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Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling by ß-Arrestin2

Division of Bone and Mineral Metabolism Harvard-Thorndike and Charles A. Dana Research Laboratories Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts 02215

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting ß-arrestin2. We examined the role of ß-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a ß-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ßarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of ß-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted ß-arrestin2-mediated endocytosis of both PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of ß-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of ß-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated ß-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with ß-arrestin2 on the cell membrane, possibly leading to uncoupling from G_s. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.

	INTRODUCTION

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PTH is a major regulator of serum calcium homeostasis and bone metabolism (1). PTH-related protein (PTHrP), first described as the hormone responsible for hypercalcemia of malignancy, is now recognized as an autocrine/paracrine factor with various biological functions in many tissues (2). Both PTH and PTHrP bind to and activate the PTH subtype 1 receptor (PTH1Rc), a member of the class II subfamily of G protein-coupled receptors (GPCRs), coupled to both G_s and G_q proteins (3). Whereas the specific biological role of the G_q-protein kinase C (PKC) signaling pathway is, at present, unclear, the cAMP-protein kinase A (PKA) signaling pathway seems to be responsible for the majority of the calciotropic and skeletal actions of PTH and PTHrP (Ref. 4 and references therein). Little is known, however, regarding the cellular and molecular mechanisms that regulate PTH1Rc activation.

Point mutations that constitutively activate the PTH1Rc, namely a heterozygous His-to-Arg mutation at position 223 (H223R) situated at the boundary between the PTH1Rc first intracellular loop and second transmembrane domain (5) and a heterozygous Thr-to- Pro mutation at position 410 (T410P) situated in the sixth transmembrane domain (6), have recently been identified. These PTH1Rc mutations are responsible for Jansen’s metaphyseal chondrodysplasia, a rare autosomal dominant disorder characterized by short-limbed dwarfism and hypercalcemia, despite normal PTH and PTHrP levels (5, 6). Transient transfection of Jansen’s PTH1Rc mutants into COS-7 cells is accompanied by a 2- to 8-fold increase in ligand-independent cAMP accumulation when compared with wild-type PTH1Rc (5, 6, 7, 8). Interestingly, a number of observations in vitro as in vivo suggest that cAMP signaling by H223R and T410P mutants becomes desensitized (7, 8, 9). Clarifying the molecular mechanisms responsible for the desensitization of G_s-mediated signaling by constitutively active PTH1Rcs provides a unique opportunity to gain insights into the regulation of PTH and PTHrP activity (4), and, more broadly, of constitutively active GPCRs.

ß-Arrestins are a family of cytoplasmic adaptor molecules that play a key role in G protein uncoupling (desensitization) and endocytosis of agonist-activated GPCRs (10, 11). We have recently demonstrated that agonist-human PTH1Rc complexes are internalized via clathrin-coated vesicles in association with ß-arrestin2 and that this mechanism is dependent on PKC activation (12). Indeed, the protein kinase inhibitor, staurosporine, inhibited ßarrestin2-mediated endocytosis of fluorescent agonists bound to the hPTH1Rc (12) and increased cAMP accumulation in response to PTH (12, 13). These observations led us to hypothesize that the cellular trafficking and cAMP signaling by constitutively active human PTH1Rcs could be directly regulated by ß-arrestins. In particular, we wanted to examine whether the constitutive activity of Jansen’s receptor mutants might result from impaired interaction with ß-arrestins and/or defective receptor internalization. To test these hypotheses, we studied cAMP accumulation by both H223R and T410P mutant as well as wild-type PTH1Rcs after overexpression of a functional ß-arrestin2-green fluorescent protein conjugate (ß-Arr2-GFP) (14). Using GFP-PTH1Rc conjugates (15) and fluorescencelabeled PTH- and PTHrP-derived agonists and antagonists (12), we were able to monitor independently the cellular trafficking of constitutively active PTH1Rc mutants, their ligands, and ß-Arr2-GFP by real-time fluorescence microscopy in living cells. By correlating the level of expression and cellular localization of the mutant receptors and ß-Arr2-GFP with their G_s-adenylyl cyclase activity, we have delineated an essential molecular mechanism regulating cAMP signaling by the human PTH1Rc.

	RESULTS

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Effects of ß-Arrestin2 on cAMP Signaling by Wild-Type and Constitutively Active Mutant PTH1Rcs
To evaluate the influence of ß-arrestin2 on cAMP signaling, a functional ß-Arr2-GFP conjugate (14) was coexpressed with either wild-type, H223R, or T410P constitutively active mutant human PTH1Rcs in COS-7 cells. Expression of ß-Arr2-GFP protein was verified by measuring GFP fluorescence in whole-cell extracts 24 h after transfection and increased proportionally to the amount of cDNA transfected (Fig. 1A). PTH1Rc expression was evaluated by Scatchard analysis of radiolabeled ¹²⁵I-PTH-(1–34) binding and did not significantly differ between cells cotransfected with ß-Arr2-GFP or control plasmid cDNA (data not shown). Moreover, the percentage of cells expressing the PTH1Rcs, as evaluated by fluorescence microscopy using a rhodamine-labeled antagonist ligand (see below), was similar in cell preparations cotransfected with the lowest and highest amount of ß-Arr2-GFP cDNA (64% ± 9% vs. 54% ± 7% of cells expressing PTH1Rcs, with 0.1 and 0.8 µg ß-Arr2-GFP cDNA/well, respectively).

