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Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutagenesis and chimeric studies have suggested that the intracellular
regions of the GPCRs, in particular the second and third intracellular
(2i and 3i) loops and sometimes the cytoplasmic tail, interact with G
proteins and mediate signal transduction (15, 16, 17, 18, 19). Sequence alignment
of various members of the GPCR superfamily shows that the acidic (Asp)
and basic (Arg) residues of the DRY triplet are highly conserved (15, 16). Whereas the Arg residue in the triplet is invariant, in a few
instances the Asp and Tyr residues are conservatively substituted with
other amino acids (15, 20). Based on their conservation, it has been
proposed that these residues have important functions in ligand binding
and/or G protein interaction and activation. In studies on the
structure/function relationships of the GnRH receptor, we examined the
roles of the conserved acidic residue Asp138, the invariant
basic residue Arg139, and the unique Ser140
residue (which is Tyr in most other GPCRs) in agonist-induced signal
transduction and receptor internalization. Few studies have explored
the roles of specific amino acids in the carboxyl-terminal portion of
the 2i loop of the GPCRs in signaling and internalization, and no
consensus sequences have been identified. We therefore evaluated the
importance of several polar residues (Ser151,
Ser153, Lys154 and Glu156) in this
region in these cellular processes (see Fig. 1
) by first making
multiple replacements (carboxyl-terminal double and quadruple
mutations, referred to as c-DM and c-QM, respectively), to be followed
by single substitutions if effects were found. These conserved and
polar residues in the GnRH receptor were replaced with other amino
acids by site-directed mutagenesis, and the expressed receptors were
analyzed for ligand binding, GnRH-stimulated inositol phosphate
production, and agonist-induced internalization of the
receptor-hormone complex.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. The conserved
Asp residue at position 138 was changed to a neutral amino acid (Asn)
to eliminate its negative charge, and to one that preserves the
negative charge (Glu) but has a longer side chain. Replacement of Asp
by Tyr or His was also performed, but these mutant receptors showed no
radioligand binding, presumably due to lack of expression. The
invariant Arg residue at position 139 was substituted with uncharged
amino acids (Gln, Ala, and Ser), and with Lys to retain the same
positive charge but with a longer side chain. The Lys139
receptor was poorly expressed and thus could not be further analyzed.
Northern blot analysis of GnRH receptor mRNA in transfected COS-7 cells
expressing mutant receptors revealed no reduction in transcript levels
or molecular size as compared with the wild type receptor (data not
shown), suggesting that the differences in cell-surface expression of
the Asp138 and Arg139 mutant receptors do not
reflect changes at the pretranslational level. The unique
Ser140 was changed to Ala. The nonconserved polar amino
acids in the carboxyl terminus of the 2i loop, namely
Ser151, Ser153, Lys154, and
Glu156, were replaced with Ala, Ala, Gln, and Gln,
respectively.
[125I]GnRH-Ag binding was measured in intact COS-7 cells
transfected with mutant or wild type GnRH receptors to determine the
expression level and the functional integrity of these receptors at the
plasma membrane. As indicated in Table 1, the wild type
and all of the detectably expressed mutant receptors bound the
radioligand with high affinity, and Scatchard analysis of the binding
data yielded linear plots, reflecting a single class of GnRH-binding
sites. Most of the modified receptors displayed similar dissociation
constants, and the Asp138 mutants had slightly increased
binding affinity. The expression levels of the DRS receptor mutants
showed more significant variations. Although alanine replacement of
Ser140 had no major effect on receptor expression, mutation
of Asp138 and Arg139 in the DRS triplet reduced
expression to approximately one-tenth and one-third to one-half of that
of the wild type receptor, respectively (Table 1). However, receptors
bearing double and quadruple mutations at the C-terminal end of the 2i
loop (c-DM and c-QM) were expressed at almost the same level as the
wild type receptor and displayed similar agonist-binding affinity.
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), the correlation between cell-surface binding
sites and the maximal inositol phosphate response was determined after
COS-7 cells were transfected with increasing amounts of the wild type
receptor cDNA. Despite the wide range of receptor expression in these
cells, there was a near-linear relationship between the measured
receptor sites and the inositol phosphate responses to GnRH stimulation
(Fig. 2). Such linearity between cell-surface receptors
and inositol phosphate responses has been also observed in COS-7 cells
transfected with angiotensin II receptors (21). This finding indicates
that valid comparisons between cells expressing mutant GnRH receptors
can be made by normalizing their inositol phosphate responses to the
number of plasma membrane-binding sites (Fig. 3B).
