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Medical Research Council Reproductive Biology Unit (A.H., M.V., A.C.H., R.S., P.L.T.), Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9EW; and Western Australian Institute for Medical Research and Keogh Institute for Medical Research (K.E.), Sir Charles Gairdner Hospital, Perth 6009, Australia
Address all correspondence and requests for reprints to: Dr. K. A. Eidne, Western Australian Institute for Medical Research, Ground Floor, B Block QE II Medical Centre, Nedlands, Perth 6009, Australia. E-mail: keidne{at}waimr.uwa.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Agonist stimulation triggers the redistribution of several GPCRs from the plasma membrane into an endosomal compartment via a clathrin- and dynamin-dependent pathway [reviewed by Ferguson et al. and Krupnick and Benovic (2, 17)]. The utilization of a clathrin-mediated pathway in agonist-induced internalization of many GPCRs has been demonstrated by means of experimental treatment with agents that perturb cellular ATP levels, pH, or ion gradients (18) and by elucidating the role of dynamin in this process (19). A GTPase dynamin acts as a force-generating molecule responsible for scission of clathrin vesicles from the plasma membrane (19) and when mutated in its GTP-binding domain can be used to selectively interfere with GPCR internalization via a clathrin-mediated pathway (20). It appears that agonist-induced receptor internalization is not associated with an increase in the number of clathrin-coated vesicles on the cell surface (21), but requires specific adaptor molecules that enhance the receptor/clathrin interaction. The nonvisual arrestins, ß-arrestin-1 (ß-arrestin) and ß-arrestin-2 (arrestin-3), have been identified as clathrin adaptors in GPCR endocytosis (22). Their binding to several agonist-activated/phosphorylated GPCRs promotes receptor targeting for internalization via the clathrin-mediated pathway [reviewed by Krupnick and Benovic (2)]. This observation has been supported by the ability of ß-arrestin dominant negative mutants to impair receptor internalization (23, 24) and by the aid of a ß-arrestin-2 construct tagged with the green fluorescent protein (GFP) (25).
The importance of the C-terminal tail as a receptor-specific
determinant for internalization can vary considerably; truncation of
the C-terminal tail in different GPCRs can impair their
internalization, have no effect, or even promote internalization (2, 17). Data obtained with the ß2-adrenergic
receptor suggested the dispensability of the C-terminal tail for
receptor/ß-arrestin interaction (23), and this region is also not
required for dynamin-dependent internalization of 
-opioid receptor
in HEK 293 cells (26). By evaluating the roles of ß-arrestin and
dynamin, different mechanisms have been reported to govern
internalization of the subtypes of muscarinic cholinergic (27, 28, 29),
opioid (30), and angiotensin II (31) receptors.
The aims of this study were, therefore, to 1) address the role of ß-arrestin in promoting GnRH-R internalization, 2) evaluate whether internalization of these receptors was dependent upon dynamin, and 3) elucidate the possible link between the C-terminal tail and the endocytotic mechanism for a given GnRH-R. To investigate this, we examined the involvement of ß-arrestin-1 and -2 and dynamin in the internalization of wild-type (WT) rat GnRH-R, catfish GnRH-R, rat TRH-R, the GnRH/TRH chimeric receptor and the GnRH-R stop codon mutants. Our results demonstrate an association between the slow internalization of the mammalian GnRH-R and the inability of the ß-arrestins to promote this process, although a dynamin-dependent mechanism is used.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression constructs
The full-length WT rat GnRH-R complementary DNA (cDNA) was in
the vector pcDNA3 (Invitrogen). The creation of three
GnRH-R stop codon mutants and the GnRH-R/TRH-R chimera has previously
been described (8). Briefly, one base in the stop codon in the WT rat
GnRH-R was added, deleted, or changed, creating the three stop codon
mutations (+1 base, -1 base, and in-frame). In all three cases the
stop codon was changed to a triplet encoding alanine. As the in-frame
stop codon mutant has previously been shown not to express (8), this
mutant was not used in the present study. The GnRH-R/TRH-R chimera was
created by cloning the C-terminal tail of the TRH-R into the C-terminal
of the rat GnRH-R after the insertion of a ClaI restriction
enzyme site; the receptor constructs are shown in Fig. 1
. Production of the GFP/ß-arrestin 1
fusion protein was performed as follows. Approximately 1.5 kb of the
ApaI/HindIII insert released from pcDNA3
containing the coding region for WT ß-arrestin-1 were subcloned into
the ApaI/HindIII site at the C-terminus of the
GFP within the pEGFPC/2 vector. The open reading frame so produced
represents the coding sequence of GFP/ß-arrestin-1. The cDNA clones
were sequenced several times using an PE Applied Biosystems (Cheshire, UK) 373A automated sequencer. Sequence
analysis was performed by means of the program GeneJockey II
(Biosoft, Cambridge, UK).
