| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Oregon Regional Primate Research Center (A.C., J.A.J., X.L., P.M.C.), Beaverton, Oregon 97006; Department of Physiology and Pharmacology (P.M.C.) Oregon Health Sciences University, Portland, Oregon 97201
Address all correspondence and requests for reprints to: P. Michael Conn, Oregon Health Sciences University, 505 N.W. 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Generation of chimeras of rGnRHR and GFP
Wild-type (wt) rGnRHR cDNA in pcDNA1 was subcloned into pcDNA3.1
at BamHI and XhoI restriction enzyme sites. One
chimera (5) of rGnRHR and GFP was constructed by overlap extension PCR
(PCR), a procedure used to join DNA fragments that contain an overlap
region (8).
To construct rGnRHR-Ctail-GFP, fusion of the N terminus of the GFP to the C terminus of the rat GnRHR with catfish GnRHR intracellular C-tail as an intermediate spacer, rGnRHR-Ctail sequence, including 5'-untranslated region and complete coding region but without the stop codon, was amplified from the rGnRHR-Ctail cDNA in pcDNA3.1, using T7 primer and a 30-mer chimeric primer (CTTGCTCACCAT/CTGTCCACTGGGTTGGTC). The rGnRHR-Ctail cDNA containing wt rGnRHR and an intracellular C-terminal tail of catfish GnRHR was constructed previously (9). The sequence for GFP including full coding region, and 3'-untranslated region was amplied from pEGFP-N1 vector using a 30-mer chimeric primer (CCCAGTGGACAG/ATGGTGAGCAAGG-GCGAG) and the pEGFP-N1 vector primer GFP-C. The DNA fragments for rGnRHR-Ctail and GFP were then used as templates in a third PCR reaction with primer set, T7, and GFP-C. The third PCR reaction produced a full-length chimeric cDNA for rGnRHR-Ctail-GFP. The chimeric cDNA was flanked by the restriction sites present in the polylinker of pcDNA3.1 vector. The cDNA was then digested with BamHI and XhoI or XbaI and subcloned into the same sites of pcDNA3.1 vector. The identity of the chimeric constructs and the correctness of the PCR-derived coding sequence was verified by Dye Terminator Cycle Sequencing according to the manufacturer’s instructions (Perkin-Elmer Corp., Foster City, CA). Large-scale plasmid DNA for transfection was prepared by double-banded CsCl gradient centrifugation. The purity and identity of plasmid DNA was further verified by restriction enzyme analysis.
Transient transfection of GH3 cells
For confocal microscopy of live cells, glass coverslips (24
x 40 mm) were immersed in 12 N HCl for 1–2 h, rinsed 3
times with sterile distilled water, then secured in a sterile 100 mm
Petri dish with dental wax. The GH3 cells were maintained
in growth medium (DMEM; Irvine Scientific, Santa Ana, CA) containing
10% FCS (HyClone Laboratories, Inc. Logan, UT) and 20
µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA) in a humidified
atmosphere (37 C) containing 5% CO2. The cells were plated
at a density of 9 x 105 cells/dish. Twenty-four hours
later, the cells were washed once with 1.5 ml OPTI-MEM (Life Technologies, Inc.) and then were transiently transfected with
0.072, 0.36, 0.72, 3.6, or 7.2 µg of cDNA for the chimeric receptor
using 10 µl Lipofectamine (Life Technologies, Inc.) in
1.5 ml OPTI-MEM/dish. Lac Z cDNA was used to bring the final DNA
concentration to 7.2 µg, so that all the transfections contained the
same amount of total DNA. Five hours later, 1.5 ml of DMEM/20% FCS was
added to each dish. Twenty-four hours after the start of the
transfection, the medium was replaced with fresh DMEM/10%
FCS/gentamicin and the cells were allowed to incubate for an additional
24 h before imaging. The cells were washed twice with warm medium
(DMEM/0.1% BSA) and then were chilled on ice and incubated with the
GnRH agonist (20 nM) conjugated to Texas Red-succinimidyl
ester, as described below, for 2 h on ice. The cells were washed
with cold medium, the coverslips containing the cells were then
transferred to a cell chamber, and 37 C medium was added to the chamber
and incubated with the live cells during confocal imaging. Images were
recorded at the indicated times after the addition of the warm
medium.