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). Normalization of the data based on receptor
number showed that the impaired responses of cells transfected with the
D138E (or D138N) receptor were attributable to
their lower expression level, and that these mutants, in fact,
activated phospholipase C more effectively than the wild type receptor
(Fig. 3B). In contrast, maximal inositol phosphate signaling by the
R139Q receptor was found to be significantly impaired (by
about 50%) even when the data were normalized for the reduced number
of binding sites (Fig. 3B). Cells transfected with the
R139S and R139A receptors also exhibited
significantly reduced (55–65% of the wild type) inositol phosphate
production after normalization for receptor number (not shown). The
InsP2/InsP3 responses mediated by the
S140A, c-DM, and c-QM mutant receptors were similar to that
of the wild type receptor (Fig. 3, A and B).
Effect of Guanosine Thiotriphosphate (GTP
S) on
[125I]GnRH Agonist Binding to Wild Type and
Mutant GnRH Receptors
The ability of the Asp138 and Arg139
mutant receptors to interact with G proteins was further evaluated by
measuring the effect of GTPS on [125I]GnRH agonist
binding to COS-7 cell membranes expressing wild type,
D138N, and R139Q mutant receptors. As shown in
Fig. 4, treatment with GTPS reduced agonist binding
to the wild type receptor by about 55%. This reduction in agonist
binding was due to a decrease in the affinity of the receptor for GnRH
and reflects the normal coupling of the activated receptor to G
protein(s). The inhibitory effect of the GTP analog on agonist binding
to the D138N receptor was essentially the same as for the
wild type receptor (Fig. 4). However, GTPS had relatively little
effect on agonist binding to the R139Q receptor (Fig. 4),
consistent with the impaired ability of this mutant to mediate inositol
phosphate production in response to GnRH stimulation (Fig. 3B).
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T3–1 cells (22, 23). The effects of mutations on
receptor internalization were evaluated by measuring the kinetics of
[125I]GnRH-Ag uptake over a period of 60 min at 37 C in
cells expressing wild type or mutant receptors. A direct comparison
between the wild type and mutant receptors was made by plotting the
percent of bound radioligand that was internalized with increasing time
of incubation (see Fig. 5, A-D). Single replacements of
Asp138 by Asn or Glu increased the rate of internalization
(Fig. 5A), and the sequestration of radioligand at 60 min was at least
100% higher than that of the wild type receptor (Fig. 5E). The
endocytotic rate constant, a cellular constant that defines the
probability of an occupied receptor being internalized in 1 min at 37
C, was also calculated using these data. Values for D138N
and D139E receptors were 200–250% higher than that of the
wild type receptor. On the other hand, mutation of Arg139
to Gln, Ala, or Ser caused much slower internalization (Fig. 5B), and
the amount of tracer sequestered after 60 min was only 47% of that of
the wild type receptor (Fig. 5E). The endocytotic rate constants were
20% of that of the wild type receptor. The kinetics and rate constant
for the internalization of S140A receptors were virtually
identical to that of the wild type receptor (Fig. 5, C and E). The
internalization kinetics of the c-DM and c-QM receptors were rapid
compared with the wild type receptor (Fig. 5D), and the amounts
sequestered at 60 min were 28% and 56% higher, respectively, than
that of the wild type receptor (Fig. 5E). The endocytotic rate
constants for the c-DM and c-QM receptors were 50–150% increased
compared with the wild type receptor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, has revealed that in addition to
their structural role, these regions are functionally important
determinants of receptor expression, signaling, and internalization.
Our findings indicate that mutations of residues Asp138,
Arg139, Ser151, Ser153,
Lys154, and Glu156 alter agonist-induced
internalization of the GnRH receptor. Most of these mutations,
including replacement of Asp138 and the combined mutations
(c-DM and c-QM), increased the endocytotic rate constants by 50–250%.