View larger version (36K): [in this window] [in a new window]   Figure 1. Desensitization of PTH-Stimulated cAMP Signaling by ß-Arrestin2 in COS-7 Cells Expressing Wild-Type PTH1Rc COS-7 cells were cotransfected with wild-type PTH1Rc and either ß-Arr2-GFP (closed bars and symbols) or a control plasmid (open bars and symbols), as described in Materials and Methods. cAMP accumulation was measured in the presence of IBMX (1 mM). A, ß-Arr2-GFP expression in cells transfected with increasing amounts of ß-Arr2-GFP cDNA was measured by spectrofluorimetry, as described in Materials and Methods. B, cAMP accumulation in cells transfected with increasing amounts of ß-Arr2-GFP or control plasmid cDNA, as indicated, and stimulated with PTH-(1–34) (100 nM) for 30 min. C and D, Kinetics of cAMP accumulation in response to PTH-(1–34) (100 nM) (C) or forskolin (10 µM) (D) in cells transfected with ß-Arr2-GFP or control plasmid cDNA (0.4 µg/well). cAMP accumulation is expressed as the percentage of maximal cAMP levels measured in the controls after 30 min [i.e. panel B, 16,452 ± 1,133 cpm/well; panel C, 14,519 ± 1,391 cpm/well; panel D, 11,851 ± 998 cpm/well]. Each point represents the mean of three separate measurements, with error bars denoting SEM. (The experiments were repeated three times with similar results.)

The ligand-independent, constitutive cAMP accumulation in COS-7 cells expressing T410P and H223R mutants was, respectively, 2.9 ± 0.1- and 5.5 ± 0.2-fold higher compared with wild-type PTH1Rc (mean ± SEM of three to five separate experiments performed in triplicate). Coexpression of ß-Arr2-GFP inhibited constitutive cAMP accumulation by H223R and T410P receptors in a dose-dependent manner (Fig. 2A). This inhibition resulted from a slower rate of cAMP accumulation (Fig. 2B). The maximal cAMP accumulation in response to PTH-(1–34) (100 nM) was comparable for mutant and wild-type PTH1Rcs, although the relative increase in cAMP accumulation above baseline was markedly blunted in cells expressing H223R and T410P (Fig. 2C), in agreement with previous observations (7, 8). Similar to wild-type receptors, ß-Arr2-GFP overexpression also significantly decreased the maximal cAMP accumulation by both mutant receptors in response to PTH (Fig. 2C).

View larger version (31K): [in this window] [in a new window]   Figure 2. Desensitization of Ligand-Independent and PTH-Stimulated cAMP Signaling by ß-Arrestin2 in COS-7 Cells Expressing Constitutively Active PTH1Rc Mutants COS-7 cells were cotransfected with constitutively active PTH1Rc mutants, H223R (left hand panels) or T410P (right hand panels), and either ß-Arr2-GFP (closed bars and symbols) or control plasmid (open bars and symbols) cDNA, as described in Materials and Methods. A, Ligand-independent, constitutive cAMP accumulation after 30 min incubation with IBMX (1 mM) in cells transfected with increasing amounts of ß-Arr2-GFP or control plasmid cDNA, as indicated. B, Kinetics of constitutive cAMP accumulation in cells transfected with ß-Arr2-GFP or control plasmid cDNA (0.4 µg/well) and incubated with IBMX (1 mM) for up to 30 min, as indicated. C, Kinetics of agonist-stimulated cAMP accumulation in cells transfected as above. In this case, cells were incubated 30 min with IBMX (1 mM), followed by PTH-(1–34) (100 nM) for various periods of time, as indicated. Data are expressed as percentage of the maximal cAMP levels measured in the controls at the end of each experiment [i.e. panel A (left), 6,711 ± 492 cpm/well; panel A (right), 3,072 ± 147 cpm/well; panel B (left), 6,474 ± 479 cpm/well; panel B (right), 2,658 ± 25 cpm/well; panel C (left), 9,026 ± 502 cpm/well; panel C (right), 8,268 ± 298 cpm/well]. Each point represents the mean of three separate measurements, with error bars denoting SEM. (The experiments were repeated three times with similar results.)

Distribution of ß-Arr2-GFP in HEK-293T Cells Expressing Constitutively Active PTH1Rc Mutants
We have previously reported that in HEK-293T cells stably expressing the human (h) PTH1Rc, receptor activation by PTH triggers the recruitment of ß-arrestin2 to the cell surface, followed by endocytosis of agonist-hPTH1Rc complexes associated with ß-arrestin2 (12). We hypothesized that ß-arrestin2 could inhibit cAMP signaling by interacting with the constitutively active PTH1Rc mutants on the cell membrane even in the absence of agonist stimulation. For this purpose, the distribution of ß-Arr2-GFP was monitored by real-time fluorescence microscopy in HEK-293T cells cotransfected with either the H223R or T410P mutants. As shown in Fig. 3, ß-Arr2-GFP was spontaneously recruited to the cell surface in cells expressing H223R or T410P (Fig. 3, a and d). This pattern differed markedly from cells expressing the wild-type PTH1Rc, in which ß-Arr2-GFP was uniformly diffused in the cytoplasm (Fig. 3g) and was mobilized to the cell membrane only after stimulation with the agonists PTH-(1–34) (Fig. 3h) or PTHrP-(1–36) (10–100 nM). Agonist stimulation further promoted redistribution of ß-Arr2-GFP from the cell membrane to the cytoplasm in cells expressing H223R and T410P as well as wild-type PTH1Rcs (Fig. 3, b, e, and i). In contrast, exposure to the antagonists Bpa2-PTHrP-(1–36) and PTH-(7–34) (both at 100–1,000 nM) was followed by a rapid redistribution of ß-Arr2-GFP from the cell membrane to the cytoplasm only in cells expressing T410P (Fig. 3f), but not H223R (Fig. 3c) or the wild-type PTH1Rcs (not shown; Ref. 12).