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Iodination of GnRH agonist
Iodinated radiolabeled GnRH analog was prepared using the
Iodogen [Pierce Chemical Co. (Rockford, IL)] method and
purified by chromatography on a Sephadex G-25 column in 0.01
M acetic acid-0.1% BSA. The specific activity of the
[125I]des-Gly10-(D-Trp6)-GnRH
was 56 µCi/µg and was calculated from self-displacement assays
using either rat pituitary homogenates or HEK 293 cells stably
expressing the WT rat GnRH-R. The specific activity of the
[125I]chicken II tracer was 90 µCi/µg and
was calculated as described previously (32).
Receptor internalization assays
The receptor internalization assay was done as described
previously (8). Briefly, cells in 24-well plates
were incubated with labeled agonist for time intervals ranging from 5
min to 2 h at 37C. At appropriate times, the surface bound
radioactivity was removed by washing with acid solution (50
mM acetic acid and 150 mM NaCl, pH 2.8). The
internalized radioactivity was determined after solubilizing the cells
in 0.2 M NaOH and 1% SDS solution. Nonspecific binding for each time
point was determined under the same conditions in the presence of 10
µM unlabeled agonist. After subtraction of nonspecific
binding, the internalized radioactivity was expressed as a percentage
of the total binding at that time interval. All time points were
performed in triplicate in at least three separate experiments. The
endocytosis rate constants (ke) were calculated
using a mathematical model described by Koenig and Edwardson (33).
Visualization of GFP/ß-arrestin
HEK 293 cells (1.5 x 106/60-mm dish)
stably expressing rat GnRH-R, TRH-R, catfish GnRH-R, or the rat
GnRH-R/TRH-R chimera were transfected with 2.5 µg GFP/ß-arrestin-1
or -2 cDNA using Superfect. After 24 h, cells were plated into
eight-well chamber slides, and treatments were carried out 48–72 h
after transfection. The cells were then fixed with 4%
paraformaldehyde, mounted with Citifluor, and sealed with coverslips.
Cells were examined under an oil immersion objective (x60) using a
Carl Zeiss LMS 510 confocal laser microscope (New York,
NY) and a filter selective for fluorescein isothiocyanate fluorescence.
Optical sections (1.0 µm) were taken, and representative sections
corresponding to the middle of the cells are presented.
Statistical analysis
Statistical significance was determined using Student’s
t test. Differences are considered significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows a schematic representation of the rat GnRH-R to
which the extended 3' tails ("ghost tails") in each of the reading
frames or the C-terminal tail of the TRH-R have been added, creating
three rat GnRH-R stop codon constructs and the chimeric GnRH-R/TRH-R.
Putative phosphorylation sites for protein kinase C and casein kinase
II sites are indicated. We have previously shown that the in-frame stop
codon mutant with the longest C-terminal tail does not express (8);
therefore, this construct was not used further. The effect of WT
ß-arrestin-1 on receptor internalization was examined in COS-7 cells,
as these cells endogenously express low levels of ß-arrestin (
70%
less ß-arrestin than HEK 293 cells) (34). The internalization
properties of the +1 base and -1 base GnRH-R stop codon constructs
were investigated in COS-7 cells transiently transfected with receptor
and control vector (pcDNA-3), receptor and WT ß-arrestin-1, receptor
and the dominant negative ß-arrestin mutant-(319–418), or receptor
and the dominant negative dynamin mutant (DynK44A). We found that
following agonist treatment (1 h), internalization of the +1 base and
-1 base mutants was not significantly different from that of the WT
rat GnRH-R, and that internalization was not affected by cotransfection
of either WT ß-arrestin-1 or ß-arrestin mutant-(319–418) (Fig. 2). However, we found that the DynK44A mutant
at this time point had an inhibitory effect on receptor internalization
(P < 0.05) for the WT rat GnRH-R as well as for each
of the expressing stop codon mutants (Fig. 2).
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). We again found that coexpression of WT
ß-arrestin-1 had no effect on the internalization of WT rat GnRH-R at
any of the time points examined. In contrast, the internalization rates
of receptors possessing a C-terminal tail (the TRH-R, catfish GnRH-R,
and the GnRH-R/TRH-R chimera) were increased by coexpression with WT
ß-arrestin-1, demonstrating the importance of a functional C-terminal
tail for ß-arrestin-dependent internalization. When cells were
cotransfected with ß-arrestin-(319–418), the effect of WT
ß-arrestin-1 was diminished or abolished for the receptors possessing
a C-terminal tail. However, the coexpression of ß-arrestin-(319–418)
had no effect on the internalization of the WT rat GnRH-R (Fig. 3).