On the other hand, substitution of Arg139 caused a
significant reduction in the rate of receptor internalization (Fig. 5E). Agonist-induced signal transduction, measured as the stimulation
of inositol phosphate production by GnRH, was largely unaffected by
mutations that increased receptor endocytosis. Conversely, mutations
that reduced internalization showed impaired signal transducing ability
(Fig. 3). The latter findings are consistent with our recent report
(20) that mutation of a highly conserved hydrophobic amino acid
(Leu147) in the 2i loop significantly impaired both
receptor-G protein coupling and receptor internalization.
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Another interesting observation was the significantly reduced
expression of receptors bearing Asp138 and
Arg139 mutations (Table 1). The low radioligand binding
capacity of cells expressing these mutant receptors could be due to
decreased receptor expression or to deficient localization of the
receptors at the cell surface. This possibility cannot be tested in the
absence of a suitable permeant ligand or a highly specific GnRH
receptor antibody. However, the agonist-binding affinity of the
Asp138 mutants was increased rather than decreased and was
largely unchanged for the Arg139Gln receptors, indicating
that these changes did not alter the integrity of the receptor. These
results also suggest that the impairment of signal generation by the
Arg139 mutant receptor was not caused by reduction of
binding affinity.
There is increasing evidence for the concept that positively charged
amino acids, located near the boundaries of transmembrane domains, are
important determinants of the topology of membrane-spanning proteins
(26, 27, 28). Because the Asp138 and Arg139
residues are located at the boundary of the third transmembrane domain
and the 2i loop, it is probable that mutation of either residue
disturbs the charge balance in this region. This in turn could disrupt
interactions and destabilize helix formation and thereby exert a
deleterious effect on receptor expression. The acidic and basic
residues in the DRY triplet are conserved in almost all GPCRs and are
located in a region that terminates as an -helical structure that
forms an extension of the third transmembrane domain (29). The
positively charged Arg residue may participate in ionic interactions
with water, G proteins, and/or charged lipid head groups. The
negatively charged Asp residue could likewise interact with G
protein(s) during receptor activation.
The highly conserved Arg residue in the DRY triplet has been shown to participate in G protein coupling. In a recent study (30), a point mutation of this residue in the m1 muscarinic receptor abolished its ability to mediate inositol phosphate production and binding of a labeled GTP analog. Mutation of the corresponding Arg residue in the m2 muscarinic receptor resulted in partial loss of receptor-G protein coupling in terms of inhibiting cAMP formation. Likewise, mutation of the corresponding Arg residue in the N-formyl peptide receptor impaired ligand binding and its ability to mobilize calcium (31). In the present study, we also observed a significant reduction in the ability of Arg139 mutants to mediate inositol phosphate formation during GnRH stimulation. The invariant Arg residue in the GnRH receptor has been suggested to be part of a conserved structural motif (I/LxxDRY/SxxI/V) in which the flanking ß-branched amino acid residues provide a hydrophobic cage that restricts its rotamer positions, thus enabling it to achieve the most favorable orientation(s) required for efficient G protein coupling (32). In regard to the structural basis of coupling specificity, this is possibly determined by cooperation between multiple regions of the receptor (15, 16, 17, 18, 19, 33).
In contrast to the above findings, no effects of Asp138
mutations on GnRH receptor signaling were observed. Previous studies
have suggested an important role for the corresponding aspartate
residue in the function of the 2A-adrenergic (34, 35),
ß2-adrenergic (36), and m1 muscarinic (37)
receptors. In these reports, substitution of aspartate by asparagine
had no influence on high-affinity agonist binding, but significantly
reduced the ability of the mutant receptors to couple to their
respective G protein/effector systems. The effects of mutation of the
corresponding Glu residue in rhodopsin have likewise been attributed to
impaired interaction with its G protein, transducin (38). Studies on
the TSH (24) and angiotensin II (25) receptors showed that multiple
mutations in the DRY region resulted in complete abolition of G protein
coupling. On the other hand, mutation of the corresponding single
acidic residue (Glu to Asp or Asn) in the LH/CG receptor did not affect
either hormone binding or signal transduction (39). The present finding
that mutation of Asp138 to Asn or Glu does not impair GnRH
receptor function is in general agreement with the data reported for
the LH/CG receptor and argues against the generality of an essential
role of this residue in signal transduction.
