ mRNA (Fig. 3b
, lane 1) which are
not found in a variety of other tissues, including liver, kidney, whole
brain, and testis (25). The size of the protected bands at 410, 304,
and 106 nucleotides corresponds to transcripts encoding: full-length
type 1a (410 nucleotides); unspliced pre-mRNA for type 1a (304
nucleotides); and type 1b GHS-R or unspliced pre-mRNA for type 1a (106
nucleotides). Using rat poly (A)+ mRNAs and allowing for
mismatches between the human and rat nucleotide sequences that generate
additional RNase digestion sites, a similar pattern of three bands
[213 (nt 884-1097; spliced type 1a mRNA), 155 (nt 729–884; spliced
type 1a mRNA) and 70 nucleotides (nt 730–800; type 1b mRNA or
unspliced type 1a pre-mRNA)] was observed in rat pituitary and
hypothalamus (Fig. 3b, lanes 2 and 3, respectively) but not in tissues
that contain no detectable GHS-R binding activity, such as liver,
kidney, or whole brain (lanes 4–6). These results were confirmed and
extended by mapping the expression of GHS-R mRNA in rat brain and
peripheral tissues by in situ hybridization, which reveals
that the strongest signals are found in hippocampal and multiple
hypothalamic nuclei and the pituitary gland (25).
A contiguous DNA fragment encoding the complete 7-TM rat ORF
(devoid of intervening sequence) was expressed in HEK-293 cells and
COS-7 cells. Radioligand binding studies were performed on membranes
prepared from transfected COS-7 cells. Binding was saturable
(Bmax of specific binding 30 pmol/mg), of high affinity
(KD = 0.7 nM) and specificity (> 90%) (Fig. 4A). As shown in Fig. 4B, both peptide and
nonpeptide GHSs compete with high affinity for the binding of
[35S]MK-0677, in a similar rank order of potency observed
for the native rat pituitary receptor and cloned human receptor
(Ki values for GHRP-2, GHRP-6, and MK-0677 are 0.5, 1.5,
and 0.7 nM, respectively). Functional activity of the
expressed receptor was determined by measuring MK-0677-dependent,
IP3-coupled mobilization of intracellular calcium and
concomitant calcium-induced aequorin bioluminescence. The rat GHS-R
expression construct was transiently transfected into an aequorin
reporter cell line [293-AEQ17; (25)]. After 40 h of expression,
the cells were charged with the essential chromophore coelenterazine
and harvested. A dose-response curve for the rat GHS-R is shown in Fig. 5 for MK-0677 concentrations ranging from
10-5 to 10-12 M giving an
EC50 value of approximately 4 nM. The rank
order potency of GHRP-2, GHRP-6, and MK-0677 in functional assays
(IP3 release) is also comparable to the EC50
values obtained in the GH release assay in primary rat pituitary cells.
One explanation for the 5-fold difference in IC50 for
MK-0677 in the radioligand binding assay and the EC50 in
the aequorin functional assay is that the high level of receptor
expression results in a large proportion of cell-surface receptors that
are not G protein-coupled and thus cannot tranduce the Ca2+
mobilization signal needed for aequorin bioluminescence. This
interpretation is corroborated by binding data showing a significant
proportion (50%) of GTP
S-insensitive binding sites. As expected,
the dose-response curve in the functional assay can be shifted as a
function of the amount of cell-surface receptor expressed using
different amounts of DNA for transfection (our unpublished
observations).
