| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Endocrine Research Unit Veterans Administration Medical Center and the Departments of Medicine and Physiology University of California San Francisco, California 94121
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Agonist-induced receptor phosphorylation appears to be a common feature of the regulation of a number of GPCRs. In the case of the ß-AR and rhodopsin, agonist binding leads to rapid phosphorylation of the cytoplasmic domain of the receptor by a GPCR kinase (GRK) (5). This phosphorylation facilitates the interaction between the receptor and an arrestin protein (6), a process that physically uncouples the receptor from its cognate G protein (7, 8). In the case of the ß-AR, arrestin binding to the phosphorylated receptor also serves to target the receptor to clathrin-coated pits and thereby to promote receptor internalization (9, 10). If the phosphorylation of the ß2-AR is reduced [either by mutagenesis of the receptor or by overexpressing a dominant negative GRK2 (11)], or if a dominant negative arrestin is overexpressed (7), agonist-induced receptor sequestration is significantly reduced. This phenotype can be rescued by overexpressing GRK2 and ß-arrestin, showing that they work synergistically and that they are both crucial for ß2-AR internalization (11, 12). A similar dependence of internalization on phosphorylation has been described for a number of other GPCRs (13, 14, 15, 16). Barak et al. (17) have recently tested a variety of GPCRs for their ability to bind a ß-arrestin-GFP fusion protein in response to agonist stimulation. They found translocation to the plasma membrane of the arrestin-green fluorescent protein (GFP) in response to 16 different ligand-activated GPCRs, suggesting that recruitment of arrestins may be a widespread mechanism for the regulation of GPCRs.
Little is known about the role of phosphorylation in the regulation of class B GPCRs, a structurally distinct GPCR subfamily that includes receptors for PTH/PTH-related peptide (PTHrP), secretin, glucagon and related peptides, calcitonin, and others. The opossum PTH receptor is rapidly phosphorylated upon agonist stimulation (18), and this phosphorylation was not evident in a C-terminally truncated receptor (termed T474) that lacks all but the membrane-proximal 16 amino acids of the lengthy cytoplasmic tail (19). This truncated, nonphosphorylated receptor was found to be well expressed at the cell surface and resembled the wild-type (wt) PTH receptor in its ability to signal to adenylyl cyclase, but displayed a marked (50%) reduction in agonist-stimulated receptor internalization (20). It is unclear whether the endocytic defect seen with this truncated receptor is related to the lack of phosphorylation, since the truncation not only prevented phosphorylation but also deleted a positive endocytic signal from the cytoplasmic tail (20). The purpose of the present study was to localize the sites of PTH receptor phosphorylation, and to express a minimally mutated, phosphorylation-deficient receptor to evaluate the role of phosphorylation in PTH receptor internalization.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
).
|
). Five of the
first six serine residues in the proximal portion of the cytoplasmic
tail were individually mutated to threonine, and the mutant receptors
were expressed in HEK-293 cells and subjected to phosphoamino acid
analysis. Only mutant receptors with substitutions S485T, S486T, or
S489T, but not receptors with S469T or S467T mutations showed the
appearance of phosphothreonine (Fig. 3).
Serine 483 was not targeted individually, but a mutant receptor was
tested in which serine residues at positions 483, 485, 486, and 489
were simultaneously mutated to threonine. This mutant receptor
displayed phosphothreonine, as expected, but also contained
phosphoserine, indicating that serine residues distal to S489 were also
phosphoacceptor sites.
