From the Department of Pharmacology, The University
of Iowa College of Medicine, Iowa City, Iowa 52242 and the
** Department of Microbiology and Immunology, Kimmel Cancer Institute,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex.
We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters.
Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway.
These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphorylation of G protein-coupled receptors (GPCRs)1 on serine and/or threonine residues is an important event in agonist-induced desensitization. GPCR phosphorylation by second messenger-dependent kinases attenuates signaling by uncoupling the receptors from their cognate G proteins, whereas phosphorylation by the G protein-coupled receptor kinases (GRKs) facilitates the interaction of the receptors with a family of inhibitory proteins called arrestins (1, 2). This phosphorylated receptor-arrestin interaction uncouples the receptors from their cognate G proteins and targets the activated receptor to clathrin-coated pits for subsequent internalization (1-4). Thus, the complex formed by the phosphorylated GPCR and arrestin serves as a common intermediate for the uncoupling of the receptor from its cognate G protein and for receptor internalization.
Using human kidney 293 cells stably transfected with the rat lutropin/choriogonadotropin receptor (rLHR) cDNA, we showed that, like many other GPCRs, the rLHR becomes phosphorylated on serine residues when the cells are stimulated with an agonist (lutropin (LH) or choriogonadotropin (CG)) (5). The identity of the kinases that mediate the agonist-induced phosphorylation of the rLHR is not known; however, neither kinase A nor kinase C can fully account for the agonist-induced phosphorylation of the rLHR (5, 6). The involvement of one of the GRKs in the agonist-induced phosphorylation of the rLHR is suggested by the finding that overexpression of GRK2, GRK4, or GRK6 enhances agonist-induced phosphorylation.2 A GRK-catalyzed phosphorylation of the rLHR is also suggested by functional studies showing that co-transfection of the rLHR with GRK2 or GRK4 diminishes the hCG-induced cAMP response (7).
By analogy with what is known about other GPCRs (see above) we proposed
that the agonist-induced phosphorylation of the rLHR was responsible
for the agonist-induced uncoupling of this receptor from its effector
system (5). Further analysis of rLHR mutants truncated at residues 653, 631, or 628 (designated rLHR-t653, rLHR-t631, or rLHR-t628) and a
full-length rLHR mutant with multiple serine substitutions (designated
rLHR-5S/TA) mapped the agonist-induced phosphorylation to a cluster
of four serine residues (Ser635, Ser639,
Ser649, and Ser652) present in the C-terminal
tail and established some functional consequences of phosphorylation of
this cluster (6, 8, 9). Rat LHR-t653, a truncated form of rLHR that
retains Ser635, Ser639, Ser649, and
Ser652, displays little or no reduction in the
agonist-induced phosphorylation, as well as a normal time course and
magnitude of agonist-induced uncoupling. On the other hand, rLHR-t631
and rLHR-t628, two truncated forms of the rLHR that lack
Ser635, Ser639, Ser649, and
Ser652, and a full-length receptor mutant in which these
four residues were simultaneously mutated to alanines (i.e.
rLHR-5S/T
A) display a 90-100% decrease in agonist-induced
phosphorylation and a delay in the rate of agonist-induced uncoupling.
The magnitude of agonist-induced uncoupling observed under
prolonged agonist stimulation is unaffected, however (8, 9).
It has been known for many years that one of the consequences of
agonist binding to the LHR is the endocytosis of the agonist-receptor complex (10). Although the endocytosis of the agonist-bound LHR has
been shown to occur via coated pits (11), the rate of endocytosis of
the agonist-receptor complex is rather slow (half-life of 60-120 min,
depending on the cell type; see Refs. 9, 10, 12, and 13), and the
majority of the agonist-receptor complex is routed to the lysosomes,
where both the agonist and the receptor are degraded (11, 14). This
pathway ultimately leads to an agonist-induced reduction in the density
of cell surface receptors by routing the receptor to a degradation
rather than a recycling pathway (14, 15). In keeping with current views
on the internalization of other GPCRs, we have shown that the
activation of the rLHR is necessary for efficient endocytosis (12, 13).