|
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
gt11 (RL1051b;
Clontech, Palo Alto, CA) were plated on Escherichia coli
strain Y1090r-. The plaques were transferred to
maximum-strength Nytran (Schleicher & Schuell, Keene, NH) denatured,
neutralized, and screened with a 1.6 kb
EcoRI/NotI fragment containing the entire coding
and untranslated regions of the swine GHS-R, clone 7–3 (20). The
membranes were incubated at 30 C in prehybridization solution [50%
formamide, 2 x Denhardts, 5 x SSPE (1 x is 0.18
M NaCl, 0.01 M NaH2PO4,
0.001 M Na2EDTA; pH 7.7), 0.1% SDS, 100
µg/ml salmon sperm DNA] for 3 h followed by overnight
incubation in hybridization solution (50% formamide, 2 x
Denhardt’s, 5 x SSPE, 0.1% SDS, 10% dextran sulfate, 100
µg/ml salmon sperm DNA) with 1 x 106 cpm/ml of
[32P]-labeled probe. The probe was labeled with
[32P]dCTP using a random priming kit (GIBCO BRL,
Gaithersburg, MD). After hybridization, the blots were washed two times
each with 2 x NaCl-sodium citrate (SSC), 0.1% SDS (at 24 C, then
37 C, and finally 55 C). A single positive clone was isolated after
three rounds of plaque purification. Phage containing the GHS-R was
eluted from plate plaques with 1 x -buffer (0.1 M
NaCl, 0.01 M MgSO4, 35 mM Tris-HCl,
pH 7.5) after overnight growth of approximately 200 pfu/150 mm dish.
After a 10-min centrifugation at 10,000 x g to remove
debris, the phage solution was treated with 1 µg/ml RNAse A and DNAse
I for 30 min at 24 C, followed by precipitation with 20% polyethylene
glycol (8000)/2 M NaCl for 2 h on ice, and collection
by centrifugation at 10,000 x g for 20 min. Phage DNA
was isolated by incubation in 0.1% SDS, 30 mM EDTA, 50
µg/ml proteinase K for 1 h at 68 C, with subsequent phenol
(three times) and chloroform (twice) extraction before isopropanol
precipitation overnight. The GHS-R cDNA insert (6.4 kb) was
subcloned from gt11 into the plasmid vector Litmus 28 (New England
Biolabs, Beverly, MA). Two micrograms of phage DNA were heated to 65 C
for 10 min, then digested with 100 U of BsiWI (New England Biolabs) at
37 C (overnight). A 6.5-kb fragment was gel purified, electro eluted,
and phenol/chloroform extracted before ligation to BsiWI-digested
Litmus 28 vector. Double-stranded DNA was sequenced on both strands on
an ABI 373 automated sequencer using the ABI PRISM dye termination
cycle sequencing ready reaction kit (Perkin Elmer; Foster City, CA).
Isolation of full-length rat hypothalamus type 1a GHS-R cDNA was
performed by screening a plasmid-based cDNA library (700,000 primary
clones) constructed in the vector pcDNA-3 (Invitrogen) using poly
(A)+ mRNA prepared from freshly dissected tissue. Two
independent positive clones were identified.
Complementary DNA Expression Studies
For sequence comparisons and functional expression studies, a
contiguous ORF for the rat GHS-R type Ia was generated by removal of
intervening sequences. PCR was used to synthesize an amino-terminal
fragment from Met-1 to Val-260 with EcoRI (5') and
HpaI (3') restriction enzyme sites, while a
carboxyl-terminal fragment was generated from Lys-261 to Thr-364 with
DraI (5') and NotI (3') restriction enzyme sites.
The construct was assembled into the mammalian expression vector pSV-7
(20) with EcoRI/NotI-digested pSV7,
EcoRI/HpaI-digested NH2-terminal
fragment, and DraI/NotI-digested C-terminal
fragment.
Transfection of COS-7 and 293-AEQ17 Cells
Transient transfections of the rat GHS-R pSV-7 construct were
conducted using the lipofectamine procedure (GIBCO BRL) according to
the manufacturer’s instructions. Transfections were performed in 60-mm
dishes (80% confluent cells) with 30 µg lipofectamine and 2.5 µg
GHS-R plasmid DNA. Receptor expression was allowed to proceed for
48–72 h.