|
|
). To determine
whether this lack of phosphorylation was due to low receptor
expression, competitive ligand-binding analysis was carried out and
receptor numbers were determined by Scatchard analysis (Fig. 5A). The wt receptor (990,000 receptors
per cell) was found to be expressed at a level only slightly greater
than that of the S(467–498)A mutant (724,000 receptors per cell) and
the S(483–498)A mutant (799,000 receptors per cell). Dose-response
studies for PTH-stimulated cAMP production were carried out with each
of these cell lines, and they were similarly sensitive to PTH
[EC50 values of 0.6 nM for the wt PTH
receptor, 0.4 nM for S(467–498)A, and 0.3 nM
for S(483–498)A] and displayed similar maximal cAMP responses (Fig. 5B). The results obtained indicate that both basal and PTH-stimulated
phosphorylation of the PTH receptor occur on serine residues between
positions 483 and 498 in the cytoplasmic tail. Moreover,
phosphorylation of these residues is not absolutely required either for
achieving high levels of receptor expression or for initiation of
receptor signaling to adenylyl cyclase. However, it is possible that
differences in signaling properties between the wt and the
phosphorylation-deficient PTH receptor would become evident under
conditions of lower levels of receptor expression.
|
|
, inset). GRK3 was overexpressed to a similar degree as GRK2
(data not shown). Overexpression of GRK2 resulted in a 3- to 4-fold
increase in agonist-induced phosphorylation of the wt PTH receptor
(Fig. 6A). To evaluate whether overexpressed GRK2 phosphorylated the
same sites as the endogenous kinase, we established HEK-293 cell lines
stably overexpressing GRK2 and the phosphorylation-deficient mutants
S(467–498)A or S(483–498)A. If overexpressed GRK2 phosphorylated the
receptor at sites different from those used by the endogenous kinase,
there should be retention of some phosphorylation in the mutant
receptors. However, neither mutant S(467–498)A nor mutant
S(483–498)A exhibited detectable phosphorylation despite the
overexpression of GRK2 (Fig. 6B). These results suggest that GRK2 and
the endogenous GRK in HEK-293 cells utilize the same phosphoacceptor
sites in the PTH receptor. Overexpression of GRK3 produced a slight
(<2-fold) increase in phosphorylation of the wt PTH receptor (data not
shown).
|
, inset). As shown in Fig. 6A, agonist-induced phosphorylation was not suppressed despite the presence
of high levels of DN-GRK2. Overexpression of another DN-GRK2 (K220R)
(23) likewise failed to suppress agonist-induced phosphorylation of the
PTH receptor (data not shown).
Relationship between Receptor Phosphorylation and Endocytosis
In HEK-293 cells expressing the wt PTH receptor,
internalization of the receptor was detected as early as 2.5 min
after the addition of agonist, and reached a maximum (40–50% of bound
radioligand) after 10–15 min (Fig. 7A).
The kinetics and extent of internalization of the
phosphorylation-deficient S(467–498)A and S(483–498)A PTH receptors
were indistinguishable from those of wt, indicating that
phosphorylation of the receptor is not required for efficient
agonist-stimulated endocytosis.
|
). This
result is similar to a previous report for the wt PTH receptor in COS-7
cells (20) and indicates that the majority of the endocytosed receptor
is internalized via a clathrin-coated pit mechanism regardless of
whether the receptor is phosphorylated. It is unclear whether the
non-sucrose-inhibitive component reflects an alternative
mechanism of internalization (e.g. caveolae) or is due to
incomplete disruption of the clathrin lattice.