Several techniques, including mutagenesis, synthetic peptides, and
antibodies, have been employed in the past to probe for signaling
function(s) in the intracellular loops of various GPCRs. Although no
general consensus has been reached concerning the location and nature
of the intracellular determinants of receptor-G protein activation, one
or more of several intracellular regions have been implicated in
various GPCRs (15, 16, 19, 33). For example, studies on
ß1-adrenergic, muscarinic, and rhodopsin receptors have
shown that the amino- and carboxyl-terminal regions of the 3i loop, and
often the amino-terminal region of the cytoplasmic tail, are important
for coupling to G proteins (19, 33, 40). In the TSH receptor, the 1i
loop and the carboxyl-terminal regions of the 2i and 3i loops are
involved in signal transduction (24). These results suggest that 1)
GPCRs contain multisite, noncontiguous intracellular determinants of
agonist-induced receptor signaling; and 2) the presence and location of
the regions involved in G protein coupling vary among individual
receptors. It has been postulated that regions of the carboxyl-terminal
tail, and the 2i and 3i loops adjacent to the transmembrane domains,
may form amphipathic -helices. These, together with charged residues
in the 2i and 3i loops, i.e. DRY (or DRS in the GnRH
receptor), may interact cooperatively to permit efficient binding to G
proteins and their subsequent activation (15).
Our findings on receptor internalization indicate that single-point
mutations of Asp138 to Asn or Glu increase receptor
endocytosis by 100%, whereas mutation of the adjacent
Arg139 residue to Gln, Ala, or Ser reduce receptor
endocytosis by almost 50%. The critical importance of Ser/Thr-rich
sequences in the 3i loop or carboxyl-terminal tail of TRH (41),
gastrin-releasing peptide (GRP) (42), muscarinic (43), and yeast
-factor receptors (44), in maintaining efficient internalization has
been demonstrated. Also, in ß2-adrenergic (45) and GnRH
(46) receptors, the aromatic amino acid in the NPXXY sequence (or its
variant) in the seventh transmembrane domain was shown to be important
in receptor internalization. The present results suggest that the Asp
and Arg residues in the Asp-Arg-Ser triad are directly or indirectly
involved in GnRH receptor internalization. Because the GnRH receptor
has no Ser/Thr-rich regions in any of its intracellular loops and lacks
a cytoplasmic tail, other residues or as yet unidentified motifs must
be involved in its internalization. Recently, a Ser-Thr-Leu sequence in
the carboxyl-terminal region of the angiotensin II receptor has been
shown to be essential for agonist-induced endocytosis (47). In
receptors for PTH and PTH-related protein, which lack the conserved
NPXXY sequence, both positive and negative regulatory elements for
internalization are present in the carboxyl-terminal tail (48). The
nature of the regions/residues that are involved in the internalization
of the GnRH receptor will be investigated in future studies to provide
more information about the structural basis for its internalization.
The impaired internalization of the Arg139 mutant receptors
suggests that more than one region of the GnRH receptor may be involved
in internalization and/or that more than one internalization pathway
exists.
In summary, this analysis of the functional significance of the residues in the Asp-Arg-Ser triplet located in the N-terminal region of the 2i loop in the GnRH receptor has provided evidence for the importance of the aspartate and arginine residues in GnRH receptor signaling and internalization. Replacement of conserved Asp138 with either Asn or Glu reduced expression levels, but the mutant receptors showed increased agonist-induced internalization and activated phospholipase C more effectively than the wild type receptor. The invariant Arg139 residue appears to play an important role both in receptor-G protein coupling and in agonist-induced internalization of the receptor. The highly conserved nature of the Asp and Arg residues in this region in almost all members of the GPCR superfamily suggests that these residues are of general importance in receptor function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Methods
Construction of Wild Type and Mutant GnRH Receptors
The 1.22-kb GnRH receptor cDNA subcloned into pcDNAI/Amp at the
XbaI site (20) was used as a template for creating
site-directed mutations according to the method of Kunkel et
al (49) using a Muta-Gene phagemid in vitro mutagenesis
kit. The sequence of the 25 mer mutagenic primer for Asp138
was 5'-GATTAGCCTGGAG/GAACGCTCCCTGGCC-3'; at the
underlined bases, codon GAC for Asp was replaced with either
GAG (for Glu) or GAA (for Asn). For Arg139, the 28 mer
mutagenic primer was: 5' GATTAGCCTGGACCAG/GCC/AGC
TCCCTGGCCATC-3'; at the underlined bases the codon CGC for
Arg was altered to CAG for Gln, GCC for Ala, or AGC for Ser. These
mutations were performed using separate primers. For
Ser140, the 20 mer mutagenic primer was:
5'-CCTGGACCGCGCCCTGGCCA-3'; at the underlined
bases the codon TCC for Ser was altered to GCC for Ala. For the double
mutation (Ser151, Ser153) at the
carboxyl-terminal (c-DM), the 49 mer mutagenic primer was:
5'-CCCCTTGCTGTACAAGCCAACGCCAAGCTTGAACAGTCTATGATCAGC-C-3';
at the underlined bases the codons AGC for Ser were altered
to GCC for Ala. For the quadruple mutation (Ser151,
Ser153, Lys154, Glu156) at the
carboxyl terminus (c-QM), the 49 mer mutagenic primer was:
5'-CCCCTTGCTGTACAA-GCCAACGCCCAGCTTCAACAGTCTATGATCAGCC-3';
at the underlined bases the codons AGC for Ser were altered
to GCC for Ala, the codon AAG for Lys was replaced with CAG for Gln,
and the codon GAA for Glu was changed to CAA for Gln. Mutations were
confirmed by the dideoxy sequencing method of Sanger et al.