Radioligand Binding Assay
The binding of [35S]MK-0677 to crude
membranes prepared from COS-7 cell transfectants was performed
essentially as described (16, 20). Crude cell membranes were prepared
on ice 48 h after transfection. Each 60-mm dish was washed twice
with 3 ml PBS, once with 1 ml homogenization buffer [50 mM
Tris-HCl (pH 7.4), 5 mM MgCl2, 2.5
mM EDTA, 30 µg/ml bacitracin]. Homogenization buffer
(0.5 ml) was added to each dish, and cells were removed by scraping and
then homogenized using a Polytron device (Brinkmann, Syosset, NY; three
bursts of 10 sec at setting 4). The homogenate was centrifuged for 20
min at 11,000 x g at 0 C, and the resulting crude
membrane pellet (chiefly containing cell membranes and nuclei) was
resuspended in homogenization buffer supplemented with 0.06% BSA (0.5
ml/60-mm dish) and kept on ice. Binding reactions were performed at 20
C for 1 h in a total volume of 0.5 ml containing: 0.1 ml membrane
suspension (25 µg protein), 10 µl [35S]MK-677
(0.05 to 1 nM; specific activity, 1200 Ci/mmol), 10 µl
competing drug, and 380–390 µl homogenization buffer. Bound
radioligand was separated by rapid vacuum filtration (Brandel 48-well
cell harvester) through GF/C filters pretreated for 1 h with 0.5%
polyethylenimine. After application of the membrane suspension to the
filter, the filters were washed three times with 3 ml each of ice-cold
50 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, 2.5 mM EDTA, and 0.015% Triton X-100,
and the bound radioactivity on the filters was quantitated by
scintillation counting. Specific binding (> 90% of total) is defined
as the difference between total binding and nonspecific binding
conducted in the presence of 50 nM unlabeled MK-0677.
Aequorin Bioluminescence Assay
The assay was carried out essentially as described with
modifications (26). Measurement of GHS-R expression in the
aequorin-expressing stable reporter cell line 293-AEQ17 was performed
using a Turner model 20E luminometer (Turner Designs, Sunnyvale, CA).
293-AEQ17 cells (60-mm dish, 8 x 105 cells plated
18 h before transfection) were transfected with 2.5 µg plasmid
DNA-30 µg lipofectamine. After approximately 40 h of expression,
the aequorin in the cells was charged for 2 h with coelenterazine
(10 µM) under reducing conditions (30 µM
reduced glutathione) in ECB buffer [140 mM NaCl, 20
mM KCl, 20 mM HEPES-NaOH (pH 7.4), 5
mM glucose, 1 mM MgCl2, 1
mM CaCl2, 0.1 mg/ml BSA], the cells were
harvested, washed, and pelleted by low speed centrifugation into
plastic luminometer tubes (75 x 12 mm, Sarstedt). ECB buffer (2.9
ml) was added without disturbing the cell pellet, and bioluminescence
measurements were triggered by the injection of 0.1 ml of 30 x
concentrated stocks of MK-0677. Recordings were followed for 2 min to
observe responses consistent with an IP3-mediated
kinetics.
Southern Blot Analysis and RNase Protection Assay (RPA)
A commercial blot (Clontech) containing
EcoRI-digested rat genomic DNA (4 µg) was hybridized
overnight with a 32P-labeled fragment of the rat GHS-R cDNA
(nt 253 to 775 of the ORF) in a solution containing 50%
formamide, 2 x Denhardts, 5 x SSPE, 0.1% SDS, 10% dextran
sulfate, 100 µg/ml salmon sperm DNA. The blot was subsequently washed
under low (4 x SSC, 55 C) and then high (0.1 x SSC, 65 C)
stringency conditions for three washes each (15 min/wash) and exposed
to X-omat film overnight at -70 C with two intensifying screens.
Synthesis of high-specific activity radiolabeled antisense probes and
the RPA was conducted using a kit (MAXIscript and HybSpeed RPA kits;
Ambion, Austin, TX) essentially as described by the manufacturer. The
antisense cRNA GHS-R probe was synthesized from a cDNA template
encompassing nt 681 to 1101 of the human GHS-R ORF inserted behind the
T7 promoter in pGEM-11Z(f)+ (Promega Biotech, Madison, WI).
Digestion of the construct with PvuI will generate a cRNA
transcript 550 nt in size with 140 nt of vector sequence. Input
poly A+ mRNA (Clontech) was 5 µg for the GHS-R probe and
0.1 µg for a control human actin probe. Precipitated fragments were
subjected to slab-gel electrophoresis (42 cm x 32 cm x 0.4
mm) in 5% acrylamide/Tris-borate-EDTA buffer containing 8
M urea. The gels were fixed, dried, and autoradiographed on
film (X-Omat; Kodak) for 3 days (GHS-R) or 18 h (actin).
