| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama 35294-0005
Address all correspondence and requests for reprints to: Jimmy D. Neill, Department of Physiology and Biophysics, BHSB 812, 1918 University Boulevard, University of Alabama, Birmingham, Alabama 35294-0005. E-mail: neill{at}uab.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit, thereby limiting its activation of the second
messenger pathway (8). We have shown that RGS3 inhibits GnRH-stimulated
inositol trisphosphate (IP3) production in cotransfected
COS-1 cells and is present in gonadotropes, suggesting a potential
regulatory role for this particular RGS in GnRH-induced desensitization
(9).
A second, more extensively studied mechanism of G protein-coupled
receptor desensitization involves the GRK/ß arrestin families of
proteins (10). This mechanism, which has been most intensively studied
for the ß2-adrenergic receptor, involves agonist-specific
induced phosphorylation of intracellular regions of the receptor that
permits ß-arrestins to bind, preventing G protein association with
the receptor (11). In a previous study (12), we reported the presence
in T3–1 gonadotropes of GRK2, -3, and -6 and hypothesized that the
GRK/ß arrestin paradigm might apply to the GnRH receptor.
Insofar as we are aware, the GRK/ß arrestin mechanism of signal modulation has not been tested for its applicability to the GnRH receptor and its associated second messenger generation. Therefore, we have determined the effects of selected GRKs and ß-arrestin-1 and -2 on GnRH-stimulated IP3 production in a heterologous cell system.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In preparation for transfections of COS-1 cells (13), we collected the cells in the afternoon from subconfluent cultures (<15 x 106 cells/T-175 flasks) using a trysin-EDTA solution (Sigma Chemical Co., St. Louis, MO) and seeded them (3 x 106 cells in 10 ml DMEM/10% FBS) into 60-mm Falcon petri dishes. The cells were incubated overnight before being transfected the next morning. One hundred microliters of Lipofectamine (Life Technologies, Gaithersburg, MD) in 700 µl OptiMEM I (Life Technologies) were mixed with 800 µl OptiMEM containing the vector with its complementary DNA (cDNA) insert to be transfected. After 15- to 30-min incubation, the Lipofectamine-DNA complexes and 6.4 ml OptiMEM were placed on the cells, which were then put in the incubator for 5–8 h before 8 ml DMEM/20% FBS was added. The cells were incubated for 20–24 h, after which the medium was aspirated and replaced with 12 ml DMEM/10% FBS. Seventy-two hours after the initiation of transfection, cells were harvested for preparation of membrane fractions for use in receptor radioassays or were treated with D-Ala6-desGly10-GnRH-ethylamide (GnRH-A) in preparation for measurements of IP3.
cDNAs for transfections
The construction of the cDNA representing the rat GnRH receptor
used for transfections of COS-1 cells has been described and validated
in detail previously (13). The cDNA bears the nucleotide sequence
encoding the HA-1 epitope tag at its 5'-end and was fused using
recombinant DNA methodology to the wild-type GnRH receptor cDNA from
which the untranslated 5'- and 3'-sequences had been removed by PCR. It
was subcloned into the eukaryotic expression vector pcDNA 3
(Invitrogen, Carlsbad, CA). After transfection and expression in
COS-1 cells, this N-terminal epitope-tagged GnRH receptor shows a
[125I]-GnRH-A binding affinity identical with that of
the wild-type receptor and mediates a robust GnRH stimulation of
IP3 production in COS-1 cells (13).
The cDNAs representing bovine GRK2 (in expression vector pBC ßARK-1) (14), bovine GRK3 (in expression vector pBC ßARK-2) (14), and human GRK6 (in expression vector pBCGRK6) (15) were provided by Dr. Jeffrey L. Benovic. cDNAs representing the ß-arrestins (in the expression vectors pCMV5-rat ß-arrestin-1 and pCMV5-rat ß-arrestin-2) (16) were provided by Dr. Robert J. Lefkowitz.
RRA for GnRH
GnRH-A was iodinated with Na125I using chloramine-T
as described by Clayton et al. (17), and purified using
carboxymethyl-cellulose and ammonium acetate. Specific radioactivities
estimated as described by Wiener and Reith (18) were about 700
µCi/µg. COS-1 cell membranes (35–75 µg protein) and 30,000 cpm
[125I]GnRH-A were mixed and incubated at 0 C for 90 min.