(50) using Sequenase II.
Transient Transfection in COS-7 Cells
Wild type and mutant GnRH receptors were transiently expressed in COS-7
cells. To measure inositol phosphate responses and internalization
kinetics, or [125I]GnRH-Ag binding to intact cells, the
cells were seeded in 24-well plates (Costar, Cambridge, MA) at a
density of 4 x 104 cells per well and cultured in
DMEM supplemented with 10% heat-inactivated FBS containing 100 U/ml of
penicillin and 100 µg/ml streptomycin (Pen-Strep) at 37 C in an
atmosphere consisting of 5% CO2-95% humidified air. At
60–70% confluence, the cells were transfected in 0.5 ml of serum-free
OPTI-MEM I medium with 1 µg of wild type or mutant plasmid DNA and
6–8 µg lipofectamine per well. For membrane-binding studies, 1
x 106 cells were cultured in 100-mm petri dishes for 3
days. Transfections were performed for 6 h using 5 ml OPTI-MEM I
containing 10 µg plasmid DNA and 16 µg/ml lipofectamine. Six hours
later, the medium was replaced with fresh medium and cultures were
maintained for 48 h before use in ligand binding, membrane
preparation, and functional assays.
Agonist Binding to Transfected Cells
The binding affinity and sites of the mutant receptors were determined
in transfected COS-7 cells incubated with 2 nM
[125I]GnRH-Ag in binding medium (M199 containing 25
mM HEPES and 0.1% BSA), in the absence or presence of
increasing concentrations of unlabeled peptide for 4 h at 4 C. The
cells were rapidly washed twice with ice-cold PBS (pH 7.4) and
solubilized in 0.2 M NaOH-1% SDS solution, and the
cell-associated radioactivity was measured by -spectrometry. All
time studies were performed in duplicate on at least three occasions,
and displacement curves were analyzed by the LIGAND program (obtained
from Dr. Peter J. Munson, National Institutes of Health, Bethesda, MD)
using a one-site model (51).
Internalization Assays
Transfected COS-7 cells were washed once with binding medium before the
addition of 2 nM 125I-labeled GnRH agonist.
Nonspecific binding was determined in the presence of 1000-fold excess
of the unlabeled GnRH agonist. After incubation at 37 C for the
indicated times, the cells were washed twice with ice-cold PBS (pH 7.4)
and incubated with 1 ml of 50 mM acetic acid-150
mM NaCl (pH 2.8) for 12 min to remove surface-bound tracer.
The acid-released radioactivity was collected to determine the
receptor-bound radioactivity, and the internalized (acid-resistant)
radioactivity was quantitated after solubilizing the cells in NaOH-SDS
solution. Radioactivities were measured by -spectrometry, and the
internalized radioligand at each timepoint was expressed as a percent
of the total (acid-resistant + acid-released) binding. The endocytotic
rate constant (52) was calculated using algorithms obtained from Dr. H.
Steven Wiley (University of Utah Medical Center, Salt Lake City, UT).