For confocal microscopy of fixed cells, the same procedure was used as described above except that the cells were transfected with 4 µg DNA/dish. Forty-eight hours after transfecting, the cells were incubated with the GnRH agonist (20 nM) conjugated to Texas Red-succinimidyl ester for 0, 5, 10, 20, 30, 60, 90, 120, 150, or 180 min at 37 C. The agonist was removed, the cells were chilled on ice and washed 2 times in cold PBS. The cells were then fixed in 4% paraformaldehyde (4 C) for 30 min. The coverslips were mounted in buffered glycerol on a glass slide for confocal imaging.
Microscopy
Cells were imaged with a Leica Corp. TCS NT
confocal system (Leica Corp. Lasertechnik, GmBH,
Heidelberg, Germany). GFP was imaged using the 488 nm excitation line
of an argon laser and an emission band pass filter of 530 ± 30
nm. Texas Red was excited with a 568 nm Kr gas laser line and imaged
through a 590 nm long pass filter. As indicated, bis-benzimine (Hoechst
33258, Molecular Probes, Inc., Eugene, OR) was used to
stain nuclei, excited with 364 nm Ar laser and imaged through a
440 ± 40 nm band pass filter. A 100x NA 1.4 PlanApochromat
objective was used for all images. In some cases, z-sections were
imaged throughout the thickness of the cell, in others, particularly in
live cell time lapse images, the section with the largest area was
chosen and imaged. The thickness of a single section was approximately
0.5 µm. Images were processed and analyzed with the use of Photoshop
(Adobe Systems Inc., San Jose, CA) and MetaMorph (Universal Imaging,
West Chester, PA).
Conjugation of a GnRH agonist to Texas Red-succinimidyl ester
We dissolved 115 µg
D-Lys6-Pro9-des-Gly10-EA-GnRH
(GnRHa, a gift from Dr. Nick Ling) in 11.5 µl of 0.1 M
sodium bicarbonate (pH 8.26). Then we dissolved 777 µg Texas Red-SE
(no. T6134, Lot no. 3471–16) were dissolved in 77 µl
dimethylsulfoxide. We mixed 100 µg GnRHa with 777 µg Texas
Red-SE and incubated at room temperature for 2 h in
the dark. Then 8.7 µl of 1.5 M hydroxylamine HCl (pH 8.6)
was added and incubated for 1 h at room temperature in the dark.
The conjugated peptide was then loaded onto a Sephadex G-25–80 column
precoated with PBS/0.3% BSA and eluted with PBS. Thirty drop fractions
were collected into glass tubes containing 20 µl PBS/0.3% BSA. RIA
and visible spectroscopy (595 nm) were used to determine which
fractions contained the conjugated peptide. The appropriate fractions
were pooled and stored at -80 C.
Bioassay of GnRHa-TxR
An inositol phosphate (IP) bioassay was used to check the
biological activity of the GnRHa-Texas Red conjugate. Briefly,
GH3 cells were plated in a 24-well Costar
(Cambridge, MA) plate at 105 cells/well. Twenty-four hours
later, the cells were washed once with 0.5 ml OPTI-MEM and then
transiently transfected with 0.8 µg plasmid chimeric receptor
DNA/well using 2 µl Lipofectamine in 0.250 ml OPTI-MEM. Five hours
later, 0.25 ml of DMEM/20% FCS was added per well. Twenty-four hours
after the start of the transfection, the medium was replaced with 0.5
ml/well DMEM/10% FCS/gentamicin, and the cells were allowed to grow.
Forty-eight hours after transfecting, the cells were washed with
DMEM/0.1% BSA, then preloaded with 0.5 ml DMEM (inositol free)
containing 4 µCi/ml [3H]inositol (NEN Life Science Products, Boston, MA) for 18 h at 37 C. After
preloading, the cells were washed with DMEM (inositol free) containing
5 mM LiCl and stimulated with the GnRH-TR conjugates for
2 h. The treatments were removed and 1 ml 0.1 M formic
acid was added to each well. The cells were frozen and thawed to
disrupt the cell membranes. IP accumulation was determined by Dowex
Anion exchange chromatography and liquid scintillation spectroscopy as
previously described (10).