Moreover, the importance of the phosphorylation of the four-serine
locus mentioned above in the endocytosis of the receptor-bound agonist was documented by the finding that cells expressing rLHR-5S/TA internalize the bound agonist at a slower rate than cells expressing the wild-type rLHR (9).
The experiments presented here were designed to determine which of the four serine residues present in this locus of the rLHR become phosphorylated and to more carefully define the role of each of these residues in the agonist-induced uncoupling and internalization of the rLHR. To this end, we constructed and analyzed four new rLHR mutants in which Ser635, Ser639, Ser649, or Ser652 was individually mutated to an alanine residue in the context of the full-length rLHR. These mutants were analyzed for phosphorylation, uncoupling, and internalization.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids and Cells--
The cloning of the rat luteal LH/CG
receptor cDNA and the template plasmid containing the full-length
coding region plus portions of the 5'- and 3'-untranslated regions of
the wild-type rLHR cDNA have been described previously (16). The
individual Ser to Ala mutants were constructed using polymerase chain
reaction strategies to alter the nucleotides coding for these residues.
The sequence of the entire region of each mutant cDNA generated by
polymerase chain reaction was verified by automated DNA sequencing. The
mutant and wild-type rLHR cDNAs were subcloned into the eukaryotic
expression vector pcDNAI/Neo (Invitrogen) for transfection. A
plasmid encoding for an HA-tagged dominant-negative mutant form of
dynamin (i.e. dynamin-K44A; see Ref. 17) was obtained from
Sandra Schmid and subcloned into pcDNA3.1 (Invitrogen) for
transfection. The expression vectors (all in pcDNA3.1) encoding for
-arrestin, arrestin-3,
-arrestin-V53D, and
-arrestin
(319-418) have also been described (4).
Intact Cell Phosphorylation Assays-- Stably transfected cells were plated in 100-mm dishes and were metabolically labeled during a 4-h incubation in phosphate-free medium containing 200 µCi/ml 32Pi. Receptor phosphorylation was ascertained after incubating the 32Pi-prelabeled cells at 37 °C with buffer only, or with 1000 ng/ml oLH for 5 min. These conditions were chosen to elicit a maximal response (5, 6, 8). One cell line expressing rLHR-wt (either 293L(wt-12) or 293L(wt-17)) and one cell line expressing one of the rLHR mutants was used in each experiment. Following lysis of the cells, the amount of wt and mutant receptor used for immunoprecipitation was equalized based on 125I-hCG binding assays (see below). Immunoprecipitations were performed using Bugs (a rabbit polyclonal antibody to the rLHR (20)) or a mixture of AntiL and R02 (two polyclonal antibodies directed against synthetic peptides derived from the known amino acid sequence of the rLHR (19, 21)). The methodology used for immunoprecipitation and SDS gels was the same as that described earlier (5, 6, 8, 19). Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens. The autoradiograms were scanned using a Bio-Rad Molecular Imaging System and captured in a digital format for presentation.
Hormone Binding and Signal Transduction Assays-- Equilibrium binding parameters for hCG were measured during an overnight incubation (4 °C) of intact cells with a fixed concentration of 125I-hCG and increasing concentrations of hCG as described previously (6, 8). Concentration-response curves for the hCG-induced increases in cAMP accumulation were obtained by measuring total cAMP levels in cells that had been incubated with at least five different concentrations of hCG for 30 min at 37 °C in the presence of a phosphodiesterase inhibitor. The different parameters that describe the concentration response curves were calculated as described elsewhere (6, 8).