View larger version (102K): [in this window] [in a new window]   Figure 3. Fluorescence Localization of ß-Arr2-GFP in HEK-293T Cells Expressing Mutant PTH1Rcs HEK-293T cells were cotransfected with mutants H223R (a–c, and j), T410P (d–f, k, and l) or wild type (g–i) PTH1Rcs and ß-Arr2-GFP. Distribution of ß-Arr2-GFP was monitored at 37 C by real-time fluorescence microscopy as described in Materials and Methods. With the H223R mutant, ß-Arr2-GFP was mobilized to the cell surface in the absence of agonist stimulation (a), and redistributed to the cytoplasm within 10 min after stimulation by PTH-(1–34) (100 nM) (b), but not with the antagonist Bpa2-PTHrP-(1–36) (1000 nM) (c). Similar observations were made with the T410P mutant before (d) and after (e) agonist stimulation, whereas Bpa2-PTHrP-(1–36) (1000 nM) also translocated ß-Arr2-GFP to the cytoplasm (f). With wild-type PTH1Rcs, ß-Arr2-GFP was uniformly diffused in the cytoplasm in the absence of agonist stimulation (g), mobilized to the cell surface (h), and further retranslocated to the cytoplasm (i) within 15 min after stimulation with PTH-(1–34) (100 nM). Staurosporine (2 µM, 30 min) did not alter the ligand-independent recruitment of ß-Arr2-GFP to the surface of cells expressing H223R or T410P (j and k, respectively), but, in the latter, prevented ß-Arr2-GFP remobilization to the cytoplasm in response to Bpa2-PTHrP-(1–36) (1,000 nM) (l). Magnification, x100–200.

Colocalization of Fluorescent Ligands with ß-Arr2-GFP in Cells Expressing Constitutively Active PTH1Rc Mutants
We next examined the endocytosis of specific fluorescent agonist and antagonist ligands (12) in HEK-293T cells coexpressing PTH1Rc mutants with ß-Arr2-GFP. The agonist Rho-PTH-(1–34) was rapidly internalized and colocalized with ß-Arr2-GFP intracellularly in cells expressing H223R (Fig. 4, a–c) and T410P (not shown), similarly to wild-type PTH1Rcs (12). The antagonist Rho-Bpa2-PTHrP-(1–36) colocalized with ß-Arr2-GFP on the cell membrane but was not rapidly internalized in cells expressing H223R (Fig. 4, d–f). Rho-Bpa2-PTHrP-(1–36) also remained localized on the cell surface in cells expressing wild-type PTH1Rcs, but did not colocalize with ß-Arr2-GFP (Fig. 4, g–i). In contrast, in cells expressing the T410P mutant a rapid redistribution of Rho-Bpa2-PTHrP-(1–36) to the cytoplasm in association with ß-Arr2-GFP was observed (Fig. 4, j–l).

View larger version (23K): [in this window] [in a new window]   Figure 4. Cellular Trafficking of Fluorescent PTH1Rc Ligands with ß-Arr2-GFP HEK-293T cells cotransfected with H223R (a–f), wild-type (g–i), or T410P (j–o) PTH1Rcs and ß-Arr2-GFP were incubated with rhodamine-labeled ligands (100 nM) and continuously monitored at 37 C for 15 min as described in Materials and Methods. In cells expressing H223R, the agonist Rho-PTH-(1–34) was internalized within 15 min (a), ß-Arr2-GFP was redistributed into the cytoplasm (b), and the two fluorescences colocalized intracellularly (yellow spots on the overlay image) (c). In contrast, the antagonist Rho-Bpa2-PTHrP-(1–36) remained bound on the cell surface (d), where clusters of ß-Arr2-GFP were also detectable (e) and colocalized with the fluorescent ligand (yellow) (f). With wild-type PTH1Rc, the antagonist Rho-Bpa2-PTHrP-(1–36) remained bound linearly to the cell surface (g), whereas ß-Arr2-GFP was evenly distributed in the cytoplasm (h). The overlay image shows the distinct distribution of the fluorescent ligand and ß-Arr2-GFP in these cells (i). With T410P, Rho-Bpa2-PTHrP-(1–36) was rapidly internalized (j), ß-Arr2-GFP was redistributed to the cytoplasm (k) where it colocalized with the fluorescent ligand (yellow spots) (l). In these cells, staurosporine (2 µM, 30 min) markedly decreased Rho-Bpa2-PTHrP-(1–36) internalization (m) and ß-Arr2-GFP redistribution to the cytoplasm (n) while maintaining their colocalization on the cell surface (yellow) (o). Magnification, x100.

Cellular Trafficking of GFP-PTH1Rc Conjugates
To directly monitor by real-time fluorescence microscopy the cellular distribution of constitutively active mutant PTH1Rcs, GFP-receptor conjugates [H223R-GFP and T410P-GFP] were generated and transiently transfected in HEK-293T cells. Cell surface receptor expression as well as constitutive and PTH-stimulated activity were verified by, respectively, radioligand binding and cAMP assays and were similar to those of the original receptor mutants (data not shown). A human wild-type PTH1Rc-GFP (generously provided by Dr. Caroline Silve, INSERM, Paris) was also used and has been characterized previously (15).

In transiently transfected HEK-293T cells, H223R-GFP was clearly localized on the cell membrane, and an intense fluorescent signal was also detected in the vicinity of the nucleus (Fig. 5, a and d). A similar distribution was observed in cells expressing wild-type PTH1Rc-GFP (Fig. 5 g). The intracellular localization of receptor-GFP conjugates is compatible with the presence of a PTH1Rc pool in a Golgi compartment, as previously observed by indirect fluorescence microscopy (12). In contrast, in cells transfected with T410P-GFP, in addition to the intracellular receptor pool, green fluorescence was mainly detectable as multiple dots localized beneath the cell surface and within the cytoplasm (Fig. 5 j). In keeping with the radioligand internalization assays (Table 2), the fluorescent agonist Rho-PTH-(1–34) was rapidly internalized in association with H223R-GFP (Fig. 5, b and c), T410P-GFP, and wild-type PTH1Rc-GFP (not shown). The fluorescent antagonist Rho-Bpa2-PTHrP-(1–36) colocalized with H223R-GFP and wild-type PTH1Rc-GFP on the cell surface, with no evidence of endocytosis of the ligand-receptor complex after 15 min at 37 C (Fig. 5, e, f, h, and i). In contrast, Rho-Bpa2-PTHrP-(1–36) initially colocalized with T410P-GFP on the cell surface (Fig. 5 k), and subsequently in the cytoplasm (Fig. 5 l).

