?-Arrestin Mediates Desensitization and Internalization but
Does Not Affect Dephosphorylation of the Thyrotropin-
releasing Hormone Receptor*
Brian W. Jones and Patricia M. Hinkle1
The G protein-coupled thyrotropin-releasing hormone (TRH)
receptor is phosphorylated and binds to ?-arrestin after agonist
exposure. To define the importance of receptor phosphorylation
and ?-arrestin binding in desensitization, and to determine
whether ?-arrestin binding and receptor endocytosis are required
for receptor dephosphorylation, we expressed TRH receptors in
fibroblasts from mice lacking ?-arrestin-1 and/or ?-arrestin-2.
Apparent affinity for [3H]MeTRH was increased 8-fold in cells
expressing ?-arrestins, including a ?-arrestin mutant that did not
permit receptor internalization. TRH caused extensive receptor
endocytosis in the presence of ?-arrestins, but receptors remained
strongly inhibited inositol 1,4,5-trisphosphate production within
10 s. At 30 min, endogenous ?-arrestins reduced TRH-stimulated
tin-2), and 84% (?-arrestins-1 and -2). In contrast, receptor phos-
phorylation, detected by the mobility shift of deglycosylated recep-
phosphorylated within 15 s of TRH addition. Receptor dephospho-
rylation was identical with or without ?-arrestins and almost com-
plete 20 min after TRH withdrawal. Blocking endocytosis with
hypertonic sucrose did not alter the rate of receptor phosphoryla-
tion or dephosphorylation. Expressing receptors in cells lacking
G?qand G?11or inhibiting protein kinase C pharmacologically did
not prevent receptor phosphorylation or dephosphorylation. Over-
expression of dominant negative G protein-coupled receptor
kinase-2 (GRK2), however, retarded receptor phosphorylation.
Receptor activation caused translocation of endogenous GRK2 to
the plasma membrane. The results show conclusively that recep-
tor dephosphorylation can take place on the plasma membrane
and that ?-arrestin binding is critical for desensitization and
The type 1 thyrotropin-releasing hormone (TRH)2receptor is a sev-
mone and prolactin release from the anterior pituitary. Upon exposure
pling to the GTP-binding proteins G?qor G?11, leading to the forma-
tion of inositol 1,4,5-trisphosphate (IP3) and the subsequent release of
Ca2?from the endoplasmic reticulum. This signaling pathway ulti-
tial for proper thyroid function and targeted deletion of the type 1 TRH
receptor results in hypothyroidism (1).
When activated, most G protein-coupled receptors (GPCRs)
undergo phosphorylation, which is followed by receptor interaction
with ?-arrestins and desensitization and endocytosis (2). Phosphoryla-
tion is carried out by second messenger-activated kinases, such as pro-
tein kinase C, or by G protein-coupled receptor kinases (GRKs). Less is
the resensitization of GPCRs. Dephosphorylation of the well studied
?2-adrenergic receptor is reported to take place in acidified endosomes
As with most GPCRs, the activity of the TRH receptor is modulated
phorylated and rapidly recruits ?-arrestin to the plasma membrane.
regions (12) and undergoes rapid and extensive internalization (7,
13–16). Following endocytosis, TRH receptors and ?-arrestin are co-
interacting with the TRH receptor (10, 19, 20). In general, GPCRs that
internalize with ?-arrestin are extensively degraded rather than recy-
cled. The TRH receptor is an exception to this rule, because it recycles
extensively following hormone withdrawal. Internalization of the TRH
receptor is inhibited by dominant negative forms of ?-arrestin and
which contains multiple phosphorylation sites. Mutant forms of ?-ar-
restin that bind in a phosphorylation-independent manner promote
agonist-independent internalization of TRH receptors lacking several
potential phosphorylation sites in the carboxyl tail (14).