Measurements of agonist-induced uncoupling in stably transfected cells were performed as follows. Cells expressing rLHR-wt or the mutant receptors were divided into two groups and incubated without (group A) or with (group B) 100 ng/ml oLH or hCG for 15 min.3 All cells were then washed with neutral and acidic buffers to remove the free and bound hormone, respectively, and each group of cells was subdivided into two groups, which were then incubated without (groups A1 and B1) or with (groups A2 and B2) 100 ng/ml hCG for 15 min at 37 °C (6, 8). Intracellular levels of cAMP were measured at the end of this incubation, and agonist-induced uncoupling was calculated as follows: ((B2Internalization Assays-- The endocytosis of 125I-hCG was measured in cells that had been briefly preincubated (i.e. 10 min at room temperature) with 40 ng/ml 125I-hCG. At the end of this preincubation, the free hormone was removed by washing, and the cells were re-incubated in fresh, hormone-free medium at 37 °C for up to 4 h. After the desired interval, the cells were placed on ice, and the medium was saved. The cells were briefly treated with an isotonic pH 3 buffer (10, 13). The radioactivity that was released by the acid treatment was considered to be surface-bound, whereas the radioactivity that remained cell-associated was considered to be internalized. The medium was precipitated with trichloroacetic acid, and the acid-insoluble and -soluble radioactivity were considered to be undegraded and degraded hormone, respectively (10, 13). Because there is little or no dissociation of the receptor-bound hCG during this incubation (10), the rate of disappearance of the receptor bound hCG can be used to measure the rate of internalization. The rates of internalization (ke) were thus calculated from the slopes of linear regression fits to plots of the ln of the surface-bound hCG versus time. The half-life of internalization (t1/2) is therefore defined as 0.693/ke.
Immunoblots-- Expression of the transfected arrestins and dynamin was ascertained by immunoblots using the ECL system of detection. The different arrestin constructs were detected using a polyclonal antibody (KEE) raised against a C-terminal 16 residue peptide (22) or a mouse monoclonal antibody (F4C1) directed against an epitope common to all known arrestins (23). The HA-tagged dynamin was detected using the 12CA5 monoclonal antibody (Boehringer Mannheim).
Hormones and Supplies-- Purified hCG (CR-127) and oLH (AFP-5551B) were obtained from the National Hormone and Pituitary Agency of the NIDDK, National Institutes of Health. 125I-hCG was prepared as described previously (24), to give a specific radioactivity of 25,000-30,000 cpm/ng. [32P]Orthophosphate was obtained from NEN Life Science Products. Phosphate-free DMEM was purchased from ICN Biomedicals (Irvine, CA). Nonidet P-40, protease inhibitors, N,N',N"-triacetylchitotriose, protein A-agarose, and bovine serum albumin were from Sigma. Okadaic acid and cypermethrin were purchased from Alexis Biochemicals (Woburn, MA). Wheat germ agglutinin agarose was from Vector Laboratories. Cell culture supplies and reagents were obtained from Corning (Corning, NY) and Life Technologies, Inc., respectively. All other materials were obtained from commonly used suppliers.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation and Functional Properties of rLHR Mutants-- Phosphoamino acid analysis and phosphorylation experiments utilizing three different C-terminal truncations of the rLHR and a full-length mutant with multiple Ser to Ala mutations have identified Ser635, Ser639, Ser649, and Ser652 as the major locus of rLHR phosphorylation in transfected cells (6, 8, 9). For the experiments presented here, we prepared four new full-length rLHR mutants in which each of these four serines was individually mutated to alanine. Each mutant cDNA (designated rLHR-S635A, rLHR-S639A, rLHR-S649A, and rLHR-S652A) was transfected into human kidney 293 cells, and clonal lines stably expressing each of the mutants were obtained. All mutant receptors bound hCG with an affinity comparable to that detected in cells expressing rLHR-wt (i.e. about 200 pM, see Table I). Of the several clonal lines obtained with each mutant, we chose those with the highest cell surface receptor density for further study (Table I).
|
Agonist-induced rLHR Phosphorylation-- The effect of individual Ser to Ala mutations on rLHR phosphorylation was determined by immunoprecipitation of the rLHR from the different stable cell lines that had been metabolically labeled with 32Pi. Each phosphorylation experiment consisted of four samples. Two samples were derived from 32Pi-labeled 293L(wt-12) or 293L(wt-17) cells incubated with or without agonist, and two samples were derived from 32Pi-labeled 293L(S635A-5), 293L(S639A-5), 293L(S649A-14), or 293L(S652A-5) cells, also incubated with or without agonist.