View larger version (26K): [in this window] [in a new window]   Figure 5. Cellular Trafficking of PTH1Rc-GFP Conjugates and Colocalization with Their Fluorescent Ligands HEK-293T cells were transfected with H223R (a–f), wild type (g–i), or T410P (j–o) PTH1Rcs-GFP conjugates and their cellular distribution monitored at 37 C before and after binding of specific fluorescent ligands, as described in Materials and Methods. In the absence of ligand, H223R-GFP was linearly distributed on the cell surface and was also present in an intracellular pool (a). The agonist Rho-PTH-(1–34) at first colocalized with H223R-GFP on the cell membrane (in yellow on the overlay image) (b), followed by internalization of agonist-receptor complexes after 15 min (c). The antagonist Rho-Bpa2-PTHrP-(1–36) also colocalized with M223R-GFP on the cell membrane (yellow) immediately after binding (e), but the ligand-receptor complex was not internalized after 15 min (f). The wild-type PTH1Rc-GFP was similarly distributed before ligand binding (g), and antagonist Rho-Bpa2-PTHrP-(1–36)–PTH1Rc-GFP complexes (yellow) also remained mostly localized on the cell surface immediately (h) and 15 min (i) after binding. In contrast, T410P-GFP was detectable as distinct fluorescent spots throughout the cytoplasm, particularly in the submembranal region, in the absence of ligand (j). Clusters of antagonist Rho-Bpa2-PTHrP-(1–36)-T410P-GFP complexes (yellow) were detectable on the cell surface immediately after binding (k) and intracellularly after 15 min (l). In these cells, staurosporine (2 µM, 30 min) markedly decreased the cytoplasmic distribution and increased the cell surface localization of T410P-GFP in the absence of ligand (m). It also decreased clustering and internalization of Rho-Bpa2-PTHrP-(1–36)-T410P-GFP complexes (yellow) immediately after binding (n) and after 15 min at 37 C (o). Magnification, x100.

Influence of Protein Kinases Activation on Cellular Trafficking and cAMP Signaling of Constitutively Active PTH1Rc Mutants
We have previously reported that the protein kinases inhibitor staurosporine, but not the selective PKA inhibitor H89, markedly decreases ß-arrestin2-mediated internalization of fluorescent agonists bound to the human PTH1Rc and significantly increases cAMP accumulation in response to its cognate agonists (12, 13). We therefore examined the effects of these pharmacological agents on the cellular distribution of constitutively active mutant PTH1Rcs, their ligands, and ß-arrestin2, as well as on cAMP signaling in HEK-293T cells. Treatment with staurosporine (2 µM for 30 min) prevented neither the ligand-independent recruitment of ß-arr2-GFP to the cell membrane in cells expressing H223R or T410P (Fig. 3, j and k), nor its mobilization in response to PTH (and PTHrP) in cells expressing wild-type PTH1Rcs (not shown). However, staurosporine decreased internalization of the fluorescent agonist Rho-PTH-(1–34) associated with ß-Arr2-GFP and with either mutant or wild-type PTH1Rc-GFP (data not shown). In addition, in cells expressing T410P, staurosporine inhibited the rapid redistribution from the cell membrane to the cytoplasm of ß-Arr2-GFP in response to the antagonist Bpa2-PTHrP-(1–36) (Fig. 3 l) as well as of the fluorescent antagonist Rho-Bpa2-PTHrP-(1–36) itself (Fig. 4, m–o). Moreover, staurosporine decreased intracellular trafficking and increased the cell surface localization of T410P-GFP, while inhibiting internalization of Rho-Bpa2-PTHrP-(1–36)-T410P-GFP complexes (Fig. 5, m–o).

Table 3 summarizes the effects of staurosporine on receptor expression and radioligand internalization, as well as constitutive and PTH-stimulated cAMP accumulation in HEK-293T cells expressing mutant and wild-type PTH1Rcs. Thus, staurosporine (2 µM, 30 min) modestly increased the cell surface expression of mutant PTH1Rcs, more significantly so for T410P (in keeping with our observations by fluorescence microscopy, above); it significantly decreased internalization of agonists with all receptor types and of antagonists with T410P; eventually, it significantly increased both constitutive and PTH-stimulated (100 nM) cAMP accumulation (up to 4-fold in cells expressing T410P) compared with untreated cells. In contrast, incubation with the selective PKA inhibitor, H89 (up to 50 µM), did not affect any of the parameters evaluated above.