Nonspecific binding was determined as counts per minute bound in the
presence of 10-6 M GnRH-A. Bound and free
[125I]GnRH-A were separated by filtration of the samples
through Whatman GFF filters (Clifton, NJ). The filters were then
counted in a four-channel 
-scintillation spectrometer (Micromedic
Systems, Horsham, PA).
Intracellular IP3 measurements
IP3 concentrations in cDNA-transfected and
GnRH-treated COS-1 cells were measured in a RRA (19) as described in
detail previously by us (13). In brief, IP3 receptors in
cell membranes were prepared from calf cerebellum. Each assay tube
contained 50 µl cerebellar membranes, 50 µl
[3H]IP3 (DuPont-New England Nuclear, Boston,
MA; 20 Ci/mmol), 300 µl assay buffer (50 mM
Tris-HCl, pH 8.3; 1 mM EDTA; and 1 mM
mercaptoethanol), and 100 µl sample (unlabeled IP3
standard or COS-1 cell extract). The standard curve was comprised of
IP3 concentrations ranging from 0.3–48.0 pmol. Bound and
free [3H]IP3 were separated by centrifugation
at 12,000–15,000 x g for 5 min. The precipitate was
dissolved in 50 µl 0.15 N NaOH and placed in a vial for
liquid scintillation spectroscopy.
In preparation for GnRH-A treatment of the transfected COS-1 cells, we removed and discarded the incubation medium, replaced it with 3 ml 0.1% BSA-DMEM, and preincubated the cells for 1 h. Then, the preincubation medium was replaced with 1 ml DMEM containing 10-7 M GnRH-A; treatments lasted 0, 10, 20, or 30 sec. IP3 was extracted from COS-1 cells by replacing the treatment medium with 1 ml cold 16.6% trichloroacetic acid. The cells were then scraped from the bottoms of the dishes, transferred to microfuge tubes, vortexed, incubated on ice for 15 min, and centrifuged for 1 min at 14,000 x g, and the supernatant was removed and allowed to stand at room temperature for 15 min. The samples were then extracted four times with 2 vol diethyl ether, and the samples were heated at 65 C for 15 min to evaporate the ether. The samples were then stored at 4 C until use in the IP3 RRA.
Antibodies
Antibodies to GRK2, -3, and -6 were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA) and used for immunocytochemistry on rat
anterior pituitary cells. They were prepared in rabbits against
synthetic peptides corresponding to the carboxyl-termini of human GRK2,
-3, and -6 (amino acids 675–689, 675–688, and 525–544,
respectively). The antibodies were affinity purified using their
respective synthetic peptides attached to a solid phase. Antibodies
against each GRK react with their cognate GRKs of mouse, rat, human,
and bovine origin, but there is no cross-reactivity among the different
GRKs.
Antiserum to rat GRK3 was provided by Dr. Robert J. Lefkowitz; it was used for Western blot analysis of rat anterior pituitary cells. The antiserum was produced in rabbits to the carboxyl-terminal 222 amino acids of rat GRK3 (amino acids 467–688) fused to GST (rabbit 7428) (20). This antiserum binds both GRK3 and -2, as there is 76% homology between the amino acid sequences of the C-termini of these two proteins (20); indeed, it detects GRK2 much more strongly than GRK3 (20).
Antiserum to rat ß-arrestin-2 was also provided by Dr. Robert J. Lefkowitz; it was used for both immunocytochemistry and Western blots. The antiserum was prepared in rabbits against a glutathione-S-transferase fusion protein containing the carboxyl-terminal 78 amino acids (amino acids 333–410) of rat ß-arrestin-2 (rabbit 7435-3) (16). The antiserum contains antibodies reacting both with ß-arrestin-2 and ß-arrestin-1 due to the 55% amino acid homology between the two proteins (16).