In earlier phosphorylation experiments, we used hCG as the agonist (5, 6, 8, 9). In more recent experiments, we switched to using oLH as the agonist because the actions of LH and CG are indistinguishable under these conditions, but the rate of dissociation of the bound oLH is much faster than that of the bound hCG (25, 26). Thus, when using oLH as the agonist, most of the bound oLH dissociates from the rLHR prior to the immunoprecipitation, whereas a substantial amount of the hCG remains receptor-bound. This turns out to be an important consideration in immunoprecipitation experiments because some of the receptor antibodies that we have started to use (see under "Materials and Methods") do not recognize the agonist-bound receptor.4 As such, when using hCG as the agonist, it is possible to misinterpret a decrease in the 32P signal as a decrease in phosphorylation, whereas in reality, the decrease is simply due to the inefficient immunoprecipitation of the hCG-receptor complex as compared with the free receptor. Following stimulation, cells were lysed, and equal amounts of the wt and mutant receptors were immunoprecipitated, resolved on SDS gels, and visualized by autoradiography as shown in Fig. 1. The results of several experiments, such as those shown in Fig. 1, were quantitated by densitometry, and these are summarized in Table II. The results obtained with rLHR-wt are in agreement with previous data showing that this receptor is phosphorylated in unstimulated cells and that agonist-stimulation elicits ~2-fold increase in the 32P signal (5, 6, 8, 9). The results summarized in Table II also show that the individual mutation of Ser635, Ser639, Ser649, or Ser652 to Ala diminishes agonist-induced phosphorylation to 57-78% of control. Because the removal or simultaneous mutation of all these four serines reduces phosphorylation to barely detectable levels (6, 8, 9), the simplest interpretation of the data shown in Table II is that all four serines are phosphorylated when cells are stimulated with agonist.
|
|
Agonist-induced Uncoupling-- The time course of agonist-induced uncoupling of the LH/CG-sensitive adenylyl cyclase in 293 cells expressing rLHR-wt suggests the existence of two phases: a fast phase, which occurs within 15 min of agonist addition and leads to a 40-60% reduction in agonist-stimulated cAMP synthesis, and a slower phase, which leads to a further 20-40% reduction in the agonist-stimulated cAMP synthesis (Fig. 2 and Refs. 8 and 9). Because the removal or mutation of Ser635, Ser639, Ser649, and Ser652 affects the time course, rather than the magnitude of agonist-induced uncoupling (cf. Fig. 2 and Refs. 8 and 9), the effect of the individual Ser to Ala mutations on this process were initially tested by rechallenging cells with agonist after a short (i.e. 15-min) preincubation with or without agonist. Experiments were also done using either 293L(wt-12) or 293L(wt-17) cells as controls, because the agonist-induced uncoupling of cells expressing rLHR-wt is independent of receptor density (8, 9).
|
Agonist-induced Internalization--
The effect of individual
serine mutations of the rLHR on the endocytosis of hCG were measured
using a protocol that allows us to follow one round of endocytosis of
the bound hormone (10, 13). The half-life of internalization of hCG in
cells expressing the rLHR-wt is slow (1-2 h, depending on the cell
type; see Refs. 9, 10, 12, and 13) when compared with the half-life of internalization of many other ligand-receptor complexes (27). In
agreement with previous data, the results summarized in Fig. 3 and Table
III show that 293 cells expressing
rLHR-wt (either 293L(wt-12) or 293L(wt-17)) internalize the bound hCG
with a half-life of 139 min. These data also show that mutation of
Ser652 had little or no effect on internalization,
mutation of Ser635 or Ser639 increased the
half-life of internalization 1.4-1.6-fold, and the mutation of
Ser649 increased the half-life of internalization 2.8-fold.