A number of critical questions remain unanswered for the TRH
uncoupled from G proteins as a consequence of phosphorylation, ?-ar-
restin association, or both. Another is whether dephosphorylation and
reactivation of the receptor require endocytosis, which would be pre-
dicted if the relevant phosphatase acts only on receptors in acidified
membrane. An additional uncertainty is whether the association of
receptor with ?-arrestin prolongs desensitization by blocking access of
advantage of fibroblasts from mice lacking ?-arrestin 1 (?Arr1KO),
?-arrestin 2 (?Arr2KO), or ?-arrestins 1 and 2 (?Arr1/2KO) and from
wild-type (wt) littermates (21) to address these questions and analyze
* This work was supported by Grant DK19974 from the National Institutes of Health (to
P. M. H.) and by a Sproull Fellowship from the University of Rochester and a National
Institutes of Health Cardiovascular Research Training Grant (to B. W. J.). The costs of
Section 1734 solely to indicate this fact.
1To whom correspondence should be addressed: Dept. of Pharmacology and Physiol-
ogy, University of Rochester Medical Center, Box 711, Rochester, NY 14642. Tel.: 585-
275-4933; Fax: 585-273-2652; E-mail: Patricia_Hinkle@urmc.rochester.edu.
pled receptor; GRK, GPCR kinase; IP3, inositol 1,4,5-trisphosphate; MEF, mouse
embryo fibroblast; wt, wild type; CHO, Chinese hamster ovary cells; HA, hemaggluti-
nin; GFP, green fluorescent protein; MeTRH, [N3-methyl-His2]TRH.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 46, pp. 38346–38354, November 18, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
38346 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 280•NUMBER 46•NOVEMBER 18, 2005
by guest on December 29, 2015
the roles of ?-arrestins in desensitization, internalization, and dephos-
phorylation of the TRH receptor.
Cell Cultures and Transfection—Mouse embryo fibroblasts (MEFs)
from mice lacking ?-arrestins were provided by Dr. Robert Lefkowitz
(Duke University, Durham, NC). G?q/11KO MEFs derived from
embryos lacking the ? subunits of Gqand G11, were provided by Dr.
Melvin Simon (California Institute of Technology, Pasadena, CA).
MEFs, COS, and CHO cells were maintained in Dulbecco’s modified
Eagle’s medium/F-12 with 5–10% fetal bovine serum and grown as
monolayers in humidified 95% air and 5% CO2at 37 °C.
We transiently transfected cells using Lipofectamine (Invitrogen) or
FuGENE (Roche Diagnostics) following manufacturers’ instructions.
the amino terminus with either hemagglutinin (HA) or FLAG epitopes
(13), ?-arrestin 2 (provided by Dr. Marc Caron, Duke University,
Durham, NC), ?-arrestin 1 (provided by Dr. Vsevolod Gurevich,
wild type and K220R GRK2 (provided by Dr. Jeffrey Benovic, Thomas
and/or HcRed (Clontech, Palo Alto, CA). When cells were transfected
with cDNA encoding arrestins, a plasmid encoding GFP, a soluble pro-
tein of similar size, was used as a control. Cells were maintained in
serum-containing media until assayed 24–48 h after transfection.
Transfection efficiencies, which were estimated by transfecting with
cDNA encoding GFP and staining nuclei with cell-permeable Hoechst
33342 (Molecular Probes, Eugene, OR), varied from 11 to 19% in ?-ar-
restin MEFs, 8–11% in G?q/11KO MEFs, and 45–55% in COS cells at
different times. We found no consistent differences in transfection effi-
ciencies between ?Arr1KO, ?Arr2KO, ?Arr1/2KO, and wt MEFs.
erated by co-transfecting with pTK-Hyg plasmid (Clontech) conferring
resistance to hygromycin B, then adding 250 ?g/ml hygromycin B
(Invitrogen) 48 h after transfection and selecting stable clones.
Radioligand Binding—To measure specific TRH binding, cells were
incubated in serum-free Dulbecco’s modified Eagle’s medium/F-12 or
Hanks’ balanced salt solution containing [3H]MeTRH (72 Ci/mmol,
PerkinElmer Life Sciences) for 30–90 min at 37 °C. Cells were then
and radioactivity was measured by liquid scintillation counting. Protein
concentrations were determined by using the Lowry method with
bovine serum albumin as a standard. For Scatchard analysis, cells were
incubated for 60 min with 0.67–20 nM [3H]MeTRH. Nonspecific bind-
dishes in all experiments.