Immunocytochemistry and immunoblotting
Rat anterior pituitary glands were dispersed using trypsin into
single cell suspensions for immunocytochemistry as previously described
(21). After attachment to glass microscope slides using
poly-L-lysine, the cells were fixed with freshly prepared
3.5% paraformaldehyde for 8 min and permeabilized with 0.1% Nonidet
P-40 for 15 min. Staining was performed with the Vectastain ABC rabbit
kit (Vector Laboratories, Burlingame, CA) using the instructions
supplied by the manufacturer. Antibodies against GRK2, -3, and -6 were
used at a concentration of 1 µg/ml, whereas antiserum against
ß-arrestin-2 was used at a dilution of 1:5000; all were incubated
with the cells overnight at room temperature. The diaminobenzidine
substrate was incubated with the cells until the appropriate level of
color had developed (usually about 1 min).
Immunoblots for GRKs and ß-arrestins were performed on rat anterior pituitary cytosol preparations. Pituitary glands from female rats were homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ) in 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 40,000 x g for 10 min; the supernate was removed and mixed with sample buffer in preparation for protein separation in SDS-PAGE. The sample was reduced by heating at 95 C for 5 min in 10% ß-mercaptoethanol and then electrophoresed on an 8% gel. Separated proteins were electroblotted onto nylon membranes (0.1-µm pore size; Immobilon-PSQ, PVDF Transfer Membrane, Millipore Corp., Bedford, MA). The membranes were then blocked in a solution of 5% nonfat dry milk in 0.05 M PBS containing 0.02% Tween-20 for 1 h. The primary antisera diluted in blocking solution (GRK3 at 1:1000 and ß-arrestin-2 at 1:200) were incubated with the strips for 1 h at room temperature. After washing the membrane three times for 10 min each time, we incubated it for 1 h at room temperature with alkaline phosphatase-conjugated goat antirabbit antiserum (Sigma) diluted 1:1000 in blocking solution. Finally, the strip was developed for 5 min in CDP-STAR Chemiluminescence Reagent (New England Nuclear Life Science Products, Boston, MA). Immunoreactive proteins were detected using the Eagle Eye II Still Videosystem together with its accompanying Eagle Sight Image Capture and Analysis Software (Stratagene, La Jolla, CA).
Data analysis
The results are presented as the mean ± SEM of
at least three independent experiments. The statistical tests were
either one- or two-way ANOVA as indicated in the figure legends.
Statistical analysis was performed using SigmaStat Statistical Software
for Windows (Jandel Scientific, San Rafael, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, in four experiments we observed
25–50% inhibitions of GnRH-stimulated IP3 production
during the 30 sec of hormone treatment. Although GRK3 expression showed
the greatest apparent inhibition, it was not significantly different
from that in the GRK2 and -6 groups (P > 0.05).
However, inhibition in all three GRK groups was significantly less than
that in the control group (P < 0.05). Calculated as
the area under the curve, inhibition by GRK3 was 56%, that by GRK2 was
23%, and that by GRK6 was 15%. The inhibition by GRKs of
GnRH-stimulated IP3 production was not due to suppression
of GnRH receptor expression, as membrane binding of
[125I]GnRH-A did not differ statistically among the
groups: control, 353 ± 47 (mean ± SEM) dpm/µg
membrane protein; GRK2, 403 ± 47; GRK3, 363 ± 59; and GRK6,
348 ± 43.
|
). For GRK2, in which 37% inhibition
was observed at 20 sec when 0.83 µg cDNA was transfected (Fig. 1
),
54% and 67% inhibitions were observed when 1.7 or 6.7 µg cDNA,
respectively, were transfected (Fig. 2). For GRK3, in which a 45%
inhibition was observed 20 sec after GnRH-A treatment began when 0.83
µg cDNA was transfected (Fig. 1), 44% and 65% inhibitions (Fig. 2)
were observed when 1.7 or 6.7 µg cDNA, respectively, were
transfected. Parenthetically, although GRK treatment significantly
suppressed GnRH-A-stimulated IP3 production in all four
groups (Fig. 2), 6.7 µg cDNA produced a statistically greater
response than 1.7 µg for GRK3, but not for GRK2. Overall, GRK2 and
GRK3 have similar quantitative effects on inhibition of GnRH-stimulated
second messenger responses. [125I]GnRH-A binding did not
differ among the groups (P > 0.05, by one-way ANOVA);
for the 1.7 µg group, values were: control, 292 ± 9 (mean
± SEM) dpm/µg membrane protein; GRK2, 317 ± 31;
and GRK3, 285 ± 29. For the 6.7 µg group, values were: control,
325 ± 23; GRK2, 285 ± 17; and GRK3, 289 ± 27. In
conclusion, these results demonstrate that overexpression of GRK2 and
-3 causes robust inhibition of GnRH-A-stimulated second messenger
responses in COS-1 cells.