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication October 18, 1996. Revision received January 16, 1997. Accepted for publication January 16, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. Mano-Otagiri, H. Ohata, A. Iwasaki-Sekino, T. Nemoto, and T. Shibasaki Ghrelin suppresses noradrenaline release in the brown adipose tissue of rats J. Endocrinol., June 1, 2009; 201(3): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chen, Z. Tang, A. Yan, W. Li, and H. Lin Molecular cloning and mRNA expression analysis of two GH secretagogue receptor transcripts in orange-spotted grouper (Epinephelus coioides) J. Endocrinol., November 1, 2008; 199(2): 253 - 265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, B. Chai, J.-y. Li, H. Wang, and M. W. Mulholland Effect of Des-acyl Ghrelin on Adiposity and Glucose Metabolism Endocrinology, September 1, 2008; 149(9): 4710 - 4716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Viani, A. Vottero, F. Tassi, G. Cremonini, C. Sartori, S. Bernasconi, B. Ferrari, and L. Ghizzoni Ghrelin Inhibits Steroid Biosynthesis by Cultured Granulosa-Lutein Cells J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1476 - 1481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Fernandez-Fernandez, M. Tena-Sempere, J. Roa, J. M. Castellano, V. M Navarro, E. Aguilar, and L. Pinilla Direct stimulatory effect of ghrelin on pituitary release of LH through a nitric oxide-dependent mechanism that is modulated by estrogen Reproduction, June 1, 2007; 133(6): 1223 - 1232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Brzozowski, P. C. Konturek, Z. Sliwowski, R. Pajdo, D. Drozdowicz, S. Kwiecien, G. Burnat, S. J. Konturek, and W. W. Pawlik Prostaglandin/Cyclooxygenase Pathway in Ghrelin-Induced Gastroprotection against Ischemia-Reperfusion Injury J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 477 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Burdyga, A. Varro, R. Dimaline, D. G. Thompson, and G. J. Dockray Ghrelin receptors in rat and human nodose ganglia: putative role in regulating CB-1 and MCH receptor abundance Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1289 - G1297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Martini, R. Fernandez-Fernandez, S. Tovar, V. M. Navarro, E. Vigo, M. J. Vazquez, J. S. Davies, N. M. Thompson, E. Aguilar, L. Pinilla, et al. Comparative Analysis of the Effects of Ghrelin and Unacylated Ghrelin on Luteinizing Hormone Secretion in Male Rats Endocrinology, May 1, 2006; 147(5): 2374 - 2382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Toshinai, H. Yamaguchi, Y. Sun, R. G. Smith, A. Yamanaka, T. Sakurai, Y. Date, M. S. Mondal, T. Shimbara, T. Kawagoe, et al. Des-Acyl Ghrelin Induces Food Intake by a Mechanism Independent of the Growth Hormone Secretagogue Receptor Endocrinology, May 1, 2006; 147(5): 2306 - 2314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Tack, I Depoortere, R Bisschops, C Delporte, B Coulie, A Meulemans, J Janssens, and T Peeters Influence of ghrelin on interdigestive gastrointestinal motility in humans Gut, March 1, 2006; 55(3): 327 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Pulman, W. M. Fry, G. T. Cottrell, and A. V. Ferguson The Subfornical Organ: A Central Target for Circulating Feeding Signals J. Neurosci., February 15, 2006; 26(7): 2022 - 2030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Davenport, T. I. Bonner, S. M. Foord, A. J. Harmar, R. R. Neubig, J.