In fact, the half-life of internalization of hCG in cells expressing
rLHR-S649A is similar to that measured in parallel experiments using
cells expressing rLHR-5S/TA, a mutant in which all four serines
were simultaneously mutated to alanines (Table III).
|
|
Roles of Arrestin and Dynamin on Agonist-induced Internalization
and Uncoupling--
In order to learn more about the structural
requirements for the agonist-induced internalization of the rLHR, we
examined the effects of nonvisual arrestins, dynamin, and mutants
thereof on the internalization of rLHR-wt, rLHR-S639A (one of the two mutants that slows down internalization and uncoupling; cf.
Figs. 2 and 3, and Tables II and III), and rLHR-S649A (a mutant that blocks internalization but does not affect uncoupling; cf.
Fig. 3 and Tables II and III). As already mentioned above, nonvisual arrestins are clathrin-binding proteins that act as adapters, linking
GPCRs to clathrin-coated pits (3). Two mutant forms of -arrestin,
-arrestin-V53D and
-arrestin(319-418), act as dominant negative
mutants of arrestin-mediated GPCR internalization because they have
reduced binding affinities for the phosphorylated GPCRs (4). Dynamin is
a GTPase that participates in the fission of endocytic vesicles from
the plasma membrane (28). Dynamin mutants that are deficient in GTP
binding (such as dynamin-K44A) block coated pit-mediated
internalization of several receptors (17, 29, 30).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Together with previous data from this laboratory (5, 6, 8, 9), the experiments presented here are consistent with a model in which four serines present in the C-terminal tail of the rLHR (Ser635, Ser639, Ser649, and Ser652) are phosphorylated in response to agonist stimulation. This accommodates the findings that the removal or mutation of Ser635, Ser639, Ser649, and Ser652 decreases basal and agonist-induced phosphorylation by at least 90% (6, 8, 9), whereas the individual serine mutations reduce agonist-induced phosphorylation only partially (Table II). However, the reduction in the magnitude of phosphorylation detected with each mutation suggests that all four serines are not phosphorylated to the same extent or that there is a hierarchy of phosphorylation sites. Additional studies to distinguish between these possibilities (such as the simultaneous mutation of groups of two or three serines at a time) were not performed because the low magnitude of the phosphorylation signal (Fig. 1 and Table II) makes the quantitation of partial changes in rLHR-phosphorylation rather difficult. Moreover, because the data presented here clearly show that the functional impact of the individual mutation of some of these serines is similar (or identical) to that observed when all four serines are mutated simultaneously (cf. Fig. 2 and Tables II and III), additional mutagenesis studies would not contribute much new information to our understanding of the functional significance of these four serines.
The functional data presented here clearly show that the attenuation of the agonist-induced uncoupling that results as a consequence of the removal (8) or mutation of Ser635, Ser639, Ser649, and Ser652 (9) can be fully reproduced by the mutation of either Ser635 or Ser639 (Table II and Fig. 2), whereas the slower rate of agonist-induced internalization detected in the multiple substitution mutant (9) can be fully reproduced by mutation of Ser649 (Table III and Fig. 3). The mutation of Ser652 had little or no effect on agonist-induced uncoupling or internalization. Taken together, these data show that the structural requirements for agonist-induced uncoupling and agonist-induced internalization of the rLHR are different. Some overlap between these structural motifs is suggested by the finding that mutation of Ser635 or Ser639, which induced maximal attenuation of uncoupling, also induced partial attenuation of internalization (Figs. 2 and 3 and Tables II and III).
One of the most interesting recent advances in our understanding of the
biology of GPCRs is the realization that the complex formed by the
phosphorylated 2-adrenergic receptor
(
2AR) and arrestin plays a pleiotropic role in the
regulation of
2AR function (3, 4, 33, 34). Thus, it is
now generally accepted that the GRK-catalyzed phosphorylation of the
2AR increases the affinity of the
2AR for
-arrestin and that the formation of the phosphorylated
2AR-arrestin complex sterically hinders the
2AR-G protein association. Because
-arrestin can also
bind clathrin with high affinity, the
2AR-arrestin
complex is targeted for internalization through clathrin-coated pits.