Role of PTH1Rc C-Terminal Cytoplasmic Domain on ß-Arrestin2 Mobilization and Ligand-Receptor Complex Endocytosis
It has been shown that, upon agonist activation, the PTH1Rc is phosphorylated at multiple sites within its intracellular C-terminal domain (18, 19). Also, PTH1Rc phosphorylation in response to PTH is markedly inhibited by staurosporine (19). To examine whether the C terminus of the human PTH1Rc is involved in the interaction with ß-arrestin2 and the internalization of ligand-receptor complexes, we generated C-terminally-truncated receptor constructs containing only an eight-amino acid residue tail (17) [H223R-del, T410P-del, and wild type PTH1Rc-del]. Cell surface expression levels and maximal PTH-stimulated (100 nM) cAMP accumulation of the wild-type PTH1Rc-del in HEK-293T cells were comparable to intact wild-type PTH1Rcs (as evaluated by, respectively, radioligand binding and cAMP accumulation assays; data not shown). In contrast, neither binding of radioiodinated PTH-(1–34) nor cAMP accumulation in response to PTH-(1–34) was detectable in HEK-293T cells transfected with H223R-del or T410P-del, indicating that these truncated receptor mutants were either not expressed or not functional (data not shown). Accordingly, fluorescence microscopy analysis was performed in HEK-293T cells coexpressing wild-type PTH1Rc-del and ß-Arr2-GFP: upon stimulation with the fluorescent agonist Rho-PTH-(1–34) (100 nM), ß-Arr2-GFP was normally recruited to the cell membrane where it colocalized with the agonist bound to the truncated receptor (Fig. 6, a–c). Rho-PTH-(1–34) was then rapidly internalized (Fig. 6, g–i). In this case however, in contrast to cells expressing intact PTH1Rcs (12), ß-Arr2-GFP rediffused immediately into the cytoplasm and did not remain associated intracellularly with the fluorescent agonist-receptor complex (Fig. 6 d–f).

View larger version (18K): [in this window] [in a new window]   Figure 6. Cellular localization of a Fluorescent Agonist with ß-Arr2-GFP in Cells Expressing a C-Terminally-Truncated Wild-Type PTH1Rc HEK-293T cells cotransfected with a wild-type PTH1Rc lacking the C-terminal cytoplasmic domain and ß-Arr2-GFP were incubated with Rho-PTH-(1–34) (100 nM) and monitored after 2 (a, b, and c) and 15 (d, e, and f) min at 37 C, as described in Materials and Methods. After 2 min, Rho-PTH-(1–34) was bound on the cell surface (a), ß-Arr2-GFP was recruited to the cell membrane (b), and the overlay image shows the colocalization (yellow) of the fluorescent ligand with ß-Arr2-GFP in these cells (c). After 15 min, Rho-PTH-(1–34) was internalized (d), and ß-Arr2-GFP has rediffused into the cytoplasm (e) and was no more colocalized with the fluorescent ligand on the overlay image (f). Magnification, x100.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The goal of this study was to investigate the role of ß-arrestin2 in regulating the cellular distribution and cAMP signaling of constitutively active mutant PTH1Rcs, H223R and T410P, recently identified as the cause of hereditary Jansen’s metaphyseal chondrodysplasia (5, 6, 7). Our study led to four major findings. First, both the constitutive activity of Jansen’s mutant receptors and their responsiveness to PTH (and PTHrP) is dependent on the level of ß-arrestin2 expression (Fig. 2). Similarly, agonist-stimulated cAMP accumulation by the wild-type PTH1Rc is inhibited by increasing expression of ß-arrestin2 (Fig. 1). Second, inhibition of cAMP signaling by the H223R and T410P mutants correlates with a ligand-independent mobilization of ß-arrestin2 to the cell membrane (Figs. 3 and 4). Third, both mutant receptors are internalized by a ß-arrestin2- and clathrin-coated vesicles-mediated process in response to their cognate agonists. However, the cellular distribution of the T410P mutant and its internalization features in response to antagonists are different from those of the H223R mutant and wild-type PTH1Rcs (Fig. 5). Fourth, neither PTH1Rc-mediated activation of protein kinases nor the receptor C-terminal cytoplasmic domain (which contains the major phosphorylation sites) is required for the receptor interaction with ß-arrestin2 on the cell membrane.

Several lines of evidence indicate that the constitutive activity of GPCRs is regulated and their responsiveness to agonists is desensitized (7, 8, 9, 21, 22, 23). Thus, in COS-7 cells transiently transfected with Jansen’s PTH1Rc mutants, ligand-independent cAMP accumulation decreases rapidly after its initial onset, whereas the relative increase of cAMP accumulation in response to PTH is blunted compared with wild-type receptors (6, 7, 8) (Fig. 2B). Moreover, transgenic models suggest that the expression and/or constitutive activity of Jansen’s receptor mutants could be downregulated in vivo (9). In this study we report that an interaction between these mutant receptors and ß-arrestin2 is one of the mechanisms regulating their constitutive activity. The ß-Arr2-GFP conjugate used in this study has previously been shown to be functionally equivalent to wild-type ß-arrestin2 in COS-7 cells (14). Kinetics of PTH-stimulated cAMP accumulation indicate that ß-arrestin2 inhibits cAMP signaling by both constitutively active mutant and wild-type PTH1Rcs through similar mechanisms. ß-Arr2-GFP overexpression had no effect on the activation of adenylyl cyclase by forskolin, suggesting that ß-arrestin2 could directly uncouple activated PTH1Rcs from G_s proteins. Recently, binding of G protein-coupled receptor kinases (GRKs) to the PTH1Rc has been shown to inhibit signaling through G_q (24). However, the acute stimulation of adenylyl cyclase by PTH was only marginally affected (24). Our observations support a distinct role for ß-arrestins in the rapid inhibition of cAMP signaling. Similarly, overexpression of ß-arrestin2 in COS cells expressing the ß2-adrenergic receptor desensitized the response to its cognate agonists (25). Hence, these results provide additional evidence for a common role of ß-arrestins in the regulation of G_s-mediated signal transduction by a number of GPCRs, including ß2-adrenergic receptors (10, 11, 25, 26), m2 muscarinic acetylcholine receptors (27), FSH receptors (28), A_2b adenosine receptor, and PGE₂ receptor (29). It should be noted, however, that other mechanisms such as the PKA-mediated activation of cAMP-phosphodiesterase and the PTH-stimulated up-regulation of regulators of G protein signaling (RGS) may also contribute to this process (30, 31).