For these calculations, correction values of 4% and 10% were used for
surface to intracellular, and intracellular to surface spillover,
respectively. Values for endocytotic rate constant for the wild type
and various mutant receptors were: wild type, 0.010;
D138E/N, 0.031–0.038; R139Q/S/A, 0.002; c-DM,
0.015; c-QM, 0.025.
Radioligand Binding to COS-7 Cell Membranes
Transfected cells were washed twice with ice-cold 10 mM
Tris-HCl (pH 7.5) containing 1 mM EDTA and scraped into 1
ml of the same medium. Cells were lysed by freezing and thawing, and
crude membranes were prepared by centrifuging the samples at
16,000 x g. The pellets were washed in the same
medium, and the protein content was measured by the BCA method (Pierce
Chemical Co., Rockford, IL). Radioligand-binding assays were conducted
using 20- to 25-µg membranes in the presence or absence of GTPS
(20). The bound radioactivity was separated by rapid filtration
followed by three washes with ice-cold PBS (pH 7.4) and measured by
-spectrometry.
Inositol Phosphate Production
COS-7 cells were labeled 24 h after transfection by incubation in
inositol-free DMEM medium containing 20 µCi/ml
[3H]inositol as described previously (20). After 24
h of labeling, cells were washed with inositol-free M199 medium and
preincubated in the same medium containing 10 mM LiCl for
30 min at 37 C, then stimulated with 100 nM GnRH for 15
min. Incubations were terminated by the addition of ice-cold perchloric
acid [5% (vol/vol) final concentration]. The inositol phosphates
were extracted as described previously (53) and separated by anion
exchange chromatography. Briefly, after neutralization, the samples
were applied to Poly-Prep columns containing Dowex AG 1-X-8 resin. The
columns were washed four times with water (3 ml/wash) and twice with
0.2 M ammonium formate in 0.1 M formic acid (3
ml/wash) to remove inositol and inositol monophosphate, respectively.
Then, InsP2 + InsP3 fractions were eluted from
the columns by washing twice with 1 M ammonium formate in
0.1 M formic acid (3 ml/wash), and their radioactivities
were measured by liquid scintillation ß-spectrometry. The mean
inositol phosphate formation of cells expressing mutant receptors was
expressed as a percentage of that mediated by the wild type receptor in
the same experiment.
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FOOTNOTES |
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Received for publication October 25, 1996. Revision received April 17, 1997. Accepted for publication May 6, 1997.
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T3–1 cells. Endocrinology 135:1165–1170[Abstract]
2A-adrenergic receptors: identification of amino acids
involved in ligand binding and receptor activation by agonists. Mol
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Z. Gaborik, G. Jagadeesh, M. Zhang, A. Spat, K. J. Catt, and L. Hunyady The Role of a Conserved Region of the Second Intracellular Loop in AT1 Angiotensin Receptor Activation and Signaling Endocrinology, June 1, 2003; 144(6): 2220 - 2228. [Abstract] [Full Text] [PDF]
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 L. Z. Krsmanovic, N. Mores, C. E. Navarro, K. K. Arora, and K. J. Catt An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion PNAS, March 4, 2003; 100(5): 2969 - 2974. [Abstract] [Full Text] [PDF]
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 E. M. F. Costa, G. Y. Bedecarrats, B. B. Mendonca, I. J. P. Arnhold, U. B. Kaiser, and A. C. Latronico Two Novel Mutations in the Gonadotropin-Releasing Hormone Receptor Gene in Brazilian Patients with Hypogonadotropic Hypogonadism and Normal Olfaction J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2680 - 2686. [Abstract] [Full Text] [PDF]
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 T. Hashizume, W.-H. Yang, C. M. Clay, and T. M. Nett Internalization Rates of Murine and Ovine Gonadotropin-Releasing Hormone Receptors Biol Reprod, March 1, 2001; 64(3): 898 - 903. [Abstract] [Full Text]
