Identification of Phosphorylation Sites in the G Protein-Coupled Receptor for Parathyroid Hormone. Receptor Phosphorylation Is Not Required for Agonist-Induced Internalization

Nicole Malecz, Tom Bambino, Margaret Bencsik and Robert A. Nissenson

Endocrine Research Unit Veterans Administration Medical Center and the Departments of Medicine and Physiology University of California San Francisco, California 94121


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G protein-coupled receptors (GPCRs) comprise a large group of intrinsic membrane proteins that are responsible for the transduction of extracellular signals to the interior of the cell. After agonist binding, the magnitude and duration of receptor signaling are tightly controlled by acute regulatory processes that include uncoupling of the receptor from its cognate G protein (desensitization) and translocation of the receptor from the plasma membrane to endocytic vesicles (internalization). The mechanisms underlying these regulatory processes have been most extensively studied for rhodopsin and the ß-adrenergic receptor (ß-AR). In both cases, perturbation of receptor regulation is associated with pathological effects in vivo, highlighting the physiological importance of these processes (1, 2, 3, 4). It is almost certain that regulation of other GPCRs is likewise physiologically significant, and it is therefore crucial to evaluate the extent to which mechanistic models based on studies with rhodopsin and the ß-AR are broadly applicable to the GPCR superfamily.

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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of Sites of Agonist-Stimulated Phosphorylation in the PTH Receptor
Initial 32P-labeling studies were carried out in human embryonic kidney (HEK)-293 cells expressing the wt PTH receptor. As previously described (18), basal phosphorylation of the receptor was detectable, and the level of phosphorylation was increased approximately 3-fold by treatment of cells with PTH. Phosphoamino analysis of the immunoprecipitated receptor revealed that phosphorylation occurred exclusively on serine residues, both basally and after treatment with PTH (Fig. 1Go).



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Figure 1. Phosphoamino Acid Analysis of 32P-Labeled wt PTH Receptor

The receptor was metabolically labeled with [32P]orthophosphoric acid, immunoprecipitated, and processed as described in Materials and Methods. The cellulose TLC plates were exposed to Kodak X-ray film at -80 C. The upper panels show receptor phosphoserine residues from cells treated with 1 µM PTH for 10 min before immunoprecipitation. The lower panels show receptor phosphoserine residues from untreated cells. The autoradiograms on the right were exposed for a shorter time; Cerenkov counting indicated that PTH treatment produced approximately a 3-fold increase in receptor phosphoserine content. The autoradiograms on the left were overexposed to emphasize the absence of phosphotyrosine and phosphothreonine. The positions of the phosphoamino acid markers are indicated.

 
These results suggested a possible mutagenesis strategy for identifying sites of receptor phosphorylation. Some serine/threonine kinases are capable of phosphorylating either serine or threonine in a given sequence context. If a mutated PTH receptor, bearing a serine to threonine transversion, contains phosphothreonine upon phosphoamino acid analysis, this would strongly suggest that the corresponding serine residue is a phosphoacceptor site in the wt receptor. Such an approach has been successfully used by Giannini et al. (21) to identify phosphorylation sites in the C5a receptor. As a guide to choosing the appropriate serine residues for mutagenesis, we took advantage of previous results from our laboratory concerning the effects of C-terminal truncation on receptor phosphorylation. Deletion of all but the proximal 16 amino acids from the cytoplasmic tail produced a receptor (T474) that was not phosphorylated either basally or in response to PTH (19), suggesting that the phosphorylated residues lie distal to residue 474 in the cytoplasmic tail. However, two other truncation mutants of the receptor, T494 and T507, did show phosphorylation. In an attempt to identify the sites of phosphorylation, serine residues proximal to position 507 were targeted for mutagenesis (Fig. 2Go). 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. 3Go). 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.



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Figure 2. Schematic Drawing of the wt PTH Receptor

The proximal portion of the wt PTH receptor’s cytoplasmic tail with all the potential phosphorylation sites are shown.