Comparison of Internalization of wild-type GnRHR and Chimeras
GH3 cells were transiently transfected using
the same method as for confocal microscopy except that 2 x
105 cells were plated per well in a 12-well plate. Each
plate served as one time point and contained wild-type rGnRHR,
rGnRHR-Ctail, and rGnRHR-Ctail-GFP transfected into the cells using 1.6
µg cDNA plus 4 µl Lipofectamine per well. Approximately 72 h
after transfection, a radioligand acid wash method was used to measure
internalization of the GnRHR. Briefly, the cells were washed twice with
warm DMEM/0.1% BSA. The cells were incubated with
125I-Buserelin (200,000 cpm/well) for 5, 10, 20, 30, 60, or
90 min. At the appropriate time, the iodinated ligand was removed, and
the plate was placed on ice. The cells were washed twice with ice-cold
PBS and 500 µl of acid solution (50 mM acetic acid, 150
mM NaCl, pH 2.8) was added to each well and incubated for
12 min on ice. To determine the surface-bound iodinated ligand, the
acid wash was collected and counted in a Packard Instrument 
counter
(Downers Grove, IL). To determine the internalized radioligand/receptor
complex, cells were solubilized in 500 µl 0.1% Triton-X 100/PBS,
collected, and counted. Nonspecific binding for all time points and all
cDNAs were determined using the same procedure but in the presence of
[10 µM] unlabeled agonist. Nonspecific binding was
subtracted from the surface-bound and internalized radioligand and the
internalized radioligand was expressed as the percent internalized of
the total bound at each time point. All time points were run in
triplicate in three experiments and were graphed as the average ±
SEM.
Data and image analysis
For bioassays, data shown are the mean of triplicate assay wells
and are presented as the mean ± SEM of replicates in
each experiment. The SEM was typically less than 10% of
the mean. Each experiment was repeated three or more times to ensure
the reproducibility of the findings.
To evaluate the rate of receptor and agonist internalization, fluorescence intensity was measured for each cell and each fluorophore at different times after activation, using MetaMorph (Universal Imaging). Fluorescence intensity, measured for every resolution element of the image, is, in principle, proportional to the number of fluorophores present in the corresponding volume element. In reality, the relation is complicated by the interference of a multitude of factors associated with optical features of the microscope, specimen, detection system, and otherwise (11). Even when all imaging parameters are kept constant, photobleaching of fluorophores, a practically unavoidable consequence of imaging, makes intensity measurements unreliable. If, however, the integrated intensity in one area, in our case the cell interior, is compared with the total intensity of fluorescence throughout the cell, their ratio will be largely unaffected by photobleaching.
In most cases only one section, the one with the largest diameter, was imaged for each cell. The thickness of each optical section was approximately 0.5 µm. The cell compartments for which fluorescence intensity was measured were modeled as two concentric spheres. The smaller one, with a diameter of 19 µm represents the interior compartment, accessible to the agonist only after internalization. The larger one, 20 µm in diameter, corresponds to the whole cell, membrane plus interior, and contains all the fluorescence associated with a cell. A 0.5 µm thick section through the middle of the two spheres, represents, in volume, 3.3% of the whole cell and 3.5% of the interior compartment. Assuming the distribution of fluorophores is isotropic, the ratio of internal to total (IN/TOTAL) fluorescence measured in a section is therefore a good approximation for the internalized fraction of fluorophore for the entire cell.
In practice, two regions of interest were drawn on the image of a section through a cell: one that contains the whole cell, the other contains the interior compartment by carefully delineating the interface between the internal side of the membrane and the cytoplasm. This technique overestimates the thickness of the membrane, which in reality is less than the resolution limit of an optical microscope, but thereby only misses a very early phase in the internalization of vesicles as soon they extend visibly into the internal compartment.