Some variations on this theme have also begun to emerge, as illustrated
by the finding that agonist stimulation of the AT1A
angiotensin (29) and the m2 muscarinic receptors (31) leads to receptor
phosphorylation, but the phosphorylated receptors are internalized by a
pathway that does not require the participation of arrestin or
clathrin-coated pits. However, overexpression of nonvisual arrestins
forces the agonist-stimulated AT1A angiotensin and m2
muscarinic receptors to be internalized via clathrin-coated pits by a
pathway that requires nonvisual arrestins (29, 31).
The data presented here for the rLHR-wt expressed in 293 cells shows
that the agonist-induced internalization of the rLHR, like that of the
2AR expressed in 293 cells, requires the participation of arrestin and clathrin-coated pits (Fig. 4). Thus, the overexpression of two dominant-negative forms of
-arrestin and a dominant-negative form of dynamin, as well a treatment with hypertonic sucrose inhibit the agonist-induced internalization of rLHR-wt. In agreement with data
obtained with the
2AR expressed in 293 cells (4), we found that
-arrestin-V53D is less effective than
-arrestin(319-418) in inhibiting the internalization of the rLHR.
However, the magnitude of the inhibition of rLHR-wt internalization
induced by overexpression of the two dominant-negative
-arrestins
and the dominant negative dynamin in 293 cells is generally lower than
those reported for the
2AR in the same cell line (4, 29,
34, 35). A more notable difference is with the effect of arrestin-3, as
the overexpression of this protein in 293 cells has little or no effect
on the internalization of the
2AR (4, 29, 34, 35), but
it enhances the internalization of the rLHR-wt about 2-fold (Fig. 4).
When considered together, these data suggest that the rLHR-wt has a low
affinity for arrestins and that the slow rate of internalization of the
rLHR-wt (t1/2 ~ 140 min; see Table III) compared
with that of the
2-AR (t1/2 < 30 min; see Ref. 29) may be a reflection, at least in part, of the weak
rLHR-arrestin interaction.
The effects of the two dominant-negative -arrestins and
dominant-negative dynamin on rLHR-S639A or rLHR-S649A were virtually indistinguishable from those detected with rLHR-wt (Fig. 4) and support
the idea that the agonist-induced internalization of these two mutants
also occurs by an arrestin- and coated pit-dependent pathway. The experiments presented here are the first to address the
involvement of nonvisual arrestins in this phenomenon. The involvement
of clathrin-coated pits in the internalization of the LHR-wt has been
previously documented using ultrastructural approaches,
however (11).
Although transient cotransfection assays have been used by others (7)
to show that GRK overexpression reduces the hCG-induced cAMP response
mediated by rLHR-wt, the same paradigm failed to reveal opposite
effects of arrestin-3 or a dominant-negative -arrestin on the
hCG-stimulated cAMP response. The overexpression of both arrestin-3 and
a dominant-negative
-arrestin had a slight stimulatory effect on
hCG-stimulated cAMP accumulation (Table IV). Although these results
suggest the existence of an arrestin-independent pathway for the
agonist-induced uncoupling of the rLHR, they may need to be interpreted
with caution because transient co-transfection assays of 293 cells have
also failed to detect an effect of
-arrestin or
-arrestin-V53D on
the agonist-induced cAMP response mediated by the
2AR
(34). Likewise, transient overexpression of
-arrestin on JEG-3 cells
has no effect on the agonist-induced desensitization of the m2
muscarinic receptor unless one of the GRKs is also co-transfected (36).
In fact,
-arrestin overexpression has been shown to enhance the
agonist-induced desensitization of the
2AR only in
stably transfected Chinese hamster ovary cells expressing large amounts of
2AR (37). On the other hand, the possibility of an
agonist-promoted but arrestin-independent pathway for rLHR-uncoupling
cannot be completely dismissed, as there are studies that demonstrate
that the GRK-catalyzed phosphorylation of rhodopsin can partially
inhibit G protein coupling in the absence of arrestin (38-40), and
there is at least one study documenting that the GRK-catalyzed
phosphorylation of the
2AR can also directly inhibit G
protein coupling in the absence of arrestin (41).