To determine whether hyperphosphorylation of the wt PTH receptor
would influence receptor endocytosis, we evaluated agonist-dependent
receptor internalization in HEK-293 cells overexpressing GRK2 (Fig. 7C). Although the PTH receptor in GRK2 overexpressing cells was
hyperphosphorylated (Fig. 6A), the initial rate and extent of receptor
internalization were unaffected.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In light of the above, an important objective of the present study was to identify the sites of phosphorylation in the PTH receptor, allowing the expression of a form of the receptor that is minimally mutated but lacks all of the phosphorylation sites. Previous studies suggested that phosphorylation occurs in the proximal portion of the cytoplasmic tail. A PTH receptor C-terminally truncated at position 474 (and thus lacking all but the proximal 16 amino acids in the cytoplasmic tail) was found to be well expressed but was not phosphorylated either basally or in response to PTH (19), whereas truncation mutants T494 and T507 were phosphorylated (E. Blind, P. Turner, and R. A. Nissenson, unpublished observations). The results of the present study demonstrate that, in HEK-293 cells, the opossum PTH receptor is phosphorylated solely on serine residues in the cytoplasmic tail despite the presence of six threonine residues. To identify these serine residues, we adopted the approach used for the C5a anaphylatoxin receptor in which candidate serine residues were mutated to threonine, and the receptor was then scanned for the presence of phosphothreonine after stimulation by agonist (21). Five of the six proximal serine residues in the cytoplasmic tail were individually converted to threonine and subjected to phosphoamino acid analysis. The serine residues identified in this way were then mutated to alanine together with two downstream serine residues to create a phosphorylation-deficient receptor. These approaches allowed us to make the following conclusions about PTH receptor phosphorylation: neither serine 467 nor serine 469 was phosphorylated, a result in accordance with the observation that T474 was not phosphorylated, since no phosphothreonine appeared if threonine was substituted for either of the two serine residues. Although this result could be explained by the inability of the kinase to recognize a threonine in these positions, the mutant receptor S(483–498)A with serine residues 467 and 469 left intact also was not phosphorylated, indicating that S467 and S469 are not sites of phosphorylation. Serine residues 485, 486, or 489 were phosphorylated in response to agonist, as shown by the presence of phosphothreonine after the threonine substitutions in those sites, identifying them as targets for a kinase activated after ligand treatment; S489 was also phosphorylated basally. A phosphorylation-deficient receptor for both the basal and the agonist-stimulated phosphorylation was obtained when all six serine residues between residues 483 and 498 were mutated to alanine, indicating that serine residues 483, 495, and 498 were also potential phosphorylation sites, although it is not yet clear whether all three of these sites are phosphorylated. The proximal part of the cytoplasmic tail is highly homologous in sequence between different species and, interestingly, these six serine residues between positions 483 and 498 are conserved in the human, rat, and opossum PTH receptor, suggesting evolutionary conservation of the phosphoacceptor sites.
Previous studies demonstrated that purified protein kinase A and protein kinase C were each capable of phosphorylating the proximal portion of the cytoplasmic tail of the PTH receptor in vitro (19), and these kinases were implicated in basal phosphorylation of the PTH receptor in intact HEK-293 cells (18). However, neither of these kinases was found to be responsible for agonist-stimulated phosphorylation of the receptor in intact HEK-293 cells (18). GRK2 was also shown to be effective in phosphorylating the cytoplasmic tail of the PTH receptor in vitro, and it was suggested that GRK2 or a related kinase was likely to mediate agonist-dependent phosphoryation in vivo (19). Since the importance of agonist-induced phosphorylation as a control mechanism for GPCR trafficking has mainly been shown for receptors phosphorylated by GRK2, and because GRK2 has been shown to be expressed in osteoblastic cells (31), it was of particular interest in the present study to assess whether the PTH receptor was a substrate for GRK2 in vivo. Comparison of the sequence surrounding the sites of phosphorylation by GRK2 in the C5a receptor (21) and the ß2-AR (5) with the sites phosphorylated in the PTH receptor does not yield an obvious consensus sequence. However, overexpression of GRK2 led to a 3- to 4-fold increase in receptor phosphorylation, indicating that GRK2 is capable of utilizing the PTH receptor as a substrate in vivo.
Two approaches were taken to assess whether GRK2 is likely to be
the endogenous kinase responsible for agonist-stimulated
phosphorylation of the PTH receptor in HEK-293 cells. First, we
evaluated whether the sites of augmented receptor phosphorylation
observed in the presence of overexpressed GRK2 were the same as those
used by the endogenous kinase. That this is the case was demonstrated
by the inability of the phosphorylation-deficient mutated PTH receptor
to be phosphorylated even in the presence of overexpressed GRK2. The
second approach tested whether the endogenous kinase could be inhibited
by altered forms of GRK2 previously shown to display dominant-negative
activity in certain cell systems. However, suppression of
agonist-stimulated PTH receptor phosphorylation was not observed with
overexpression of either the K220R mutant, which is no longer
catalytically active, or the C-terminal GRK2 fragment, which binds the
ß
-subunits of the G-protein. It is not clear whether this
result represents a failure of the dominant-negative constructs to
effectively suppress endogenous GRK2 activity, or whether the relevant
endogenous kinase is an enzyme distinct from GRK2.