|
The results are shown in Fig. 3. As
expected from the results depicted in Figs. 1 and 2, GRK2 and GRK3
transfections alone inhibited GnRH-stimulated IP3
production significantly (P < 0.05 vs. the
control group). The inhibition for both GRKs was 37%, which compares
favorably with the 37% and 45% inhibitions observed 20 sec after the
initiation of GnRH-A treatment with GRK2 and GRK3, respectively (Fig. 1), where 0.8 µg GRK cDNAs was also transfected. Transfections of the
ß-arrestins alone did not result in significant inhibitions of
hormone-stimulated intracellular IP3 levels (Fig. 3).
Cotransfections of the GRKs together with the ß-arrestins resulted in
statistically significant inhibition relative to the control value
(P < 0.05) and to that with ß-arrestins alone
(P < 0.05), but not when compared with that produced
by GRKs alone (P > 0.05). However, the GRK2 and
ß-arrestin-2 group approached statistically significant inhibition
relative to that with GRK2 alone (P = 0.06; Fig. 3).
[125I]GnRH binding did not differ significantly among the
nine groups (P > 0.05, by one-way ANOVA): control,
137 ± 19 (mean ± SEM) dpm/µg membrane
protein; GRK2, 147 ± 25; GRK3, 157 ± 28; ß-arrestin-1,
167 ± 20; ß-arrestin-2, 128 ± 37; GRK2 plus
ß-arrestin-1, 176 ± 21; GRK2 plus ß-arrestin-2, 136 ±
33; GRK3 plus ß-arrestin-1, 155 ± 21; and GRK3 plus
ß-arrestin-2, 121 ± 28.
|
), which approached but did not reach statistical significance
(P = 0.06), prompted us to examine their effects in
greater detail. Instead of measuring IP3 levels once (20
sec) after treatment with GnRH-A (Fig. 3), we increased the number of
measurements to three at 10, 20, and 30 sec (Fig. 4). GRK2 cDNA (0.8 µg) and
ß-arrestin-2 cDNA (6.7 µg) alone significantly inhibited
(P < 0.05) IP3 production relative to the
control (Fig. 4); however, the two treatment groups were not
significantly different (P > 0.05) from each other.
The combined expression of GRK2 and ß-arrestin-2 inhibited
IP3 production (P < 0.05) more than either
alone (Fig. 4). Expressed as the area under the curve, ß-arrestin-2
inhibited IP3 production by 20%, GRK2 inhibited by 31%,
and GRK2 plus ß-arrestin-2 inhibited by 49%. As in earlier
experiments, binding of [125I]GnRH-A did not differ
significantly (P > 0.05) among the groups: control,
115 ± 21 dpm/µg membrane protein (mean ±
SEM); GRK2, 129 ± 25; ß-arrestin-2, 101 ± 12;
and GRK2 plus ß-arrestin-2, 95 ± 15.
|
).
Compared with the normal rabbit Ig control, GRK2 antibodies stained all
of the cells darkly (Fig. 5A). GRK3 antibodies also stained all of the
cells, but the staining was less intense than with GRK2 (Fig. 5). GRK6
presented a peculiar pattern of staining; the cytoplasm was stained
lightly, but still more intensely than in the control group, whereas
the nucleoli were intensely stained (Fig. 5A). ß-Arrestins also are
expressed in all rat pituitary cells, as shown by greater staining with
ß-arrestin antiserum than with the normal rabbit serum control (Fig. 5B).
|
) at about
110 and 55 kDa are unknown, but have been detected previously with this
antiserum (22).
|
) using the antiserum prepared against rat ß-arrestin-2 (16).
This antiserum cross-reacts with both ß-arrestin-1 and -2; thus, we
assume that the approximately 45-kDa band (Fig. 6) is ß-arrestin-2,
and the approximately 50-kDa band (Fig. 6) is ß-arrestin-1, as these
are their reported relative molecular masses in the rat (16). The
identity of the minor immunoreactive band at about 40 kDa (Fig. 6) is
unknown. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
protein and hence activate phospholipase Cß1,
including the muscarinic m1, m3, and m5 receptors (24); the substance P
(NK1) receptor (26); endothelin A and B receptors (27); and the
thrombin receptor (28), among others.