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T. Madigou, E. Mañanos-Sanchez, S. Hulshof, I. Anglade, S. Zanuy, and O. Kah Cloning, Tissue Distribution, and Central Expression of the Gonadotropin-Releasing Hormone Receptor in the Rainbow Trout (Oncorhynchus mykiss) Biol Reprod, December 1, 2000; 63(6): 1857 - 1866. [Abstract] [Full Text]
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 N. S. Olesnicky, A. J. Brown, Y. Honda, S. L. Dyos, S. J. Dowell, and L. A. Casselton Self-Compatible B Mutants in Coprinus With Altered Pheromone-Receptor Specificities Genetics, November 1, 2000; 156(3): 1025 - 1033. [Abstract] [Full Text]
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R. D. Price and E. Sanders-Bush RNA Editing of the Human Serotonin 5-HT2C Receptor Delays Agonist-Stimulated Calcium Release Mol. Pharmacol., October 1, 2000; 58(4): 859 - 862. [Abstract] [Full Text]
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 S. H. Hoffmann, T. t. Laak, R. Kühne, H. Reiländer, and T. Beckers Residues within Transmembrane Helices 2 and 5 of the Human Gonadotropin-Releasing Hormone Receptor Contribute to Agonist and Antagonist Binding Mol. Endocrinol., July 1, 2000; 14(7): 1099 - 1115. [Abstract] [Full Text]
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B. E. Troskie, J. P. Hapgood, R. P. Millar, and N. Illing Complementary Deoxyribonucleic Acid Cloning, Gene Expression, and Ligand Selectivity of a Novel Gonadotropin-Releasing Hormone Receptor Expressed in the Pituitary and Midbrain of Xenopus laevis Endocrinology, May 1, 2000; 141(5): 1764 - 1771. [Abstract] [Full Text] [PDF]
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A. E. Alewijnse, H. Timmerman, E. H. Jacobs, M. J. Smit, E. Roovers, S. Cotecchia, and R. Leurs The Effect of Mutations in the DRY Motif on the Constitutive Activity and Structural Instability of the Histamine H2 Receptor Mol. Pharmacol., May 1, 2000; 57(5): 890 - 898. [Abstract] [Full Text]
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A. Scheer, T. Costa, F. Fanelli, P. G. De Benedetti, S. Mhaouty-Kodja, L. Abuin, M. Nenniger-Tosato, and S. Cotecchia Mutational Analysis of the Highly Conserved Arginine within the Glu/Asp-Arg-Tyr Motif of the alpha 1b-Adrenergic Receptor: Effects on Receptor Isomerization and Activation Mol. Pharmacol., February 1, 2000; 57(2): 219 - 231. [Abstract] [Full Text]
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A. Heding, M. Vrecl, A. C. Hanyaloglu, R. Sellar, P. L. Taylor, and K. A. Eidne The Rat Gonadotropin-Releasing Hormone Receptor Internalizes via a {beta}-Arrestin-Independent, but Dynamin-Dependent, Pathway: Addition of a Carboxyl-Terminal Tail Confers {beta}-Arrestin Dependency Endocrinology, January 1, 2000; 141(1): 299 - 306. [Abstract] [Full Text] [PDF]
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H.-O. Chung, Q. Yang, K. J. Catt, and K. K. Arora Expression and Function of the Gonadotropin-releasing Hormone Receptor Are Dependent on a Conserved Apolar Amino Acid in the Third Intracellular Loop J. Biol. Chem., December 10, 1999; 274(50): 35756 - 35762. [Abstract] [Full Text] [PDF]
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M. Blomenröhr, A. Heding, R. Sellar, R. Leurs, J. Bogerd, K. A. Eidne, and G. B. Willars Pivotal Role for the Cytoplasmic Carboxyl-Terminal Tail of a Nonmammalian Gonadotropin-Releasing Hormone Receptor in Cell Surface Expression, Ligand Binding, and Receptor Phosphorylation and Internalization Mol. Pharmacol., December 1, 1999; 56(6): 1229 - 1237. [Abstract] [Full Text]
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L. L. K. Elias, A. Huebner, G. D. Pullinger, A. Mirtella, and A. J. L. Clark Functional Characterization of Naturally Occurring Mutations of the Human Adrenocorticotropin Receptor: Poor Correlation of Phenotype and Genotype J. Clin. Endocrinol. Metab., August 1, 1999; 84(8): 2766 - 2770. [Abstract] [Full Text]
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F. Fanelli, P. Barbier, D. Zanchetta, P. G. de Benedetti, and B. Chini Activation Mechanism of Human Oxytocin Receptor: A Combined Study of Experimental and Computer-Simulated Mutagenesis Mol. Pharmacol., July 1, 1999; 56(1): 214 - 225. [Abstract] [Full Text]