Identifying the sites of agonist-stimulated PTH receptor phosphorylation allowed us to take multiple approaches to address the role of phosphorylation in receptor endocytosis. Overexpression of GRK2, which enhanced PTH-stimulated receptor phosphorylation, had no influence on the rate or extent of receptor internalization. Conversely, expression of dominant-negative forms of GRK2 did not influence receptor internalization although, as discussed above, we have not evaluated directly whether GRK2 activity is suppressed in these cell lines. Most importantly, targeted mutations of serine residues in the proximal portion of the cytoplasmic tail of the PTH receptor abolished PTH-stimulated receptor phosphorylation but had no effect on the kinetics of receptor endocytosis. When mutations are introduced, there is always the possibility that the effects seen are caused by the altered amino acid sequence; i.e. an unphosphorylated wt PTH receptor may behave differently than a mutant receptor with alanines in place of serines. However, by narrowing the potential phosphorylation sites down to six, we kept the necessary mutations to a minimum, and there was no evidence that introduction of alanine residues at those sites produced any alterations in receptor expression or signaling.
These results demonstrate that, in HEK-293 cells, agonist-stimulated PTH receptor internalization occurs by a mechanism that is not dependent on receptor phosphorylation. The mechanism underlying PTH receptor internalization, therefore, is distinct from that proposed for the ß-AR and certain other class I GPCRs, which involves phosphorylation of the receptor by a GRK followed by arrestin binding to the phosphorylated receptor (6, 7). It is likely that the structural determinants of PTH receptor endocytosis include sequences (e.g. Y-X-X-hydrophobic; L-L) known to mediate the association of other membrane proteins with components of clathrin-coated pits (32). Indeed, the sequence Y-G-P-M lies within a region of the cytoplasmic tail of the PTH receptor that has previously been shown to be important for optimal PTH-stimulated internalization (20). Our findings appear to differ from those obtained by Fukayama et al. (33), who reported that expression of a dominant-negative form of GRK2 (K220R) reduced acute agonist-induced down-regulation of the endogenous PTH receptor in SaOS-2 human osteosarcoma cells. It is conceivable that phosphorylation of the PTH receptor by GRK2 is involved in PTH-stimulated receptor internalization in SaOS-2 cells, but not in HEK-293 cells. Alternatively, the high level of overexpression of the PTH receptor in HEK-293 cells may have masked an effect of K220R GRK2 in the present study. It is also possible that K220R GRK2 may influence acute PTH receptor endocytosis in SaOS-2 cells independently from any effect it may have on receptor phosphorylation. The availability of phosphorylation-deficient, mutated forms of the PTH receptor, such as those described in the present studies, will be useful in more clearly defining the role of phosphorylation in receptor trafficking and signaling.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Site-Directed Mutagenesis
Oligonucleotide-mediated mutagenesis was performed using the
site-directed mutagenesis kit (CLONTECH, Palo Alto, CA) according to
the instructions of the manufacturer using a
BglII/AflIII selection primer. Mutagenic primers
were designed to introduce the mutation of choice and either added or
removed a restriction site, to facilitate identification of mutant
clones.