 


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Figure 3. Phosphoamino Acid Analysis of 32P-Labeled PTH Receptor Mutants

Individual serine residues in the cytoplasmic tail of the PTH receptor were replaced by threonine residues, and each mutant receptor was expressed in HEK-293 cells. In the S(483–489)T mutant all serine residues between position 483 and 489 were changed to threonine. Cells were metabolically labeled with [32P]orthophosphoric acid for 2 h and incubated for 10 min either without (basal) or with 1 µM PTH (+PTH). After immunoprecipitation the receptor was subjected to phosphoamino acid analysis as described in Materials and Methods. The phosphoserine and phosphothreonine signals are shown.

 
In an attempt to create a phosphorylation-deficient receptor, two alanine mutants were studied, one in which all serine residues between position 467 and 498 [S(467–498)A] and another in which all serine residues between positions 483 and 498 [S(483–498)A] were converted to alanine. When expressed in HEK-293 cells, both of these mutant receptors lacked detectable phosphorylation both basally and in response to PTH (Fig. 4Go). 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. 5AGo). 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. 5BGo). 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.



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Figure 4. Phosphorylation of the wt and Mutant PTH Receptors Expressed in HEK-293 Cells

The mutants tested were S(467–498)A, a mutant in which all serine residues between positions 467 and 498 were converted to alanine, and S(483–498)A, a mutant in which all serine residues between positions 483 and 498 were converted to alanine. Phosphorylation was carried out in cells treated without (basal) or with 1 µM PTH, as described in Materials and Methods. After labeling, the receptors were immunoprecipitated and evaluated by SDS-PAGE. The mol wt of the PTH receptor is indicated.

 


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Figure 5. Comparison of wt and Phosphorylation-Deficient PTH Receptors in HEK-293 Cells

A, Estimates of functional levels of receptor expression and of receptor affinity determined by Scatchard analysis of competitive ligand binding data, as described in Materials and Methods. B, Dose-response curves for bPTH(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 )-stimulated cAMP production, and the resulting EC50 values. Data are expressed as the mean ± SEM of triplicate samples. The results are representative of three independent experiments.

 
Investigation of the Involvement of Known GRKs in PTH Receptor Phosphorylation
Two approaches were taken to explore the possible role in PTH receptor phosphorylation of GRKs known to be important for other GPCRs (8). First, GRKs 2 or 3 were overexpressed in HEK-293 cells stably expressing the wt PTH receptor. The cDNAs encoding the GRKs were subcloned into an episomal vector (pCEP4) and transfected into HEK-293 cells; successfully transfected cells were selected in the presence of hygromycin, as described in Materials and Methods. Under these conditions, overexpression of GRK2 was readily apparent by Western blotting (Fig. 6AGo, 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. 6AGo). 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. 6BGo). 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).



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Figure 6. Effects of GRK2 on PTH Receptor Phosphorylation in HEK-293 Cells

A, Cells stably transfected with the wt PTH receptor alone or with the wt PTH receptor and either GRK2 or a DN-GRK2 were metabolically labeled with [32P]orthophosphoric acid either without (basal) or in the presence of 1 µM PTH. Receptors were immunoprecipitated and then separated by SDS-PAGE, and the respective bands were excised and phosphorylation was quantified by Cerenkov counting. The inset shows a Western blot performed with a polyclonal GRK2 antibody in untransfected cells (lane 1), and in cells transfected with a plasmid-encoding GRK2 (lane 2) or DN-GRK2 (lane 3). B, Phosphorylation of wt and mutant PTH receptors in cells overexpressing GRK2. The cells were metabolically labeled with [32P]orthophosphoric acid, after which the PTH receptor was immunoprecipitated and separated by SDS-PAGE, and autoradiograms were developed. The mol wt of the PTH receptor is indicated.