We have recently reported that agonist stimulation of wild-type PTH1Rc promotes a rapid relocalization of ß-arrestin2 from the cytoplasm to the cell membrane (12). To support the hypothesis that constitutively active PTH1Rc mutants induce a ligand-independent mobilization of ß-arrestin2, we monitored the cellular localization of ß-Arr2-GFP by fluorescence microscopy in living cells (12, 14). Expression of Jansen’s receptor mutants in HEK-293T cells is indeed associated with a ligand-independent recruitment of ß-Arr2-GFP to the cell membrane (Figs. 3 and 4). Similarly, the _1b adrenergic receptor mutant A293E, which constitutively activates G_q in COS-7 cells, promotes ß-arrestin2 mobilization independently of the agonist, epinephrine, in HEK-293 cells (32). These findings support the emerging idea that desensitization in response to spontaneous receptor activity may occur as a result of ligand-independent interactions between constitutively active GPCRs and ß-arrestins on the cell membrane. Thus, rapid inhibition of G_s-mediated signal transduction and targeting of ligand-PTH1Rc complexes to clathrin-coated vesicles (12), both mediated by ß-arrestin2, may be two chronologically and mechanistically related, but functionally distinct, phenomena.

Two sets of experiments were performed to gain insights into the molecular basis of the interaction between ß-arrestin2 and the PTH1Rc. First, pharmacological inhibition of protein kinases activity by staurosporine [which is known to inhibit phosphorylation of the activated PTH1Rc (19)] did not prevent the constitutive colocalization of Jansen’s receptor mutants with ß-Arr2-GFP on the cell membrane. Second, truncation of the cytoplasmic tail of the wild-type human PTH1Rc [which contains most of the receptor’s phosphorylation sites (17, 18, 19)] did not affect agonist-induced translocation of ß-Arr2-GFP to the cell membrane (Fig. 6). Of note, C-terminal deletions of the rat and opossum PTH1Rc have previously been reported to result in functional receptors (17, 20). Taken together, our data suggest that recruitment of ß-arrestin2 by activated PTH1Rcs is independent from receptor phosphorylation and, more broadly, from the receptor C-terminal cytoplasmic domain. Moreover, a fluorescent agonist was rapidly internalized in cells expressing the Cterminally-truncated, wild-type human PTH1Rc (Fig. 6). In agreement with the latter observation, proximal deletions of the opossum PTH1Rc C terminus decreased, but did not abolish, internalization of agonist radioligands (17). However, we found that in cells expressing PTH1Rc-del, ß-Arr2-GFP did not colocalize with the fluorescent agonist intracellularly, suggesting that the receptor cytoplasmic tail may provide stabilization of ligand-receptor-ß-arrestin2 complexes during endocytosis. Similar conclusions have been drawn regarding the secretin receptor (33, 34) and a chimeric angiotensin II type 1A receptor containing the cytoplasmic tail of the ß2-adrenergic receptor (35).

Using GFP-tagged PTH1Rc mutants and fluorescent agonists and antagonists, we were able to monitor independently the cellular distribution of both constitutively active PTH1Rcs and their ligands by fluorescence microscopy in living HEK-293T cells. Our results indicate important similarities, but also differences, between the H223R and T410P mutants. In the absence of ligand, wild-type and H223R PTH1Rcs are mostly localized on the cell membrane as well as in an intracellular pool. The latter observation is in agreement with our previous data obtained by indirect fluorescence microscopy in HEK cells stably expressing a human C-Tag-PTH1Rc (12). The precise function of this cytoplasmic pool of receptors is presently unknown. Agonist binding to either receptor mutant is followed by rapid and extensive endocytosis, as shown by both fluorescence microscopy and radioligand internalization, and this process can be inhibited by disrupting clathrin-coated lattices with sucrose (Table 2). Therefore, the usual mechanisms of ß-arrestin2-mediated endocytosis of agonist-PTH1Rc complexes previously described (12) appear to be operative with both constitutively active mutant PTH1Rcs. In agreement with these findings, rapid internalization of constitutively active LH/human CG receptor mutants (36, 37) and _1b adrenergic receptor mutants (32) also occurs in response to their cognate agonists.

However, in contrast to the H223R mutant, T410P-GFP was also broadly localized in the cytoplasm of unstimulated HEK-293T cells. Similarly, localization of the constitutively active _1d-adrenoreceptor in intracellular compartments has been recently reported (38). In addition, occupancy of the T410P mutant by antagonists, i.e. in the absence of stimulation of intracellular signaling, was rapidly followed by the appearance of antagonist-receptor-ß-arrestin2 complexes in the cytoplasm (Figs. 4 and 5). Taken together, these observations indicate that the T410P mutation of the PTH1Rc is functionally unique and clearly differs from the H223R mutation (although both result in constitutive receptor activity). They suggest that the T410P mutant PTH1Rc has an increased level of spontaneous intracellular trafficking. These findings support the hypothesis that an altered intracellular trafficking of T410P might explain its lower constitutive activity compared with H223R (7).

The molecular mechanisms underlying the higher constitutive trafficking of the T410P mutant are, at present, not fully clear. Current models of GPCR signaling and distribution suggest that constitutive activity might be associated with constitutive phosphorylation (39, 40, 41). Unfortunately, in the absence of high-affinity antibodies against the human PTH1Rc, our attempt to directly investigate phosphorylation of Jansen’s receptor mutants remained elusive. Moreover, C terminus truncation of H223R and T410P mutants resulted in severely reduced receptor expression and/or functionality, precluding their direct examination by fluorescence microscopy. Nevertheless, staurosporine apparently decreased intracellular trafficking of the T410P mutant, both spontaneously and after antagonist binding, resulting in its predominant localization on the cell membrane (Figs. 4 and 5). In turn, staurosporine, but not the specific PKA inhibitor H8M89, significantly increased both constitutive and agonist-stimulated cAMP accumulation by the PTH1Rc mutants, more significantly so with T410P (Table 3). Accordingly, one might speculate that PTH1Rc-mediated activation of protein kinases, particularly PKC, plays a role in the constitutive intracellular trafficking of the T410P mutant. In this regard, it is interesting to note that T410P has been reported to signal normally through Gq, whereas H223R does not (5, 6, 7, 8).