Generation of PTH Receptor and PTH Receptor Mutant-Expressing
Cell Lines
Opossum PTH/PTHrP receptor cDNA in pcDNA1 (kindly provided by
Drs. H. Jueppner and G. V. Segre; Endocrine Unit, Massachusetts
General Hospital, Boston, MA), and the respective receptor mutants
obtained by site-directed mutagenesis were subcloned into the
expression vector pCEP4 or pcDNA3–1, amplified in Escherichia
coli HB101 and column purified (Plasmid maxi Kit, QIAGEN,
Chatsworth,CA) for transfection. HEK-293 cells were maintained in DMEM
containing 10% FCS and antibiotics (100 U/ml penicillin and 100
µg/ml streptomycin) in 5% CO2 at 37 C. Calcium
phosphate-mediated transfection was performed as previously described
(34). After an overnight incubation at 37 C, cells were washed twice
with calcium-magnesium-free PBS, grown for another day in DMEM, and
then subjected to selection with 200 µg/ml hygromycin for at least 3
weeks. Pooled hygromycin-resistant clones were used for all the
experiments using pCEP4 vectors. HEK cell lines stably expressing the
wt PTH receptor were isolated as follows. Cells were transfected (as
described above) with cDNA encoding the wt PTH receptor cloned into
pcDNA3–1, and selection was carried out in the presence of 400 µg/ml
G418 until distinct colonies appeared. Clonal lines were isolated by
limiting dilution, and a line displaying high levels of PTH receptor
expression (assessed by ligand binding assays and by Western blotting)
was chosen for further studies.
Generation of GRK2- and -3 Expressing HEK-293 Cell Lines
cDNAs for GRK2 and -3 (kindly provided by Dr. J. Benovic) were
subcloned into pCEP4 or, in the case of GRK2, also into pcDNA3–1.
Transfection was carried out as described above; individual colonies
were lifted individually from the culture dish and allowed to expand
further in the presence of G418; the selection of positive clones was
carried out as described above. Hygromycin-resistant pools of cells
(pCEP4) or G418-resistant clonal cell lines (pcDNA3–1) were evaluated
by Western blotting for GRK expression. Studies were carried out with
cells estimated to express GRKs at a level exceeding endogenous levels
by at least 10-fold.
Ligand Binding and Internalization
PTH receptor binding and internalization studies were carried
out as previously described (20). In brief, HEK 293 cells were grown
for 3 days in 35-mm wells to confluence and incubated in 1 ml of DMEM
containing 20 mM HEPES, 0.1% BSA (DHB), 60,000 cpm of
125I-labeled human (h)PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and varying
concentrations of unlabeled bovine (b)PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Under these
conditions, the concentration of labeled hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) added was
approximately 0.1 nM. After a 1-h incubation at room
temperature, cells were washed, collected in 1 ml of 0.8 M
NaOH, and bound [125I]hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was assessed. In
studies for Scatchard analysis these conditions were modified in that
incubations were carried out at 4 C for at least 2 h in 12-well
plates in a volume of 0.5 ml. Receptor affinity and number were
calculated using the GraphPad Prism program. For internalization
studies, the cells were incubated at room temperature for varying times
with [125I]hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) amide and washed twice in
ice-cold PBS, and surface-bound ligand was extracted by two 5-min
incubations on ice with 50 mM glycine buffer, pH 3.0,
containing 0.1 M NaCl. The internalized radioligand was
then extracted by exposing cells to 0.8 M NaOH. Receptor
internalization is expressed as the percent of cell-associated
radioligand remaining after acid washing. To disrupt clathrin lattices,
cells were treated with 0.45 M sucrose in DHB for 1 h
at 37 C before binding and internalization studies.
cAMP Assay
For the assay of cAMP levels, cells prepared as described above
were incubated in 1 ml DHB, 1 mM isobutylmethylxanthine,
and various concentrations of bovine (b)PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) for 10 min at room
temperature. Cells were then washed twice in ice-cold PBS, and the
cellular cAMP was extracted with 1.5 ml 95% ethanol and quantified by
RIA (35).
32P Labeling and Immunoprecipitation
Labeling and immunoprecipitation were carried out as previously
described (18). Briefly, HEK-293 cells were grown to confluence in
35-mm wells, washed in phosphate- and serum free DMEM, and incubated in
the same media for 30 min. To label the intracellular ATP pools, cells
were incubated in 0.7 ml phosphate and serum-free DMEM, 20
mM HEPES, and 200 µCi [32P]orthophosphoric
acid (5 mCi/ml) for 2 h. PTH was added to the appropriate wells 10
min before the end of the 2-h period. Cells were lysed by adding 170
µl RIPA buffer/well (150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS)
supplemented with phosphatase inhibitors: 300 nM okadaic
acid, 10 mM tetrasodium pyrophosphate, 0.1 mM
sodium orthovanadate, and 10 mM sodium fluoride, and rocked
intermittently for 1 h at 4 C. The PTH receptor was
immunoprecipitated with a monoclonal anti-PTH receptor antibody
immobilized on Sepharose beads. Immunoprecipitates were subjected to
SDS-PAGE, and the radiolabeled receptor was visualized by
autoradiography and quantitated by Cerenkov counting. Gel loading was
normalized to levels of receptor expression, determined by Scatchard
analysis and/or by Western blotting.