-P. Pin, M. Spedding, M. Kojima, and K. Kangawa International Union of Pharmacology. LVI. Ghrelin Receptor Nomenclature, Distribution, and Function Pharmacol. Rev., December 1, 2005; 57(4): 541 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zizzari, H. Halem, J. Taylor, J. Z. Dong, R. Datta, M. D. Culler, J. Epelbaum, and M. T. Bluet-Pajot Endogenous Ghrelin Regulates Episodic Growth Hormone (GH) Secretion by Amplifying GH Pulse Amplitude: Evidence from Antagonism of the GH Secretagogue-R1a Receptor Endocrinology, September 1, 2005; 146(9): 3836 - 3842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. G. Smith Development of Growth Hormone Secretagogues Endocr. Rev., May 1, 2005; 26(3): 346 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kojima and K. Kangawa Ghrelin: Structure and Function Physiol Rev, April 1, 2005; 85(2): 495 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gaytan, C. Morales, M. L. Barreiro, P. Jeffery, L. K. Chopin, A. C. Herington, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Expression of Growth Hormone Secretagogue Receptor Type 1a, the Functional Ghrelin Receptor, in Human Ovarian Surface Epithelium, Mullerian Duct Derivatives, and Ovarian Tumors J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1798 - 1804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Jeffery, R. P. Duncan, A. H. Yeh, R. A. Jaskolski, D. S. Hammond, A. C. Herington, and L. K. Chopin Expression of the Ghrelin Axis in the Mouse: An Exon 4-Deleted Mouse Proghrelin Variant Encodes a Novel C Terminal Peptide Endocrinology, January 1, 2005; 146(1): 432 - 440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, T. R. Lin, Y. Hu, Y. Fan, L. Zhao, E. L. Stuenkel, and M. W. Mulholland Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus J. Physiol., September 15, 2004; 559(3): 729 - 737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-P. Chanoine and A. C. K. Wong Ghrelin Gene Expression Is Markedly Higher in Fetal Pancreas Compared with Fetal Stomach: Effect of Maternal Fasting Endocrinology, August 1, 2004; 145(8): 3813 - 3820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Luque, R. D. Kineman, S. Park, X.-D. Peng, F. Gracia-Navarro, J. P. Castano, and M. M. Malagon Homologous and Heterologous Regulation of Pituitary Receptors for Ghrelin and Growth Hormone-Releasing Hormone Endocrinology, July 1, 2004; 145(7): 3182 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, L. Zhao, T. R. Lin, B. Chai, Y. Fan, I. Gantz, and M. W. Mulholland Inhibition of Adipogenesis by Ghrelin Mol. Biol. Cell, May 1, 2004; 15(5): 2484 - 2491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wierup, S. Yang, R. J. McEvilly, H. Mulder, and F. Sundler Ghrelin Is Expressed in a Novel Endocrine Cell Type in Developing Rat Islets and Inhibits Insulin Secretion from INS-1 (832/13) Cells J. Histochem. Cytochem., March 1, 2004; 52(3): 301 - 310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Camina, M. C. Carreira, S. El Messari, C. Llorens-Cortes, R. G. Smith, and F. F. Casanueva Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a Endocrinology, February 1, 2004; 145(2): 930 - 940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gaytan, M. L. Barreiro, J. E. Caminos, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, R. Paniagua, M. Nistal, F. F. Casanueva, et al. Expression of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in Normal Human Testis and Testicular Tumors J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 400 - 409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Malagon, R. M. Luque, E. Ruiz-Guerrero, F. Rodriguez-Pacheco, S. Garcia-Navarro, F. F. Casanueva, F. Gracia-Navarro, and J. P. Castano Intracellular Signaling Mechanisms Mediating Ghrelin-Stimulated Growth Hormone Release in Somatotropes Endocrinology, December 1, 2003; 144(12): 5372 - 5380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kaiya, M. Kojima, H. Hosoda, S. Moriyama, A. Takahashi, H. Kawauchi, and