In conclusion, this study addresses the role of ß-arrestin2 in the regulation of both cAMP signaling and cellular localization of human PTH1Rcs. Taken together, our observations provide important insights into the regulatory mechanisms of PTH (and PTHrP) activity. In addition, they support an emerging concept about the direct role of ß-arrestins in the regulation of constitutive GPCRs activity. The fact that naturally occurring, constitutively active GPCR mutants are likely subjected to regulation in vivo (9, 21, 22, 42) highlights the potential physiological relevance of this regulatory mechanism. These results may lead to the development of novel molecular strategies to address a number of disorders associated with constitutively active GPCR mutants (22, 29).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Peptide Synthesis and Radioligand Preparation
The syntheses, purification, and characterization of PTHrP-(1–36)NH₂ [PTHrP-(1–36)], [Nle^8,18,Tyr³⁴]bPTH-(1–34)NH₂ [PTH-(1–34)], [Nle^8,18,Lys¹³(N-5-carboxymethylrhodamine),L-2-Nal²³,Arg^26,27,Tyr³⁴]bPTH-(1–34)NH₂ [Rho-PTH-(1–34)], [Bpa²,Ile⁵,Arg¹¹,Lys¹³,Tyr³⁶]PTHrP-(1–36)NH₂ [Bpa2-PTHrP-(1–36)], and [Bpa²,Ile⁵,Arg¹¹,Lys¹³(N-5-carboxymethylrhodamine),Tyr³⁶]PTHrP-(1–36)NH₂ [Rho-Bpa2-PTHrP-(1–36)], were carried out as previously described (12). The pure products were characterized by analytical HPLC, electron-spray mass spectrometry, and amino acid analysis. Radioiodination and HPLC purification of PTH-(1–34) and Bpa2-PTHrP-(1–36) were carried out as reported previously (43).

Expression Vectors Encoding Constitutively Active and Wild-Type Human PTH1Rcs, ß-Arr2-GFP
Expression plasmids encoding Jansen’s mutant receptors T410P and H223R were the generous gift of Dr. H. Juppner (Massachusetts General Hospital, Boston, MA) (5, 6). T410P PTH1Rc mutant full-length cDNA was subcloned into the mammalian expression vector pZeoSv2(+) (Invitrogen, Carlsbad, CA) using the ApaI and EcoRI polylinker restriction sites. H223R PTH1Rc mutant full-length cDNA was amplified by PCR using primers forward 5'-GCGATGGGGACCGCCCGG and reverse 5'-TCACATGACTGTCTCCCA. The 2-kb PCR product was directly ligated into the mammalian expression vector pCDNA3.1-TOPO (Invitrogen). The previously cloned full-length wild-type human PTH1Rc cDNA (44) was subcloned into the HindIII and NotI restriction sites of pZeoSv2(+). Receptor-GFP conjugates (H223R-GFP and T410P-GFP) were generated by subcloning the mutant receptors’ cDNA into HindIII and SacII restriction sites of the pEGFP-N1 vector (CLONTECH Laboratories, Inc. Palo Alto, CA). A human wild-type PTH1Rc-GFP (generously provided by Dr. Caroline Silve, INSERM, Paris) was also used and has previously been characterized (15). Truncated receptors lacking the whole C-terminal cytoplasmic domain but for eight proximal amino acid residues, i.e. del472593^stop (H223R-del, T410P-del, and wild-type PTH1Rc-del) were generated by PCR using primers forward as above and reverse 5'-CTTGATCTCAGCTTGTAC. The 1.6-kb PCR product was directly ligated into the mammalian expression vector pCDNA3.1-TOPO (Invitrogen). Integrity and orientation of the final constructs were verified by dideoxynucleotide sequencing and endonuclease restriction of unique SphI and SpeI sites in the H223R and T410P mutants, respectively (H. Juppner, personal communication). A p(S65T)-N3 plasmid containing the sequence of ß-Arr2-GFP was a generous gift from Dr. M. Caron (Duke University, Durham, NC) and has been previously characterized (14, 45).

Transfection Procedure
COS-7 cells (a generous gift from Dr. S. Goldring, Beth Israel Deaconess Medical Center, Boston, MA) and human embryonic kidney cells (HEK-293T, a generous gift from Dr. O. Behar, Massachusetts General Hospital, Boston, MA) were plated at 1.5 x 10⁵ cells per coverslip on 25-mm glass coverslips for fluorescence microscopy experiments and at 1.0 x 10⁵ cells per well in 24-well plastic dishes (Corning, Inc., Corning, NY) for adenylyl cyclase, radioligand binding, and radioligand internalization assays, as previously described (12). Twenty-four hours after plating, transient transfections were carried out using Fugene (Roche Molecular Biochemicals, Indianapolis, IN) reagent and constructs cDNA in a 2:1 to 3:1 ratio (vol/wt). Typically, 2.0 µg and 0.33 µg of, respectively, PTH1Rc and ß-Arr2-GFP plasmid cDNA, or 0.5–1.0 µg of PTH1Rc-GFP plasmid cDNA were transfected per coverslip, whereas 0.4 µg of PTH1Rc plasmid cDNA and variable amounts of ß-Arr2-GFP or control plasmid cDNA, as indicated in the figure legends, were transfected per 24-well dish. All subsequent experiments were performed 24 h after transfection.