Phosphoamino Acid Analysis
The 32P-labeled receptor was excised from the dried
SDS-polyacrylamide gel, and protein hydrolysis was carried out in 200
µl of 6 M HCl/band at 110 C for 90 min. The hydrolyzed
protein was then transferred to a new tube and dried in a Speed Vac
centrifuge. The dried residue was resolubilized in 5 µl of buffer 1
at pH 1.9 [formic acid-acetic acid-H2O; 22:78:900
(vol/vol/vol)], spotted on a thin layer cellulose plate (Merck) and
separated by electrophoresis for 40 min at 1300 V followed by a second
electrophoresis in buffer 2 at pH 3.5 [pyridine-acetic
acid-H2O; 5:50:945 (vol/vol/vol)] for 15 min at 1000 V in
the orthogonal direction. Phosphoserine, phosphothreonine, and
phosphotyrosine (0.5 mg each) were included in the sample and made
visible after the separation by ninhydrin staining. The dried thin
layer plates were subjected to autoradiography.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by the Medical Research Service of the Department of Veterans Affairs and by NIH Grant DK-35323 (to R.A.N.). Dr. Nissenson is a Research Career Scientist of the Department of Veterans Affairs.
Received for publication June 15, 1998. Revision received August 25, 1998. Accepted for publication August 28, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  B. Wang, Y. Yang, A. B. Abou-Samra, and P. A. Friedman NHERF1 Regulates Parathyroid Hormone Receptor Desensitization: Interference with {beta}-Arrestin Binding Mol. Pharmacol., May 1, 2009; 75(5): 1189 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. U. Miedlich and A. B. Abou-Samra Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E665 - E671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Wang, X. Chen, X. Zhang, and L. Ma Phosphorylation State of {micro}-Opioid Receptor Determines the Alternative Recycling of Receptor via Rab4 or Rab11 Pathway Mol. Endocrinol., August 1, 2008; 22(8): 1881 - 1892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wang, A. Bisello, Y. Yang, G. G. Romero, and P. A. Friedman NHERF1 Regulates Parathyroid Hormone Receptor Membrane Retention without Affecting Recycling J. Biol. Chem., December 14, 2007; 282(50): 36214 - 36222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Bounoutas, H. Tawfeek, L. F. Frohlich, U.-i. Chung, and A. B. Abou-Samra Impact of Impaired Receptor Internalization on Calcium Homeostasis in Knock-In Mice Expressing a Phosphorylation-Deficient Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Endocrinology, October 1, 2006; 147(10): 4674 - 4679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ueda, N. F. Neel, E. Schutyser, D. Raman, and A. Richmond Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells Cancer Res., June 1, 2006; 66(11): 5665 - 5675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Neve Novel Features of G Protein-Coupled Receptor Kinase 4 Mol. Pharmacol., March 1, 2006; 69(3): 673 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Barthet, F. Gaven, B. Framery, K. Shinjo, T. Nakamura, S. Claeysen, J. Bockaert, and A. Dumuis Uncoupling and Endocytosis of 5-Hydroxytryptamine 4 Receptors: DISTINCT MOLECULAR EVENTS WITH DIFFERENT GRK2 REQUIREMENTS J. Biol. Chem., July 29, 2005; 280(30): 27924 - 27934. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-Q. Liu, S. Tan, M. C. Mendel, R. J. Murrills, B. M. Bhat, B. Schlag, R. Samuel, J. J. Matteo, R. de la Rosa, K. Howes, et al. Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors J Biomol Screen, June 1, 2005; 10(4): 304 - 313. [Abstract] [PDF]
|
|
|
|
|
|