K. Kangawa Peptide Purification, Complementary Deoxyribonucleic Acid (DNA) and Genomic DNA Cloning, and Functional Characterization of Ghrelin in Rainbow Trout Endocrinology, December 1, 2003; 144(12): 5215 - 5226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Holst, A. Cygankiewicz, T. H. Jensen, M. Ankersen, and T. W. Schwartz High Constitutive Signaling of the Ghrelin Receptor--Identification of a Potent Inverse Agonist Mol. Endocrinol., November 1, 2003; 17(11): 2201 - 2210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kawamura, N. Sato, J. Fukuda, H. Kodama, J. Kumagai, H. Tanikawa, A. Nakamura, Y. Honda, T. Sato, and T. Tanaka Ghrelin Inhibits the Development of Mouse Preimplantation Embryos in Vitro Endocrinology, June 1, 2003; 144(6): 2623 - 2633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.L. Barreiro, J.S. Suominen, F. Gaytan, L. Pinilla, L.K. Chopin, F.F. Casanueva, C. Dieguez, E. Aguilar, J. Toppari, and M. Tena-Sempere Developmental, Stage-Specific, and Hormonally Regulated Expression of Growth Hormone Secretagogue Receptor Messenger RNA in Rat Testis Biol Reprod, May 1, 2003; 68(5): 1631 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Tannenbaum, J. Epelbaum, and C. Y. Bowers Interrelationship between the Novel Peptide Ghrelin and Somatostatin/Growth Hormone-Releasing Hormone in Regulation of Pulsatile Growth Hormone Secretion Endocrinology, March 1, 2003; 144(3): 967 - 974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gaytan, M. L. Barreiro, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Immunolocalization of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in the Cyclic Human Ovary J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 879 - 887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hosoda, M. Kojima, T. Mizushima, S. Shimizu, and K. Kangawa Structural Divergence of Human Ghrelin. IDENTIFICATION OF MULTIPLE GHRELIN-DERIVED MOLECULES PRODUCED BY POST-TRANSLATIONAL PROCESSING J. Biol. Chem., January 3, 2003; 278(1): 64 - 70. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hosoda, M. Kojima, and K. Kangawa Ghrelin and the Regulation of Food Intake and Energy Balance Mol. Interv., December 1, 2002; 2(8): 494 - 503. [Abstract] [Full Text]
|
|
|
|
 |
|
 V. Bodart, M. Febbraio, A. Demers, N. McNicoll, P. Pohankova, A. Perreault, T. Sejlitz, E. Escher, R.L. Silverstein, D. Lamontagne, et al. CD36 Mediates the Cardiovascular Action of Growth Hormone-Releasing Peptides in the Heart Circ. Res., May 3, 2002; 90(8): 844 - 849. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Makino, H. Hosoda, K. Shibata, I. Makino, M. Kojima, K. Kangawa, and T. Kawarabayashi Alteration of Plasma Ghrelin Levels Associated With the Blood Pressure in Pregnancy Hypertension, March 1, 2002; 39(3): 781 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Murata, Y. Okimura, K. Iida, M. Matsumoto, H. Sowa, H. Kaji, M. Kojima, K. Kangawa, and K. Chihara Ghrelin Modulates the Downstream Molecules of Insulin Signaling in Hepatoma Cells J. Biol. Chem., February 8, 2002; 277(7): 5667 - 5674. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tena-Sempere, M. L. Barreiro, L. C. Gonzalez, F. Gaytan, F.-P. Zhang, J. E. Caminos, L. Pinilla, F. F. Casanueva, C. Dieguez, and E. Aguilar Novel Expression and Functional Role of Ghrelin in Rat Testis Endocrinology, February 1, 2002; 143(2): 717 - 725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Date, M. Nakazato, S. Hashiguchi, K. Dezaki, M. S. Mondal, H. Hosoda, M. Kojima, K. Kangawa, T. Arima, H. Matsuo, et al. Ghrelin Is Present in Pancreatic {alpha}-Cells of Humans and Rats and Stimulates Insulin Secretion Diabetes, January 1, 2002; 51(1): 124 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hattori, T. Saito, T. Yagyu, B.