The ratio IN/TOTAL fluorescence for each fluorophore was measured at increasing times after activation and the values were plotted using Sigmaplot 3.0 (Jandel Scientific, SPSS, Inc., Chicago, IL). Data were fitted with a second order polynomial function: b0+b1·t-b2·t2.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The
potency is somewhat higher than observed for GnRH itself; this is
expected since the underived agonist itself is more potent and the
addition of a bulky group (i.e. Texas Red) at the
six-position stabilizes the active (i.e. receptor binding)
configuration. The chimeric protein consisting of a rat GnRH receptor,
a C-terminus linker from the catfish GnRH receptor and green
fluorescent protein (GnRHR-C-tail-GFP) is expressed in GGH3
cells and appears at the membrane. Cells transfected with the
GnRHR-C-tail-GFP construct display green fluorescence visible in
epi-fluorescence microscopy. Only approximately 30% of cells exhibit
fluorescence, and the total intensity varies from cell to cell,
corresponding to different expression levels of the receptor (Fig. 1B).
|
and D). The overlap of the red and green
labels, electronically adjusted to normalize intensities, is shown in
yellow (Fig. 1E).
Comparison of internalization rates
To compare the rate of internalization of WT GnRHR along with
chimeras the C-tail or C-tail-GFP and acid wash protocol was used. The
data (Fig. 2) show that the initial
internalization rate of GnRHR-C-tail-GFP is somewhat slower than WT
GnRHR or GnRHR-C-tail (0–30 min). Thereafter, (25–90 min) the lines
are nearly parallel suggesting similar rates.
|
, F–H). The
size of internalized vesicles varies largely from cell to cell and
within the same cell, from diameters close to the resolution limit,
2–300 nm, to slightly larger than 2 µm. Vesicles detach from the
membrane and move deeper inside the cell, eventually accumulating in
the perinuclear region. The internalization process continues for about
2–3 h, at which time no visible agonist remaining is bound to the
membrane (Fig. 3, A and B). At no time,
however, does all the receptor disappear from the external
membrane.
|
) for up to 3 h, or to a GnRH antagonist
(5).
After internalization, the receptor-agonist complex
(yellow colored) dissociates and free (red
colored) agonist as well as free (green colored) receptor
can be seen discretely distributed inside the cell (Figs. 3B and 4).
Particles with different ratios of receptor to agonist (assessed by the
degree of overlap of the green and red label)
coexist for at least 3 h, the longest time examined in this study.
Some of the larger vesicles have a visible hole in the middle,
suggesting that the agonist is still bound to the membrane surrounding
the vesicle; others show a clear green ring surrounding a homogeneously
red stained interior, suggesting that the agonist has separated from
the membrane bound receptor and is filling up the vesicle core (Fig. 3, D and E).
|
We monitored the receptor-agonist complex internalization in live cells
by imaging single cells over a period of time (Fig. 1, E and H) or from
different cells in a culture, at various times after activation by
agonist (Fig. 3, A and B). In additional studies, we examined cells
fixed after exposure to the agonist for various times. Fixed cells can
be exposed to more laser light than live cells, without visible damage.
Figure 4 shows a stereo pair of the
three-dimensional reconstruction of a cell expressing GnRHR-C-tail-GFP,
fixed 60 min after activation by GnRHa-TxR and whose nucleus was
stained with bis-benzimine.
Internalization rate
Internalization of the receptor and agonist were determined
separately, analyzing the green and the red
channel independently. Results are expressed as ratio of fluorescence
measured in an area of interest that excludes the membrane, the cell
interior determined as in Fig. 5E, to the
total fluorescence, including the membrane.
|
gives information
on the rate of internalization and recycling of agonist and receptor.
The curves are best fitted with second order linear equations. The
first term, b0, describes the fraction of fluorescence
inside the cell at time zero, contributed by autofluorescence,
background due to electronic and optical noise, and, in the case of
GnRHR-C-tail-GFP construct, of the expressed receptor waiting to be
transported to the membrane. The coefficient of the first order term, b1, corresponds to the rate of internalization, representing the fraction of total receptor and respectively agonist associated with one cell, that is internalized each minute, in one cell. The internal fraction is not a linear function of time, for both receptor and agonist. The coefficient of the second order term, b2, is a measure of the decrease in the observed internalization due to mechanisms that quench fluorescence in various compartments, or by the recycling of fluorophores back to the cell membrane.