ß-Arr2-GFP Conjugate Protein Expression
COS-7 cells grown in 24-well plastic dishes and transfected with increasing amounts of ß-Arr2-GFP cDNA, as described above, were lysed 24 h later with 100 µl of a solution containing Tris-PO₄ (25 µM)/dithiothreitol (2 µM)/EDTA (2 µM) (Sigma, St. Louis, MO)/Glycerol 10%/Triton X-100 1%. The lysate was briefly centrifuged and GFP fluorescence measured in the supernatant by spectrofluorimetry (Analyst 96.384, LJT Biosystems, Sunnyvale, CA) using 475-nm and 530-nm excitation and emission wavelengths, respectively.

Radioreceptor Binding and Internalization Assays
Radioreceptor binding assay was carried out as reported (46) using HPLC-purified ¹²⁵I-[Nle^8,18,Tyr³⁴]bPTH-(1–34)NH₂ [¹²⁵I-PTH-(1–34)] as radioligand. Curves were drawn by CA-Cricket Graph III (version 1.0; Computer Associates, Islandia, NY), and cell surface receptor expression was estimated from Scatchard analysis, as described previously (47). Radioligand internalization assays (17) were performed by incubating the cells for 30 min at room temperature in 250 µl of DMEM/10% FBS containing approximately 100 x 10³ cpm (0.1 nM) of either the agonist ¹²⁵I-PTH-(1–34) or the antagonist ¹²⁵I-[Bpa²,Ile⁵,Arg¹¹,Lys¹³,Tyr³⁶]PTHrP-(1–36)NH₂ [¹²⁵I-Bpa2-PTHrP-(1–36)]. Under these conditions, specific binding represented 10–20% of the total radioligand. Cells were then washed twice with ice-cold PBS, and surface-bound radioligand was extracted by two consecutive treatments with 500 µl of an ice-cold solution containing 50 mM glycine/100 mM NaCl (pH 3). Cells were then lysed with 0.1 M NaOH to measure internalized radioligand. In some experiments, cells were preincubated 30 min and maintained in hypertonic medium (0.45 M sucrose) during incubation with the radioligands, to inhibit clathrin-coated vesicles-mediated internalization, as previously demonstrated (12). Nonspecific binding and internalization were determined in parallel experiments in which radioligand binding was competed by 1 µM unlabeled PTH-(1–34).

Adenylyl Cyclase Assay
cAMP accumulation was determined in subconfluent cell cultures in the presence of the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (1 mM), as previously described (46). Kinetics of constitutive cAMP accumulation in cells expressing Jansen’s mutant receptors were obtained by incubating the cells with IBMX (1 mM) at 37 C for various periods of time, as indicated in the figure legends. Kinetics of PTH-stimulated cAMP accumulation were evaluated in cells preincubated 30 min with IBMX (1 mM) and subsequently exposed to PTH-(1–34) (100 nM) in the presence of IBMX for variable periods of time. In all cases, the reaction was stopped by adding 1.2 M trichloroacetic acid, and cAMP was isolated by the two-column chromatographic method.

Fluorescence Microscopy
Cellular distribution of fluorescent ligands, receptor-GFP conjugates, and ß-Arr2-GFP was assessed by real-time fluorescence microscopy as previously described (12). Briefly, transfected cells grown on glass coverslips were rinsed in PBS and mounted in an open-air, temperature-controlled block on a Nikon epifluorescence microscope (Diaphot 300, Nikon, Melville, NY). ß-Arr2-GFP or PTH1Rcs-GFP distribution was first analyzed in the absence of ligand by monitoring the cells maintained in PBS/BSA 0.1% at 37 C for up to 30 min. Effects of agonists and antagonists on ß-Arr2-GFP or PTH1Rcs-GFP localization were evaluated by subsequently adding the various ligands (100 to 1,000 nM) in the same buffer and by monitoring redistribution of the green fluorescence after 2, 5, 10, and 15 min. Dual fluorescence microscopy to colocalize ß-Arr2-GFP (and PTH1Rcs-GFP, respectively) and rhodamine-labeled ligands was performed by preincubating cells with ice-cold PBS/BSA 1% (blocking of nonspecific binding) followed by incubation with the fluorescent ligand for 10 min on ice or at room temperature, as indicated. The unbound ligand was then removed by carefully rinsing the coverslips with PBS, and the cells were warmed to 37 C for continuous microscopy monitoring. Images of the distribution of ß-Arr2-GFP (PTH1Rcs-GFP, respectively) and rhodamine-labeled ligands were acquired sequentially, i.e. within a 10-sec interval and in the same cellular plan, using fluorescein and rhodamine filters, respectively. Overlay images were generated using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

Statistics
Comparisons between experimental groups were performed by two-factor ANOVA using Statview (SAS Institute, Inc., Cary, NC).

	ACKNOWLEDGMENTS

 
We wish to thank particularly Dr. M. Rosenblatt for his intellectual, human, and financial support to the authors and their work. We thank Drs. L. Suva, M. Chorev, and J. Alexander for helpful discussions and critical reading of this manuscript. We also thank Drs. M. Caron for kindly providing us with ß-Arr2-GFP cDNA; H. Juppner for Jansen’s mutant receptor cDNAs; and Caroline Silve for hPTH1Rc-GFP cDNA.

	FOOTNOTES

 
Address requests for reprints to: Dr. S. Ferrari, Beth Israel Deaconess Medical Center, Division of Bone and Mineral Metabolism (HIM 944), 77 Ave Louis Pasteur, Boston, Massachusetts 02215. E-mail: sferrar2{at}caregroup.harvard.edu

This work was supported, in part, by Grant RO1-DK-47940 from the National Institute of Diabetes, Digestive and Kidney Diseases to M.R. S.L.F. is supported by a postdoctoral fellowship grant from the Swiss National Science Foundation and the Fondation des Bourses de Recherche en Médecine et Biologie.

Received for publication March 17, 2000. Accepted for publication October 11, 2000.

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