-H. Jiang, K. Kitagawa, and C. Inagaki GH, GH Receptor, GH Secretagogue Receptor, and Ghrelin Expression in Human T Cells, B Cells, and Neutrophils J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4284 - 4291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Petersenn, A. C. Rasch, M. Penshorn, F. U. Beil, and H. M. Schulte Genomic Structure and Transcriptional Regulation of the Human Growth Hormone Secretagogue Receptor Endocrinology, June 1, 2001; 142(6): 2649 - 2659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Lasko, A. I. Korytko, W. B. Wehrenberg, and L. Cuttler Differential GH-releasing hormone regulation of GHRH receptor mRNA expression in the rat pituitary Am J Physiol Endocrinol Metab, April 1, 2001; 280(4): E626 - E631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Gualillo, J. E. Caminos, M. Blanco, T. Garcia-Caballero, M. Kojima, K. Kangawa, C. Dieguez, and F. F. Casanueva Ghrelin, A Novel Placental-Derived Hormone Endocrinology, February 1, 2001; 142(2): 788 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Date, M. Kojima, H. Hosoda, A. Sawaguchi, M. S. Mondal, T. Suganuma, S. Matsukura, K. Kangawa, and M. Nakazato Ghrelin, a Novel Growth Hormone-Releasing Acylated Peptide, Is Synthesized in a Distinct Endocrine Cell Type in the Gastrointestinal Tracts of Rats and Humans Endocrinology, November 1, 2000; 141(11): 4255 - 4261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Nass, J. Gilrain, S. Anderson, B. Gaylinn, A. Dalkin, R. Day, M. Peruggia, and M. O. Thorner High Plasma Growth Hormone (GH) Levels Inhibit Expression of GH Secretagogue Receptor Messenger Ribonucleic Acid Levels in the Rat Pituitary Endocrinology, June 1, 2000; 141(6): 2084 - 2089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tivesten, E. Bollano, K. Caidahl, V. Kujacic, X. Y. Sun, T. Hedner, A. Hjalmarson, B.-A. Bengtsson, and J. Isgaard The Growth Hormone Secretagogue Hexarelin Improves Cardiac Function in Rats after Experimental Myocardial Infarction Endocrinology, January 1, 2000; 141(1): 60 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. C. Palyha, S. D. Feighner, C. P. Tan, K. K. McKee, D. L. Hreniuk, Y.-D. Gao, K. D. Schleim, L. Yang, G. J. Morriello, R. Nargund, et al. Ligand Activation Domain of Human Orphan Growth Hormone (GH) Secretagogue Receptor (GHS-R) Conserved from Pufferfish to Humans Mol. Endocrinol., January 1, 2000; 14(1): 160 - 169. [Abstract] [Full Text]
|
|
|
|
|
|
V. Locatelli, G. Rossoni, F. Schweiger, A. Torsello, V. De Gennaro Colonna, M. Bernareggi, R. Deghenghi, E. E. Müller, and F. Berti Growth Hormone-Independent Cardioprotective Effects of Hexarelin in the Rat Endocrinology, September 1, 1999; 140(9): 4024 - 4031. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. D. Feighner, C. P. Tan, K. K. McKee, O. C. Palyha, D. L. Hreniuk, S. Pong, C. P. Austin, D. Figueroa, D. MacNeil, M. A. Cascieri, et al. Receptor for Motilin Identified in the Human Gastrointestinal System Science, June 25, 1999; 284(5423): 2184 - 2188. [Abstract] [Full Text]
|
|
|
|
|
|
N. Shah, W. S. Evans, C. Y. Bowers, and J. D. Veldhuis Tripartite Neuroendocrine Activation of the Human Growth Hormone (GH) Axis in Women by Continuous 24-Hour GH-Releasing Peptide Infusion: Pulsatile, Entropic, and Nyctohemeral Mechanisms J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2140 - 2150. [Abstract] [Full Text]
|
|
|
|
|
|