For the example in Fig. 5, the time zero ratio of the internal
vs. total fluorescence intensity is 20% for the agonist and
32% for the receptor. The difference may reflect, at least partly, the
amount of receptor synthesized and not yet transported to the
membrane.
The coefficient of the first order term, is similar for the agonist 3.8·10-3 min-1 and receptor, 4.3·10-3 min-1. The coefficient of the second order is much larger for the receptor 3.0·10-5 min-2 than for the agonist, 1.4·10-5 min-2. This means that the observed internalization of the receptor is slowed more than that rate of the agonist, consistent with the idea that the receptor might be recycled back to the membrane after it releases the agonist that remains inside the cell.
To see whether repeated irradiation of a cell during a time lapse
experiment affects the rates measured, different cells within a similar
population were imaged at various times and analyzed (Fig. 6A). The results of the regression are
similar to those for the single cell in Fig. 4: b0 is 20%
and 41%, b1 is 5.0·10-3 min-1
and 3.7·10-3min-1, b2 is
1.63·10-5 min-2 and 2.0·10-5
min-2 for agonist and receptor respectively (Table 1).
|
|
Results for three different transfection concentrations are presented
in Fig. 6, A-C. In spite of large cell-to-cell variations in
fluorescence intensity and distribution at different times, the
regressions have remarkably similar patterns. For Fig. 6D, all cells
measured, regardless of the concentration of DNA used for their
transfection, were pooled in 10 min intervals and averaged. The
resulting curve gives better regression coefficients and values similar
to those for individual cases. We conclude that changing the
concentration of cDNA in the transfection solution does not affect the
measured internalization rates for the GnRH receptor and agonist. This
could be due to the fact that the number of receptors expressed by the
cell does not affect the internalization rate. Table 1 summarizes the
results of the regressions for all samples investigated.
For all samples, the ratio internal/total fluorescence at time zero is larger for the receptor, with values between 30% and 40%, than for the agonist, around 20%. The internal fraction of agonist increases faster than that of the receptor, suggesting that the latter is transported back to the membrane. The two curves intersect around the 120-min time point.
Independent processing of agonist and receptor
The initial internalization pattern for receptor and agonist are
quite similar. The fraction of agonist inside the cell continues to
increase for at least 2 h. The receptor, however, shows a peak at
approximately 80 min after activation, after which the internal
fraction starts decreasing. After approximately 2 h, there is a
larger fraction of agonist than of receptor inside the cells. This
could be due either to the faster destruction of GnRHR-GFP inside cells
or to the increase of their numbers on the membrane. As mentioned
before, even at longer times after activation, when all visible agonist
has been internalized, there still are free receptors on the membrane.
This may suggest that the decrease of the internal fraction of
receptors is due to the increase of their number on the membrane,
outside the internal space. The appearance of free receptors on the
membrane may result either from the recycling of previously
internalized receptors, or from the transport to the membrane of newly
synthesized protein.
To distinguish between these two possibilities, cells were preincubated
with cycloheximide to prevent new protein synthesis, and exposed to the
same activation protocol as the nontreated cells, Fig. 7. Internalization occurs vigorously for
the agonist, but there is very little free receptor inside the cell.
The receptors on the membrane are, however, never depleted, but rather
they accumulate on the membrane at levels close to those at the onset
of activation. These results suggest that, after internalization, the
receptors are freed of agonist and transported back to the membrane,
whereas the agonist is retained inside the cell for at least 3 h,
the longest time investigated. Linear regression analysis of
internalization data (Table 1) shows that the internalization of
agonist is not modified by treatment with cycloheximide. For the
receptor, the internalization rate also appears unmodified. The change
of the internalization rate with time, due to modifying factors such as
appearance of newly synthesized receptors in the cell interior and the
transport back to the membrane of previously internalized, is however
strongly affected by cycloheximide, changing from an average of
2.69·10-5 min-2 for nontreated cells to
0.88·10-5 min-2, after treatment.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GnRHR internalization has been an area of substantial challenge in the absence of generally available antisera for the receptor. Alternative approaches have relied on the GnRHR derivative described in the manuscript as well as epitope-tagged receptor (12). The absence of the long cytoplasmic tail usually associated with receptors of the seven transmembrane G protein-coupled superfamily has made it attractive to consider the result of production of chimeras in which the tail is lengthened by various additions (9, 13, 14). Although all modifications produced changes in patterns of receptor regulation, the relation appears complex. Although it is a limitation of the present study that the C-terminal modification needed to prepare a fluorescent derivative may modify processing, the GnRHR-C-tail-GFP, binds and internalizes on a similar time course as observed by other means of measuring this process.