 
The results of the above studies suggest a possible role for GRK2 in PTH receptor phosphorylation. In the second approach, we used a C-terminal fragment of GRK2 that has previously been shown to function as a dominant inhibitor of GRK2 catalytic activity (22). A cDNA encoding this dominant-negative (DN) GRK2 was subcloned into pCEP4 and was transfected into HEK-293 cells stably expressing the wt PTH receptor. Western blotting confirmed successful expression of the DN-GRK2 (Fig. 6Go, inset). As shown in Fig. 6AGo, 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. 7AGo). 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.



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Figure 7. Internalization of PTH Receptors in HEK-293 Cells

A, Time course for agonist-stimulated internalization of the wt PTH receptor and phosphorylation-deficient PTH receptor mutants. B, Effect of hypertonic sucrose on PTH receptor internalization. HEK-293 cells expressing wt PTH receptors or phosphorylation-deficient PTH receptor mutants were incubated in the absence or presence of 0.45 M sucrose (to disrupt clathrin lattices), and internalization was measured after 10 min of exposure to [125I]PTHrP. C, Lack of effect of overexpression of GRK2 on agonist-stimulated internalization of the wt PTH receptor. Internalization was assessed as non-acid-extractable radioligand uptake (see Materials and Methods). Similar results were obtained in three independent experiments each with triplicate data points.

 
Disruption of clathrin lattices by treatment of HEK-293 cells with hypertonic sucrose inhibited the internalization of both the wt and phosphorylation-deficient PTH receptors by 60–70% (Fig. 7BGo). 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. 7CGo). Although the PTH receptor in GRK2 overexpressing cells was hyperphosphorylated (Fig. 6AGo), the initial rate and extent of receptor internalization were unaffected.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endocytosis of GPCRs in response to agonist binding is a widespread phenomenon that is important in the down-regulation of responsiveness that accompanies chronic exposure to agonist. For most GPCRs, endocytosis appears to occur through a classic clathrin-coated pit mechanism (24, 25), although a few exceptions have been noted (e.g. 26). At least two structural features in the cytoplasmic domain of GPCRs directly contribute to receptor endocytosis. First, the cytoplasmic domain of some GPCRs contains sequences (e.g. Y-X-X-hydrophobic; L-L) that mediate the interaction of the receptor with clathrin-associated adaptor proteins (27, 28). Second, agonist-stimulated phosphorylation of the cytoplasmic domain of GPCRs can facilitate their association with clathrin-coated pits. In the case of the ß-AR, agonist binding leads to phosphorylation of the receptor by a GRK, which in turn facilitates the association of an arrestin protein with the receptor. Evidence suggests that the arrestin-phosphoreceptor complex then associates with clathrin-coated pits through the direct binding of arrestin to clathrin (9). However, not all GPCRs depend on agonist-induced phosphorylation for their internalization: a cholecystokinin receptor with a truncated cytoplasmic tail internalized normally (29), as did a secretin receptor with a truncation that eliminated phosphorylation (30). A limitation of these studies is the use of truncation to eliminate the sites of phosphorylation, since truncation may also induce conformational changes in the receptor, thereby altering its accessibility or its affinity for the endocytic machinery.

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{gamma}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Bovine 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) and 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) were obtained from Bachem (Torrance, CA). Human embryonic kidney (HEK) 293 cells and the expression vector pCEP4 were obtained from Invitrogen (San Diego, CA). Carrier-free [32P] orthophosphoric acid was obtained from New England Nuclear Corp. (Boston, MA). All oligonucleotides were synthesized and purified at University of California San Francisco Biomolecular Resource Facility.

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
 
We thank Dr. Jeffrey Benovic for cDNAs for GRK2 and 3 and Dr. Robert J. Lefkowitz for the minigene of the carboxyl terminus of GRK 2.


    FOOTNOTES
 
Address requests for reprints to: Robert A. Nissenson, Endocrine Research Unit, Department of Medicine, University of California, San Francisco, Veterans Affairs Medical Center, 4150 Clement Street (111N), San Francisco, California 94121-1598. E-mail: Chicago{at}itsa.ucsf.edu

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.


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 RESULTS
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 MATERIALS AND METHODS
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