E. E. Muller, V. Locatelli, and D. Cocchi Neuroendocrine Control of Growth Hormone Secretion Physiol Rev, April 1, 1999; 79(2): 511 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kaji, S. Tai, Y. Okimura, G. Iguchi, Y. Takahashi, H. Abe, and K. Chihara Cloning and Characterization of the 5'-Flanking Region of the Human Growth Hormone Secretagogue Receptor Gene J. Biol. Chem., December 18, 1998; 273(51): 33885 - 33888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Giustina and J. D. Veldhuis Pathophysiology of the Neuroregulation of Growth Hormone Secretion in Experimental Animals and the Human Endocr. Rev., December 1, 1998; 19(6): 717 - 797. [Abstract] [Full Text]
|
|
|
|
|
|
M. Korbonits, R. A. Jacobs, S. J. B. Aylwin, J. M. Burrin, P. L. M. Dahia, J. P. Monson, J. Honegger, R. Fahlbush, P. J. Trainer, S. L. Chew, et al. Expression of the Growth Hormone Secretagogue Receptor in Pituitary Adenomas and Other Neuroendocrine Tumors J. Clin. Endocrinol. Metab., October 1, 1998; 83(10): 3624 - 3630. [Abstract] [Full Text]
|
|
|
|
|
|
D. F. Carmignac, P. A. Bennett, and I. C. A. F. Robinson Effects of Growth Hormone Secretagogues on Prolactin Release in Anesthetized Dwarf (dw/dw) Rats Endocrinology, August 1, 1998; 139(8): 3590 - 3596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Briard, V. Guillaume, C. Frachebois, M. Rico-Gomez, N. Sauze, C. Oliver, and A. Dutour Endotoxin Injection Increases Growth Hormone and Somatostatin Secretion in Sheep Endocrinology, June 1, 1998; 139(6): 2662 - 2669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. F. Adams, B. Huang, M. Buchfelder, A. Howard, R. G. Smith, S. D. Feighner, L. H. T. van der Ploeg, C. Y. Bowers, and R. Fahlbusch Presence of Growth Hormone Secretagogue Receptor Messenger Ribonucleic Acid in Human Pituitary Tumors and Rat GH3 Cells J. Clin. Endocrinol. Metab., February 1, 1998; 83(2): 638 - 642. [Abstract] [Full Text]
|
|
|
|
|
|
S. D. Feighner, A. D. Howard, K. Prendergast, O. C. Palyha, D. L. Hreniuk, R. Nargund, D. Underwood, J. R. Tata, D. C. Dean, C. P. Tan, et al. Structural Requirements for the Activation of the Human Growth Hormone Secretagogue Receptor by Peptide and Nonpeptide Secretagogues Mol. Endocrinol., January 1, 1998; 12(1): 137 - 145. [Abstract] [Full Text]
|
|
|
|
|
|
P. A. Bennett, G. B. Thomas, A. D. Howard, S. D. Feighner, L. H. T. Van der Ploeg, R. G. Smith, and I. C. A. F. Robinson Hypothalamic Growth Hormone Secretagogue-Receptor (GHS-R) Expression Is Regulated by Growth Hormone in the Rat Endocrinology, November 1, 1997; 138(11): 4552 - 4557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zheng, A. Bailey, M.-H. Jiang, K. Honda, H. Y. Chen, M. E. Trumbauer, L. H.T. Van der Ploeg, J. M. Schaeffer, G. Leng, and R. G. Smith Somatostatin Receptor Subtype 2 Knockout Mice Are Refractory to Growth Hormone-Negative Feedback on Arcuate Neurons Mol. Endocrinol., October 1, 1997; 11(11): 1709 - 1717. [Abstract] [Full Text]
|
|
|
|
|
|
H. Hosoda, M. Kojima, H. Matsuo, and K. Kangawa Purification and Characterization of Rat des-Gln14-Ghrelin, a Second Endogenous Ligand for the Growth Hormone Secretagogue Receptor J. Biol. Chem., July 14, 2000; 275(29): 21995 - 22000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kaiya, M. Kojima, H. Hosoda, A. Koda, K. Yamamoto, Y. Kitajima, M. Matsumoto, Y. Minamitake, S. Kikuyama, and K. Kangawa Bullfrog Ghrelin Is Modified by n-Octanoic Acid at Its Third Threonine Residue J. Biol. Chem., October 26, 2001; 276(44): 40441 - 40448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Bodart, M. Febbraio, A. Demers, N. McNicoll, P. Pohankova, A. Perreault, T. Sejlitz, E. Escher, R.L. Silverstein, D. Lamontagne, et al. CD36 Mediates the Cardiovascular Action of Growth Hormone-Releasing Peptides in the Heart Circ. Res., May 3, 2002; 90(8): 844 - 849. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