The GnRH receptor has been described as activating the
Gq protein (29) and subsequently phospholipase Cß1 (4)
to generate the second messengers IP3 and Ca+2
(4). According to the results of the present study, the
GRK/ß-arrestin paradigm may include the GnRH receptor, as GRK2 and -3
and ß-arrestin-2 suppress GnRH-stimulated IP3 production
in a transfected heterologous cell system. This inhibition was robust,
approaching 70% when relatively high amounts of GRK2 or -3 cDNAs were
transfected; moreover, as expected from previous studies (11),
coexpression of GRK2 and ß-arrestin-2 produced a greater inhibition
than either alone. These results coupled with our finding of endogenous
expression of GRKs and ß-arrestins in rat anterior pituitary gland
strongly suggest an involvement of the GRK/ß-arrestin paradigm in
setting the GnRH responsiveness of cells expressing the GnRH
receptor.
The substrate specificity of GRKs for various receptors except GRK1 remains problematical (11). GRK1 (rhodopsin kinase) is expressed primarily in the retina, so its actions seem to be restricted to rhodopsin (30). GRK2 and -3 are widely distributed and are active at phosphorylating numerous different seven-transmembrane receptors (11, 24, 25), so it is difficult to identify specific GRK/receptor pairs that interact in vivo. One instance where GRK3 vs. GRK2 specificity is apparent is odorant-induced second messenger production in rat nasal cilia; GRK3 antibodies, but not GRK2 antibodies, block second messenger production (31). Monoclonal antibodies directed at GRK2/3 and GRK4–6 were used to show that GRK2/3, but not GRK4–6, was involved in angiotensin AT1 receptor phosphorylation (32). This ablation approach using antibodies to the GRKs is powerful, and it or antisense cDNA ablation approaches (33) are needed to sort out the specific GRKs involved in altering GnRH responsiveness in pituitary gonadotropes.
The arrestin family is comprised of four cytoplasmic proteins, two of which are restricted to the retina (25), and two of which show a wide distribution in mammalian tissues (11, 24). The latter two are ß-arrestin-1 and -2 (ß-arrestin-2 also is known as arrestin-3); they bind to intracellular loops of G protein-coupled receptors that have been phosphorylated by the GRKs (11, 24, 25). There is very little discrimination among seven-transmembrane receptors by the two types of arrestins (34). One reported instance of ß-arrestin specificity is the unique presence of ß-arrestin-2 in olfactory receptor neurons; antibodies to ß-arrestin-2 were shown to abolish odorant-induced desensitization (23). The results of our studies showing that ß-arrestin-2, but not ß-arrestin-1, overexpression in the presence of GRK2 overexpression inhibited GnRH-stimulated IP responses in COS-1 cells need to be repeated under more stringent conditions. Even if repeatable, much more rigorous approaches than that used here will be necessary to show specificity for ß-arrestin-2.
Because our results suggest that the GRK/ß arrestin paradigm (11)
applies to GnRH receptor desensitization, we should be able to observe
phosphorylation of the GnRH receptor. However, we have been unable to
demonstrate its phosphorylation in a circumstance where pretreatment
with GnRH had desensitized GnRH-stimulated IP3 production
in COS-1 cells (13). Similarly, we have been unable to demonstrate
phosphorylation of the GnRH receptor after suppression of
GnRH-stimulated IP3 production by GRK2 and ß-arrestin-2
(unpublished data). In both instances, we expressed an HA1
epitope-tagged GnRH receptor for immunoprecipitation studies after
labeling the cells with 32Pi. Further studies
are needed to resolve this discrepancy with the GRK/ß-arrestin
paradigm. Parenthetically, the argument (35) that the GnRH receptor is
not phosphorylatable because it lacks a C-terminal, cytoplasmic tail
(3) is countermanded by the observations that the
2A-adrenergic (36) and the m2 muscarinic (37) receptors
are phosphorylated on sites in their third intracellular loops during
desensitization, and that the angiotensin AT1A (38) and TSH
(39) receptors, also desensitized by GRK phosphorylation, are still
desensitized after removal of their cytoplasmic tails.