L. Wang, S. Liu, L. D. Quarles, and R. F. Spurney Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E826 - E834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Jorgensen, L. Martini, T. W. Schwartz, and C. E. Elling Characterization of Glucagon-Like Peptide-1 Receptor {beta}-Arrestin 2 Interaction: A High-Affinity Receptor Phenotype Mol. Endocrinol., March 1, 2005; 19(3): 812 - 823. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Estall, B. Yusta, and D. J. Drucker Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization Mol. Biol. Cell, August 1, 2004; 15(8): 3673 - 3687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. B. Sneddon, C. E. Magyar, G. E. Willick, C. A. Syme, F. Galbiati, A. Bisello, and P. A. Friedman Ligand-Selective Dissociation of Activation and Internalization of the Parathyroid Hormone (PTH) Receptor: Conditional Efficacy of PTH Peptide Fragments Endocrinology, June 1, 2004; 145(6): 2815 - 2823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. B. Sneddon, C. A. Syme, A. Bisello, C. E. Magyar, M. D. Rochdi, J.-L. Parent, E. J. Weinman, A. B. Abou-Samra, and P. A. Friedman Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50) J. Biol. Chem., October 31, 2003; 278(44): 43787 - 43796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tumova, R. M. Iwasiow, and M. Tiberi Insight into the Mechanism of Dopamine D1-like Receptor Activation. EVIDENCE FOR A MOLECULAR INTERPLAY BETWEEN THE THIRD EXTRACELLULAR LOOP AND THE CYTOPLASMIC TAIL J. Biol. Chem., February 28, 2003; 278(10): 8146 - 8153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Humrich, C. Bermel, T. Grubel, U. Quitterer, and M. J. Lohse Regulation of Phosducin-like Protein by Casein Kinase 2 and N-terminal Splicing J. Biol. Chem., February 7, 2003; 278(7): 4474 - 4481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chauvin, M. Bencsik, T. Bambino, and R. A. Nissenson Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and {beta}-Arrestin Mol. Endocrinol., December 1, 2002; 16(12): 2720 - 2732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Chen, J.-C. Dai, and E. M. Greenfield Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1432 - C1440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bisello, M. Chorev, M. Rosenblatt, L. Monticelli, D. F. Mierke, and S. L. Ferrari Selective Ligand-induced Stabilization of Active and Desensitized Parathyroid Hormone Type 1 Receptor Conformations J. Biol. Chem., October 4, 2002; 277(41): 38524 - 38530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Castro, F. Dicker, J.-P. Vilardaga, C. Krasel, M. Bernhardt, and M. J. Lohse Dual Regulation of the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Signaling by Protein Kinase C and {beta}-Arrestins Endocrinology, October 1, 2002; 143(10): 3854 - 3865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Mason, L. B. Kozell, and K. A. Neve Regulation of Dopamine D1 Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation Mol. Pharmacol., April 1, 2002; 61(4): 806 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-P. Vilardaga, C. Krasel, S. Chauvin, T. Bambino, M. J. Lohse, and R. A. Nissenson Internalization Determinants of the Parathyroid Hormone Receptor Differentially Regulate beta -Arrestin/Receptor Association J. Biol. Chem., March 1, 2002; 277(10): 8121 - 8129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. W. Tawfeek, F. Qian, and A. B. Abou-Samra Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling Mol. Endocrinol., January 1, 2002; 16(1): 1 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Horikawa, B. D. Gaylinn, C. E. Lyons Jr., and M. O. Thorner Molecular Cloning of Ovine and Bovine Growth Hormone-Releasing Hormone Receptors: The Ovine Receptor Is C-Terminally Truncated Endocrinology, June 1, 2001; 142(6): 2660 - 2668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Desai, H. April, C. Nwaneshiudu, and B. Ashby Comparison of Agonist-Induced Internalization of the Human EP2 and EP4 Prostaglandin Receptors: Role of the Carboxyl Terminus in EP4 Receptor Sequestration Mol. Pharmacol., April 13, 2001; 58(6): 1279 - 1286. [Abstract] [Full Text]