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). The presence of the dominant negative
DynK44A mutant reduced internalization for all of the receptors,
indicating utilization of a dynamin-dependent internalization pathway
regardless of whether the receptor possesses a C-terminal tail. The
coexpression of WT ß- arrestin-1 had no effect on internalization
in the presence of the DynK44A mutant. The WT dynamin coexpression had
no effect on receptor internalization (data not shown).
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). The coexpression of
ß-arrestin-(319–418) decreased the basal agonist-induced
internalization of the TRH-R to some extent, slightly decreased the
internalization of the GnRH-R/TRH-R and catfish GnRH-R, and had no
effect on WT rat GnRH-R internalization. The addition of both dominant
negative forms of ß-arrestin and dynamin did not synergistically
inhibit receptor internalization (Fig. 5).
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). The WT rat GnRH-R endocytosis rate
constants calculated for all of the different experiments were not
significantly different. However, the endocytosis rate constants for
the TRH-R, catfish GnRH-R, and the GnRH-R/TRH-R chimera all increased
significantly (P < 0.05) when the receptor was
coexpressed with WT ß-arrestin-1. This effect was inhibited when
ß-arrestin-(319–418) was coexpressed with WT ß-arrestin-1 and
receptor. The endocytosis rate constants for all of the studied
receptors were significantly (P < 0.05) reduced by
coexpression of DynK44A. Constants derived from experiments in which
receptor was coexpressed with DynK44A and WT ß-arrestin-1 are not
shown, as they are similar to constants from experiments with only
receptor and DynK44A.
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, A–F) or
GFP/ß-arrestin-2 (Fig. 6, G–J). Before agonist stimulation the
distribution of GFP/ß-arrestins is mainly cytosolic (Fig. 6, A, C, G,
and I), as observed for all receptors studied (only unstimulated TRH-R-
and WT rat GnRH-R-expressing cells are shown). After agonist
stimulation for 3 min, GFP/ß-arrestin-1 (Fig. 6, B, E, and F) and
GFP/ß-arrestin-2 (Fig. 6H) translocated from the cytosol to the cell
membrane only in the cells expressing a receptor with a C-terminal
tail. In the cells expressing the WT rat GnRH-R (Fig. 6, C and D, I and
J), GFP/ß-arrestin-1 (Fig. 6D) and GFP/ß-arrestin-2 (Fig. 6J)
remained cytosolic even after agonist stimulation for 10 min.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The abilities of different receptors to use either the ß-arrestin-dependent or -independent pathway was further confirmed by 1) results obtained with the dominant negative ß-arrestin mutant [ß-arrestin-(319–418)] and 2) visualization of the cellular distribution of GFP-linked ß-arrestins. By cotransfecting the cells with the ß-arrestin-(319–418) mutant, we were able to substantially reduce the WT ß-arrestin-1-promoted increase in the ke value of the TRH-R, catfish GnRH-R and GnRH-R/TRH-R chimera. However, there was no effect on internalization of the WT rat GnRH-R or that of the stop codon mutants.
To further support these data we also employed the GFP fusion tag
approach. The successful fusion of ß-arrestins with GFP has been
achieved by linking the C-terminus of both ß-arrestin-2 (25) and
ß-arrestin-1 (12, 40, 41) with the N-terminus of the GFP. Here, we
report that a functional GFP/ß-arrestin-1 construct can also be
obtained by inserting the ß-arrestin-1 into the C-terminus of the
GFP. Using confocal microscopy, time- and agonist-dependent
redistribution of GFP/ß-arrestin-1 to the plasma membrane was
observed for all receptors examined except the mammalian form of the
GnRH-R. Although only modest discrimination in the specificity of
nonvisual arrestins binding to GPCRs has been observed in
vitro (42), a recent study suggested involvement of
ß-arrestin-2, but not ß-arrestin-1 in GnRH-R desensitization (43).
Therefore, we wished to establish whether ß-arrestin-2 also has a
specific effect on GnRH-R internalization. By employing the
GFP/ß-arrestin-2 construct, which has been used to demonstrate
ß-arrestin translocation to the plasma membrane for a number of
ligand-activated GPCRs (25, 44), the same results were obtained as
those for GFP/arrestin-1. This observation excludes the possibility
that the two forms of nonvisual arrestin may have different effects on
GnRH-R internalization, and it is also consistent with internalization
assay results. The disparity in the effects of ß-arrestin on
desensitization vs. internalization has also been reported
for the m2 muscarinic acetylcholine receptor (mAChR) (27). The
overexpression of the ß-arrestin-1 selectively interferes with
-opioid receptor/G protein coupling (45), but probably not with the
internalization, as this receptor subtype remains on the plasma
membrane after agonist activation (30).