When taken together, the effects of arrestin overexpression on the agonist-induced uncoupling (Table IV) and internalization (Fig. 4) of the rLHR-wt, as well as the effects of individual Ser mutations on agonist-induced uncoupling (Fig. 2 and Table II) and internalization of the rLHR (Fig. 2 and Table III) are consistent with the following two models.
Model 1 assumes that Ser635, Ser639, and
Ser649 are involved in the interaction of the rLHR with
arrestin, but the role of Ser649 in this interaction is
more important than that of Ser635 and Ser639.
This model also assumes that arrestin is involved in the
agonist-induced internalization of the rLHR but not in the
agonist-induced uncoupling of the rLHR. Model 1 accommodates all the
findings presented here. Thus, the mutation of Ser635,
Ser639, or Ser649 slows down internalization,
but the effect of S649A mutation is more pronounced than those of the
S635A or S639A mutation (Fig. 3 and Table III). Furthermore, arrestin-3
overexpression does not enhance the internalization of rLHR-S649A to
the same level as that detected with rLHR-wt or rLHR-S639A (Fig. 4)
because the S649A mutation reduces the binding affinity for arrestin.
Because Ser635, Ser639, and Ser649
are all involved in the receptor-arrestin interaction, Model 1 is also
consistent with the finding that the dominant-negative -arrestins
inhibit the internalization of rLHR-wt, rLHR-S639A, and rLHR-S649A
(Fig. 4). The lack of effect of the S649A mutation and the effect of
the S635A and S639A mutations on agonist-induced uncoupling (Table II)
are accommodated by the assumption that uncoupling is
arrestin-independent. This assumption is, in turn, supported by our
inability to demonstrate an effect of arrestin-3 or dominant-negative
-arrestins on the agonist-induced cAMP accumulation (Table IV) under
conditions similar to those used to detect effects on agonist-induced
internalization (Fig. 4).
Model 2 assumes that Ser635 and Ser639 are
involved in the interaction of the rLHR with arrestin, and
Ser649 is involved in the interaction of the rLHR with
another protein (protein X). This model also assumes that the
agonist-induced uncoupling of the rLHR is mediated by arrestin, and the
agonist-induced internalization of the rLHR is mediated by arrestin and
protein X. The attenuation of agonist-induced uncoupling detected in
rLHR-S635A and rLHR-S639A and the lack of effect of rLHR-S649A (Table
II) can thus be explained by a reduced interaction of rLHR-S635A and rLHR-S639A with arrestin and a normal interaction of rLHR-S649A with
arrestin. Conversely, the strong inhibition of internalization detected
in rLHR-S649A and the partial effect of rLHR-S635A and rLHR-S639A (Fig.
3 and Table III) can be explained by a reduced interaction with protein
X (rLHR-S649A) and a reduced interaction with arrestin (rLHR-S635A and
rLHR-S639A). In this model, arrestin-3 overexpression does not enhance
the internalization of rLHR-S649A to the same level as that detected
with rLHR-wt or rLHR-S639A (Fig. 4), because the S649A mutation is
predicted to reduce the binding of rLHR to protein X, and the
interaction of the rLHR with both protein X and with arrestin is needed
for internalization. The inhibitory effect of dominant-negative
-arrestins on the internalization of rLHR-wt, rLHR-S639A, and
rLHR-S649A (Fig. 4) can also be accommodated by the assumption that the
rLHR-arrestin interaction is partially responsible for internalization.
What Model 2 cannot explain, however, is the lack of effect of
arrestin-3 and a dominant-negative
-arrestin on agonist-induced cAMP
accumulation (Table IV). If this paradigm measures uncoupling (see
above), then Model 2 predicts an inhibitory effect of arrestin-3 and a stimulatory effect of the dominant-negative
-arrestin on hCG-induced cAMP accumulation.