Finally, the possibility should be considered that the GRK/ß-arrestin paradigm, rather than being related to desensitization, instead mediates the LH secretory responsiveness levels of gonadotropes induced by gonadal steroids and manifested at different stages of the estrous cycle (40). A similar idea was earlier considered for thrombin action (28); namely, that the rate of thrombin receptor desensitization may set the gain in thrombin receptor signaling and, hence, define a cell’s initial sensitivity to thrombin.
In summary, we have provided preliminary evidence in our studies that the GRK/ß-arrestin paradigm of desensitization of G protein-coupled, seven-transmembrane receptors can modulate GnRH receptor signaling. However, more evidence is needed to unequivocally establish such a role. First, we need to provide evidence that the many other characteristics of this paradigm, such as phosphorylation of the receptor and translocation of the GRKs from cytoplasm to membrane (11, 24, 25) to name just two, also apply to the GnRH receptor. Second, we need to ablate the GRKs and ß-arrestins using antibodies (32) or antisense DNA approaches (33) on pituitary gonadotropes to demonstrate that receptor signaling is altered. These studies are underway.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 21, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
i3, is a member of a protein family with a highly
conserved core domain. Proc Natl Acad Sci USA 92:11916–11920T3–1 cell
line. Endocrinology 137:3942–3947[Abstract]
T3–1 cells due to uncoupling of inositol
1,4,5-trisphosphate generation and Ca2+ mobilization.
J Biol Chem 271:23711–237172A-adrenergic receptor confer short term
agonist-promoted desensitization. Evidence for a receptor
kinase-mediated mechanism. J Biol Chem 267:4740–4746This article has been cited by other articles:
 |
|
 |
|
  J. Zhou, M. F. A. Livak, M. Bernier, D. C. Muller, O. D. Carlson, D. Elahi, S. Maudsley, and J. M. Egan Ubiquitination is involved in glucose-mediated downregulation of GIP receptors in islets Am J Physiol Endocrinol Metab, August 1, 2007; 293(2): E538 - E547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Jones, G. J. Song, E. K. Greuber, and P. M. Hinkle Phosphorylation of the Endogenous Thyrotropin-releasing Hormone Receptor in Pituitary GH3 Cells and Pituitary Tissue Revealed by Phosphosite-specific Antibodies J. Biol. Chem., April 27, 2007; 282(17): 12893 - 12906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Hassan and D Mason Mechanisms of desensitization of the adrenocorticotropin response to arginine vasopressin in ovine anterior pituitary cells J. Endocrinol., January 1, 2005; 184(1): 29 - 40. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gaudreau, C. Le Gouill, M.-H. Venne, J. Stankova, and M. Rola-Pleszczynski Threonine 308 within a Putative Casein Kinase 2 Site of the Cytoplasmic Tail of Leukotriene B4 Receptor (BLT1) Is Crucial for Ligand-induced, G-protein-coupled Receptor-specific Kinase 6-mediated Desensitization J. Biol. Chem., August 23, 2002; 277(35): 31567 - 31576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Lin, H.-y. Wang, and C. C. Malbon Gravin-mediated Formation of Signaling Complexes in beta 2-Adrenergic Receptor Desensitization and Resensitization J. Biol. Chem., June 16, 2000; 275(25): 19025 - 19034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Tseng and X.-Y. Zhang Role of G Protein-Coupled Receptor Kinases in Glucose-Dependent Insulinotropic Polypeptide Receptor Signaling Endocrinology, March 1, 2000; 141(3): 947 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. D. Neill, L. C. Musgrove, L. W. Duck, and J. C. Sellers High Efficiency Method for Gene Transfer in Normal Pituitary Gonadotropes: Adenoviral-Mediated Expression of G Protein-Coupled Receptor Kinase 2 Suppresses Luteinizing Hormone Secretion Endocrinology, June 1, 1999; 140(6): 2562 - 2569. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Bunemann and M M. Hosey G-protein coupled receptor kinases as modulators of G-protein signalling J. Physiol., May 15, 1999; 517(1): 5 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
,
P = 0.06 for the comparison of GRK2 plus
ß-arrestin-2 vs. GRK2.