|
|
|
|
|
|
P. H. Carter, B. D. Petroni, R. C. Gensure, E. Schipani, J. T. Potts Jr., and T. J. Gardella Selective and Nonselective Inverse Agonists for Constitutively Active Type-1 Parathyroid Hormone Receptors: Evidence for Altered Receptor Conformations Endocrinology, April 1, 2001; 142(4): 1534 - 1545. [Abstract] [Full Text]
|
|
|
|
|
|
S. L. Ferrari and A. Bisello Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling by {beta}-Arrestin2 Mol. Endocrinol., January 1, 2001; 15(1): 149 - 163. [Abstract] [Full Text]
|
|
|
|
|
|
L. Min and M. Ascoli Effect of Activating and Inactivating Mutations on the Phosphorylation and Trafficking of the Human Lutropin/Choriogonadotropin Receptor Mol. Endocrinol., November 1, 2000; 14(11): 1797 - 1810. [Abstract] [Full Text]
|
|
|
|
|
|
J. A. Olivares-Reyes, S. Jayadev, L. Hunyady, K. J. Catt, and R. D. Smith Homologous and Heterologous Phosphorylation of the AT2 Angiotensin Receptor by Protein Kinase C Mol. Pharmacol., November 1, 2000; 58(5): 1156 - 1161. [Abstract] [Full Text]
|
|
|
|
|
|
A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 M. Mannstadt, H. Juppner, and T. J. Gardella Receptors for PTH and PTHrP: their biological importance and functional properties Am J Physiol Renal Physiol, November 1, 1999; 277(5): F665 - F675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Ferrari, V. Behar, M. Chorev, M. Rosenblatt, and A. Bisello Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY J. Biol. Chem., October 15, 1999; 274(42): 29968 - 29975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. d. F. M. Lazari, X. Liu, K. Nakamura, J. L. Benovic, and M. Ascoli Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor Mol. Endocrinol., June 1, 1999; 13(6): 866 - 878. [Abstract] [Full Text]
|
|
|
|
|
|
Z. Huang, T. Bambino, Y. Chen, J. Lameh, and R. A. Nissenson Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein Endocrinology, March 1, 1999; 140(3): 1294 - 1300. [Abstract] [Full Text]
|
|
|
|
|
|
K. Berrada, C. L. Plesnicher, X. Luo, and M. Thibonnier Dynamic Interaction of Human Vasopressin/Oxytocin Receptor Subtypes with G Protein-coupled Receptor Kinases and Protein Kinase C after Agonist Stimulation J. Biol. Chem., August 25, 2000; 275(35): 27229 - 27237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Hanyaloglu, M. Vrecl, K. M. Kroeger, L. E. C. Miles, H. Qian, W. G. Thomas, and K. A. Eidne Casein Kinase II Sites in the Intracellular C-terminal Domain of the Thyrotropin-releasing Hormone Receptor and Chimeric Gonadotropin-releasing Hormone Receptors Contribute to beta -Arrestin-dependent Internalization J. Biol. Chem., May 18, 2001; 276(21): 18066 - 18074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Li, X. Liu, L. Min, and M. Ascoli Mutations of the Second Extracellular Loop of the Human Lutropin Receptor Emphasize the Importance of Receptor Activation and De-emphasize the Importance of Receptor Phosphorylation in Agonist-induced Internalization J. Biol. Chem., March 9, 2001; 276(11): 7968 - 7973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-P. Vilardaga, M. Frank, C. Krasel, C. Dees, R. A. Nissenson, and M. J. Lohse Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein J. Biol. Chem., August 31, 2001; 276(36): 33435 - 33443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Olivares-Reyes, R. D. Smith, L. Hunyady, B. H. Shah, and K. J. Catt Agonist-induced Signaling, Desensitization, and Internalization of a Phosphorylation-deficient AT1A Angiotensin Receptor J. Biol. Chem., October 5, 2001; 276(41): 37761 - 37768. [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 |