Another murine GnRH-GFP construct has been described that uses a shorter space (15), and, when expressed in CHO cells is transported to the membrane, binds hormone and is capable of signal transduction. Binding GnRH affected lateral diffusion within the membrane, but the authors did not investigate internalization. The values we measured for the rate of internalization of GnRHR-C-tail-GFP and GnRHa-TxR by confocal microscopy, around 0.5%/min, are very similar to previously reported values for the human GnRHR and for a chicken mutant where the C terminus was truncated (14). Likewise, the internalization rates of GnRHR, GnRHR-C-tail, and GnRHR-C-tail GFP (by acid wash) are similar to other reports and to the confocal data herein.
The results of this study extend our understanding of the receptor-ligand internalization process by suggesting independent processing of these two moieties and recycling of the receptor. Understanding the basis of the recycling process is a significant feature of the clinical utility of GnRH agonists and antagonists and, accordingly, warrants additional consideration.
|
Footnotes |
|---|
Received February 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Y. Ko, H. Guo, N. Barengo, and H. Naora Inhibition of Ovarian Cancer Growth by a Tumor-Targeting Peptide That Binds Eukaryotic Translation Initiation Factor 4E Clin. Cancer Res., July 1, 2009; 15(13): 4336 - 4347. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Finch, C. J. Caunt, S. P. Armstrong, and C. A. McArdle Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors Am J Physiol Cell Physiol, January 1, 2009; 297(3): C591 - C600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Pawson, E. Faccenda, S. Maudsley, Z.-L. Lu, Z. Naor, and R. P. Millar Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization Endocrinology, March 1, 2008; 149(3): 1415 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Hull, A. Ohnishi, K. Moto, Y. Kawasaki, R. Kurata, M. G. Suzuki, and S. Matsumoto Cloning and Characterization of the Pheromone Biosynthesis Activating Neuropeptide Receptor from the Silkmoth, Bombyx mori: SIGNIFICANCE OF THE CARBOXYL TERMINUS IN RECEPTOR INTERNALIZATION J. Biol. Chem., December 3, 2004; 279(49): 51500 - 51507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. W. Tawfeek and A. B. Abou-Samra Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E704 - E710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Leanos-Miranda, A. Ulloa-Aguirre, T. H. Ji, J. A. Janovick, and P. M. Conn Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3360 - 3367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Castro-Fernandez, J. A. Janovick, S. P. Brothers, R. A. Fisher, T. H. Ji, and P. M. Conn Regulation of RGS3 and RGS10 Palmitoylation by GnRH Endocrinology, April 1, 2002; 143(4): 1310 - 1317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Patel, U. Kumar, D. C. Lamb, J. S. Eid, M. Rocheville, M. Grant, A. Rani, T. Hazlett, S. C. Patel, E. Gratton, et al. Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells PNAS, March 5, 2002; 99(5): 3294 - 3299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. Oksche, G. Boese, A. Horstmeyer, J. Furkert, M. Beyermann, M. Bienert, and W. Rosenthal Late Endosomal/Lysosomal Targeting and Lack of Recycling of the Ligand-Occupied Endothelin B Receptor Mol. Pharmacol., June 1, 2000; 57(6): 1104 - 1113. [Abstract] [Full Text]
|
|
|
|
|
|
J. J. Blum, M. C. Reed, J. A. Janovick, and P. M. Conn A mathematical model quantifying GnRH-induced LH secretion from gonadotropes Am J Physiol Endocrinol Metab, February 1, 2000; 278(2): E263 - E272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cornea, J. A. Janovick, G. Maya-Nunez, and P. M. Conn Gonadotropin-releasing Hormone Receptor Microaggregation. RATE MONITORED BY FLUORESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., January 12, 2001; 276(3): 2153 - 2158. [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 |