Experiments are now being planned to directly measure the interaction of the rLHR (and mutants thereof) with arrestin and to search for other proteins that may interact with the C-terminal tail of the rLHR and affect its internalization. We will not be able to differentiate between the two models described above until one or both of these strategies are successful. Regardless of which of the two models proposed above is correct, it is clear that the agonist-induced uncoupling and internalization of the rLHR are affected by mutations of different serine residues. This finding mirrors the results presented in a recent publication on the m2 muscarinic receptor, in which it was concluded that the agonist-induced uncoupling and internalization of this GPCR are mediated by the distinct Ser/Thr clusters present in the third intracellular loop (42). Although the study on the m2 receptor did not define the importance of individual Ser/Thr residues, it clearly showed that the phosphorylation of only one of the two clusters promotes uncoupling, whereas the phosphorylation of both clusters is needed to promote agonist-induced internalization. Thus, the data from these two studies, conducted with different GPCRs, lead to basically the same conclusion, that the agonist-induced uncoupling and internalization are mediated by distinct Ser/Thr residues.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank JoEllen Fabritz and Ann Martin for expert technical assistance and other members of the Ascoli laboratory for helpful suggestions throughout the course of these studies. We also thank Dr. Deborah L. Segaloff for critically reading the manuscript, Allen Spiegel (National Institutes of Health) for the KEE antibody, Larry Donoso (Wills Eye Hospital) for the F4C1 antibody, and Sandra Schmid (Scripss Research Institute) for the dynamin-K44A plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA-40629 (to M. A.) and GM-47417 (to J. L. B.). The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by National Institutes of Health Grant DK-25295.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship (Fundaçao De Amparo A Pesquisa Do Estado De São Paulo, 96/1454-8) from the State of Sao Paulo (Brazil).
¶ Supported by Training Grant DK-07018 from the National Institutes of Health.
Partially supported by a fellowship from the Lalor
Foundation.
Supported by Training Grant DK-07705 from the National
Institutes of Health.
§§ Established Investigator of the American Heart Association.
¶¶ To whom correspondence should be addressed: Dept. of Pharmacology, 2-512 BSB, The University of Iowa, Iowa City, IA 52242-1109. Tel.: 319-335-9907; Fax: 319-335-8930; E-mail: mario-ascoli{at}uiowa.edu.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; LHR, lutropin/choriogonadotropin receptor;
rLHR, rat LHR; wt, wild-type; GRK, G protein-coupled receptor kinase;
LH, lutropin; CG, choriogonadotropin; hCG, human CG;
2AR,
2-adrenergic receptor; oLH, ovine
lutropin; HA, hemagglutinin.
2 M. d. F. M. Lazari and M. Ascoli, unpublished observations.
3 All data were combined because no differences were noted when oLH or hCG were used.
4 Immunoprecipitation experiments (not shown) using 293L(wt-12) or 293L(wt-17) cells metabolically labeled with [35S]methionine showed that Bugs, a polyclonal antibody to the rLHR used in previous phosphorylation experiments (5, 6, 8, 9), can immunoprecipitate equivalent amounts of receptor from cells that had been preincubated with buffer only, hCG, or oLH. Similar experiments done with some of the newer antibodies (antiL and RO2; see under "Materials and Methods") showed that these antibodies immunoprecipitate equivalent amounts of receptor from [35S]methionine-prelabeled cells that had been preincubated with buffer only or oLH. The amount of receptor immunoprecipitated from cells preincubated with hCG was somewhat lower, however.
5
In agreement with other studies (31),
preliminary experiments (not presented) performed with -arrestin or
arrestin-3 showed that these two enhanced the internalization of hCG to
the same extent. Most of the experiments presented here were done using arrestin-3.
6 Complete dose-response curves for the effects of hCG on cAMP accumulation in the transiently transfected cells were not obtained. It should be noted, however, that in the case of the rLHR, agonist-induced uncoupling is due mostly (or entirely) to a decrease in the maximal cAMP response, rather than to changes in the sensitivity of this response (9). As such, the use of a single, maximally effective concentration of hCG is a reliable indicator of uncoupling (7, 9).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|