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K. K. Arora, H.-O. Chung, and K. J. Catt Influence of a Species-Specific Extracellular Amino Acid on Expression and Function of the Human Gonadotropin-Releasing Hormone Receptor Mol. Endocrinol., June 1, 1999; 13(6): 890 - 896. [Abstract] [Full Text]
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A. Pizard, A. Blaukat, W. Muller-Esterl, F. Alhenc-Gelas, and R. M. Rajerison Bradykinin-induced Internalization of the Human B2 Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail J. Biol. Chem., April 30, 1999; 274(18): 12738 - 12747. [Abstract] [Full Text] [PDF]
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C. M. Niswender, S. C. Copeland, K. Herrick-Davis, R. B. Emeson, and E. Sanders-Bush RNA Editing of the Human Serotonin 5-Hydroxytryptamine 2C Receptor Silences Constitutive Activity J. Biol. Chem., April 2, 1999; 274(14): 9472 - 9478. [Abstract] [Full Text] [PDF]
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M. Vrecl, L. Anderson, A. Hanyaloglu, A. M. McGregor, A. D. Groarke, G. Milligan, P. L. Taylor, and K. A. Eidne Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of {beta}-Arrestin on Internalization Kinetics Mol. Endocrinol., December 1, 1998; 12(12): 1818 - 1829. [Abstract] [Full Text]
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X. Lin, J. A. Janovick, and P. M. Conn Mutations at the Consensus Phosphorylation Sites in the Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor: Effects on Receptor Ligand Binding and Signal Transduction Biol Reprod, December 1, 1998; 59(6): 1470 - 1476. [Abstract] [Full Text]
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K. K. Arora, L. Z. Krsmanovic, N. Mores, H. O'Farrell, and K. J. Catt Mediation of Cyclic AMP Signaling by the First Intracellular Loop of the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., October 2, 1998; 273(40): 25581 - 25586. [Abstract] [Full Text] [PDF]
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L. Z. Krsmanovic, N. Mores, C. E. Navarro, S. A. Saeed, K. K. Arora, and K. J. Catt Muscarinic Regulation of Intracellular Signaling and Neurosecretion in Gonadotropin-Releasing Hormone Neurons Endocrinology, October 1, 1998; 139(10): 4037 - 4043. [Abstract] [Full Text] [PDF]
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Y.-M. Sun, R. P. Millar, H. Ho, M. C. Gershengorn, and N. Illing Cloning and Characterization of the Chicken Thyrotropin-Releasing Hormone Receptor Endocrinology, August 1, 1998; 139(8): 3390 - 3398. [Abstract] [Full Text] [PDF]
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A. Heding, M. Vrecl, J. Bogerd, A. McGregor, R. Sellar, P. L. Taylor, and K. A. Eidne Gonadotropin-releasing Hormone Receptors with Intracellular Carboxyl-terminal Tails Undergo Acute Desensitization of Total Inositol Phosphate Production and Exhibit Accelerated Internalization Kinetics J. Biol. Chem., May 8, 1998; 273(19): 11472 - 11477. [Abstract] [Full Text] [PDF]
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A. Ulloa-Aguirre, D. Stanislaus, V. Arora, J. Vaananen, S. Brothers, J. A. Janovick, and P. M. Conn The Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor Couples the Receptor to Gs- and Gq/11-Mediated Signal Transduction Pathways: Evidence from Loop Fragment Transfection in GGH3 Cells Endocrinology, May 1, 1998; 139(5): 2472 - 2478. [Abstract] [Full Text] [PDF]
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J. Ballesteros, S. Kitanovic, F. Guarnieri, P. Davies, B. J. Fromme, K. Konvicka, L. Chi, R. P. Millar, J. S. Davidson, H. Weinstein, et al. Functional Microdomains in G-protein-coupled Receptors. THE CONSERVED ARGININE-CAGE MOTIF IN THE GONADOTROPIN-RELEASING HORMONE RECEPTOR J. Biol. Chem., April 24, 1998; 273(17): 10445 - 10453. [Abstract] [Full Text] [PDF]
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R. D. Price, D. M. Weiner, M. S. S. Chang, and E. Sanders-Bush RNA Editing of the Human Serotonin 5-HT2C Receptor Alters Receptor-mediated Activation of G13 Protein J. Biol. Chem., November 21, 2001; 276(48): 44663 - 44668. [Abstract] [Full Text] [PDF]
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