Inositol Phosphate Accumulation—To measure total inositol phos-
phate accumulation, we labeled transfected cells either immediately or
24 h after transfection with 2–5 ?Ci/ml myo-[3H]inositol overnight in
F-10 media with 5% fetal bovine serum. Cells were treated with 10 mM
LiCl with or without TRH at 37 °C. Dishes were placed on ice, washed
formic acid at 4 °C. [3H]Inositol phosphates were subsequently isolated
by ion exchange chromatography (22).
Radioreceptor Assay of IP3—IP3mass was measured by treating
35-mm dishes with 1 ?M TRH in Dulbecco’s phosphate-buffered saline
solution for various times at 37 °C. Dishes were then placed on ice, and
IP3was extracted by addition of 5% trichloroacetic acid. Samples were
extracted with 3:1 trifluorotrichloroethane:octylamine, and IP3was
(Buckinghamshire, UK) according to the manufacturer’s instructions.
Immunoprecipitation and Deglycosylation—CHO cells expressing
HA-tagged TRH receptor in 35-mm dishes or MEFs transfected in
60-mm dishes were treated as detailed. Immunoprecipitation of recep-
ml of lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% Triton
biochem) and phosphatase inhibitors (10 mM sodium fluoride, 10 mM
sodium pyrophosphate, and 100 nM sodium orthovanadate). After cen-
trifugation, supernatants were incubated 12–18 h with 1:5000 mono-
clonal HA11 antibody (Covance, Berkeley, CA). After 1-h incubation
with protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA),
samples were washed four times with 1 ml of lysis buffer. Deglycosyla-
tion was performed using peptide N-glycosidase F (New England Bio-
labs, Beverly, MA) exactly as instructed by the manufacturer, and the
erol, pH 6.8).
Electrophoresis and Immunoblotting—Immunoprecipitated and degly-
cosylated proteins were separated on a 10% polyacrylamide gel by SDS-
Phosphorylation/Dephosphorylation—To measure TRH receptor
phosphorylation, we treated transfected cells with 100 nM TRH for
either 5 or 45 min, washed them three times with saline to remove
excess agonist, then allowed the cells to recover for different times
before they were harvested. We immunoprecipitated HA-tagged TRH
receptors, deglycosylated, ran SDS-PAGE, and observed the mobility
shift due to TRH receptor phosphorylation. To quantify and compare
dephosphorylation rates in different cell types, the relative mobility of
TRH receptor bands was measured using NIH Image version 1.63 or
graphing distance traveled versus density were generated for each lane.
The mobility of the peak density was determined, and the relative
mobility of TRH receptors in each lane was defined by assigning a value
of 0 to the distance traveled by untreated TRH receptors and a value of
Alkaline Phosphatase Treatment—To confirm that TRH receptor
up-shift is due to phosphorylation, we immunoprecipitated HA-tagged
TRH receptors from stably transfected CHO cells and incubated them
with 0, 4, 20, or 100 units/ml calf intestine alkaline phosphatase (Cal-
biochem) for 1 h at 37 °C. Samples were then deglycosylated and sepa-
rated by SDS-PAGE as described above.
Internalization—To follow internalization of the TRH receptor, we
measured the acid resistance of specifically bound [3H]MeTRH. Cells
were incubated in 5 nM [3H]MeTRH then washed on ice with ice-cold
saline. Surface ligand was extracted with ice-cold acid/salt buffer (0.2 M
solubilizing the cells in 0.1% SDS. Internalization was also measured by
an enzyme-linked immunosorbent assay using minor modifications of
the procedure described by Song and Hinkle (24).
We also imaged receptors in ?Arr1/2KO MEFs stably expressing a
and HcRed. Cells were plated on ECL (Upstate, Chicago, IL)-coated
coverslips, transfected, stimulated with 1 ?M TRH, and imaged on a
fluorescence microscope as described previously (9).
Translocation of GRK2—CHO cells stably transfected with TRH
receptor in 6-cm dishes were incubated for 1 h in Hanks’ balanced salt
NOVEMBER 18, 2005•VOLUME 280•NUMBER 46JOURNAL OF BIOLOGICAL CHEMISTRY 38347
by guest on December 29, 2015
Brian W. Jones and Patricia M. Hinkle
2005, 280:38346-38354. J. Biol. Chem.
Thyrotropin-releasing Hormone Receptor
Dephosphorylation of the
Internalization but Does Not Affect
-Arrestin Mediates Desensitization and
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M502918200 originally published online September 23, 2005
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