The next question we asked was whether the inhibitory modulators of clathrin-mediated endocytosis could also affect internalization of receptors that use the ß-arrestin-independent pathway. For these studies we examined the effect of the dominant negative dynamin DynK44A mutant on internalization kinetics. DynK44A coexpression resulted in impaired internalization of all receptor types examined, reflected in the decreased endocytosis rate constants. Similarly, the ß-arrestin-promoted enhancement of the TRH-R, catfish GnRH-R, and GnRH-R/TRH-R chimera internalization rates was impaired by mutant dynamin DynK44A coexpression. These results provide evidence that the rat GnRH-R uses a ß-arrestin-independent, but dynamin-dependent, pathway, suggesting that involvement of ß-arrestin is not a prerequisite for the use of the dynamin-dependent pathway via clathrin-coated vesicles. However, only partial inhibition of WT rat GnRH-R endocytosis by overexpressed DynK44A could suggest that distinct subpopulations of receptors internalize via different pathways. This could relate to the dual coated pit pathway hypothesis, which predicts the existence of two distinct (stage 1 and stage 2) clathrin-mediated pathways that operate in parallel (46). However, due to the lack of any perturbing agent that affects the stage 1 pathway, its existence remains largely speculative.
In view of recent findings that establish a role for dynamin in the internalization of caveolae (47, 48), dynamin dependence of GPCR endocytosis can no longer be considered to imply a clathrin-mediated event. However, it has not been shown that internalized caveolae ever fuse with endosomes from coated pits (49). Therefore, it is unlikely that the GnRH-R undergoes endocytosis by caveolae, as its colocalization with the transferrin receptors, a well established marker for endocytosis via clathrin-coated pits, has been demonstrated recently (12). Characterization of the internalization pathway for a number of other GPCRs has also unveiled a greater complexity in the choice of endocytotic pathway used. Three subtypes of the mAChR (m1, m3, and m4) use the same internalization pathway as rat GnRH-R, arrestin independent but dynamin dependent (28, 29), whereas the m2 mAChR subtype preferentially uses an arrestin-independent internalization pathway, although it can enter the arrestin-dependent pathway in the presence of overexpressed arrestins (27). Internalization of m2 receptor subtype is also differentially regulated by dynamin in different cell types, HEK-293 and CHO as opposed to COS-7 (29). The results obtained for the AT1A receptor showed that this receptor internalizes via a ß-arrestin- and dynamin-independent pathway; however, it can be mobilized to the dynamin-dependent pathway upon overexpression of ß-arrestin (31). It could be assumed that utilization of a particular pathway can underlie a distinct function of receptor internalization. Supporting this, evidence has been provided that internalization can play a part in 1) the reestablishment of ß2-adrenergic receptor function (50) and 2) prolonged desensitization of the m4 mAChR (51), two GPCRs that use different internalization pathways after agonist activation.
In summary, our results demonstrate that the rat GnRH-R expressed in a heterologous cell system uses a dynamin-dependent, but ß-arrestin-independent, internalization mechanism and that the receptor domain conferring arrestin dependency can be added from another GPCR to the rat GnRH-R, thereby augmenting endocytosis. The implication of these results for the pituitary gonadotrope remains to be investigated.
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Acknowledgments |
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Footnotes |
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2 Supported by a Royal Society/NATO Postdoctoral Fellowship.
Received May 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell line.
Endocrinology 136:4864–4871[Abstract]
and opioid receptors are differentially regulated by
dynamin-dependent endocytosis when activated by the same alkaloid
agonist. J Biol Chem 272:27124–27130 and but not µ opioid
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O. Benard, Z. Naor, and R. Seger Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone J. Biol. Chem., February 9, 2001; 276(7): 4554 - 4563. [Abstract] [Full Text] [PDF]
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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]
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K. M. Kroeger, A. C. Hanyaloglu, R. M. Seeber, L. E. C. Miles, and K. A. Eidne Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor. DETECTION IN LIVING CELLS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., April 13, 2001; 276(16): 12736 - 12743. [Abstract] [Full Text] [PDF]
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), 1.0 µg
WT ß-arrestin-1 (
), or 1.0 µg WT ß-arrestin-1 and 4.0 µg
ß-arrestin-(319–418) (
) in COS-7 cells. Cells were incubated with
the appropriate labeled agonist for the indicated time. Surface-bound
radioactivity was then determined as the radioactivity that could be
removed by acid wash. Internalized radioactivity was determined after
solubilization of the cells as described in Materials and
Methods. A curve has been fitted to the data points, as
described by Koenig and Edwardson (
), or 1.0 µg WT
ß-arrestin-1 and 4.0 µg DynK44A (
) in COS-7 cells. Cells were
incubated with the appropriate labeled agonist for the indicated time.
Surface-bound radioactivity was then determined as the radioactivity
that could be removed by acid wash. Internalized radioactivity was
determined after solubilization of the cells as described in
Materials and Methods. A curve has been fitted to the
data points as described by Koenig and Edwardson (
