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Originally published In Press as doi:10.1074/jbc.M202544200 on July 9, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38524-38530, October 11, 2002
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Selective Ligand-induced Stabilization of Active and Desensitized Parathyroid Hormone Type 1 Receptor Conformations*

Alessandro BiselloDagger §, Michael Chorev, Michael Rosenblatt, Luca Monticelli||, Dale F. Mierke||, and Serge L. Ferrari**DaggerDagger

From the Dagger  Department of Medicine, Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the  Department of Medicine, Bone and Mineral Metabolism Unit, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02215, the || Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, and the ** Department of Internal Medicine, Division of Bone Diseases, University Hospital, 1211 Geneva 14, Switzerland

Received for publication, March 15, 2002, and in revised form, June 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For many G protein-coupled receptors, agonist-induced activation is followed by desensitization, internalization, and resensitization. In most cases, these processes are dependent upon interaction of agonist-occupied receptor with cytoplasmic beta -arrestins. The ligand-induced intramolecular rearrangements of the receptor responsible for the desensitized versus active conformational states, which dictate both the pharmacological properties of ligands and the biological activity of G protein-coupled receptors, have not been fully elucidated. Here, we identify specific interactions between parathyroid hormone (PTH)-related protein and the human PTH type 1 receptor (PTH1Rc) and the related receptor conformational changes that lead to beta -arrestin-2-mediated desensitization. PTH-related protein analogs modified at position 1 induced selective stabilization of the active G protein-coupled state of the receptor, resulting in lack of beta -arrestin-2 recruitment to the cell membrane, sustained cAMP signaling, and absence of ligand-receptor complex internalization. Mechanistically, the ligands modified at position 1, interacting with the extracellular end of helix VI of PTH1Rc, produced a translocation of transmembrane helices V and VI that differed from that induced by the cognate agonist, resulting in significantly different conformations of the third intracellular loop. These results show how specific interactions between PTH1Rc and its ligands may stabilize distinct conformational states, representing either the active G protein-coupled or a desensitized beta -arrestin-coupled receptor state. In addition, they establish that sustained biological activity of PTH1Rc may be induced by appropriately designed agonist ligands.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-coupled receptors (GPCRs)1 represent a major class of membrane-bound proteins that mediate a wide variety of biological functions, including sensitivity to light and odorants, endocrine and cardiovascular control, and neurotransmission. The ligand-induced intracellular signaling of GPCRs is tightly regulated by several mechanisms. For numerous GPCRs, desensitization involves translocation of arrestins from the cytosol to the cell membrane, their direct interaction with agonist-activated GPCRs, and consequent inhibition of G protein coupling (1, 2). Additionally, arrestin-mediated internalization of agonist-receptor complexes through clathrin-coated vesicles and subsequent receptor recycling to the cell membrane are responsible for the recovery of cellular responsiveness to agonists (resensitization). Receptor desensitization and resensitization accomplish the fundamental physiological role of modulating the cellular responses to both acute and chronic stimulation. An important implication is that not only signal transduction per se, but also the mechanisms regulating signal transduction have a profound influence in determining the pathophysiological processes mediated by GPCRs. Many important questions remain regarding the relationship between receptor occupancy, signaling, and desensitization. In particular, the structural features that differentiate the active and desensitized states and how extracellular interactions with the ligand translate into intracellular events (such as G protein activation and interaction with arrestins) have not been fully characterized.

Parathyroid hormone (PTH) is a major regulator of serum calcium homeostasis and bone metabolism (3). 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 (4). Both PTH and PTHrP bind to and activate the PTH type 1 receptor (PTH1Rc), a member of the class II subfamily of GPCRs coupled to both Gs and Gq proteins (5). As for many other GPCRs, interaction of activated PTH1Rc with beta -arrestin-2 is a primary mechanism for rapid desensitization of cAMP signaling and further internalization of ligand-receptor complexes (6, 7). Several lines of evidence suggest that recruitment of beta -arrestin-2 to the cell membrane and its association with activated PTH1Rc are largely (if not completely) independent of intracellular signaling and receptor phosphorylation (7-9). Therefore, the increased affinity of agonist-occupied PTH1Rc for beta -arrestin-2 and the consequent reduction in coupling with G proteins may result from specific conformational states distinct from those of the active G protein-bound receptor. To characterize the specific interactions with agonists that stabilize these distinct functional states, we developed analogs of the biologically active N-terminal fragment of PTHrP (i.e. PTHrP-(1-36)) with selective pharmacological profiles. Analysis of the signaling properties of these analogs combined with fluorescence monitoring of the cellular distribution and extensive molecular modeling of the ligand-receptor complexes allowed us to identify specific interactions between ligand and receptor and the related conformational changes within PTH1Rc that are associated with beta -arrestin-2-mediated desensitization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide Synthesis and Radioligand Preparation-- The synthesis, purification, and characterization of PTHrP-(1-34)-NH2 (PTHrP-(1-34)), PTHrP-(2-36)-NH2 (PTHrP-(2-36)), [Bpa1,Ile5,Arg11,13,Tyr36]PTHrP-(1-36)-NH2 (Bpa1-PTHrP-(1-36)), [Bpa1,Ile5,Arg11,Lys13(Nepsilon -5- carboxymethylrhodamine),Tyr36]PTHrP-(1-36)-NH2 (Rho-Bpa1- PTHrP-(1-36)), bovine [Nle8,18,Tyr34]PTH-(1-34)-NH2 (PTH-(1-34)), and bovine [Nle8,18,Lys13(Nepsilon -5-carboxymethylrhodamine),L-2-Nal23, Arg26,27,Tyr34]PTH-(1-34)-NH2 (Rho-PTH-(1-34)) were carried out as previously described (6). The pure products were characterized by analytical HPLC, electron spray mass spectrometry, and amino acid analysis. Radioiodination and HPLC purification of PTH-(1-34), PTHrP-(2-36), and Bpa1-PTHrP-(1-36) were carried out as reported (6).

Cell Culture and Transfection Method-- Human embryonic kidney cells (HEK-293) and HEK-293 cells stably expressing human PTH1Rc (clone C-21) were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum as described (10). Cells were plated at 1.5 × 105 cells/coverslip on 35-mm glass coverslips for fluorescence microscopy experiments and at 1.0 × 105 cells/well in 24-well plastic dishes (Corning Inc., Corning, NY) for adenylyl cyclase, radioligand binding, and radioligand internalization assays and transfected as previously described (7). All subsequent experiments were performed 24 h after transfection.

Radioreceptor Binding and Internalization Assays-- Radioreceptor binding and internalization assays were carried out as reported (7) using HPLC-purified radioiodinated compounds. The binding affinities (Kd) of each PTHrP analog were calculated from Scatchard analysis of radioreceptor binding assays (using each radioiodinated compound as tracer and competition with each unlabeled analog) as described previously (6, 7).

Adenylyl Cyclase, Intracellular Calcium, and Inositol Phosphate Assays-- cAMP accumulation was determined in subconfluent cell cultures in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine as previously described (7). Briefly, ligand-stimulated cAMP accumulation was measured in cells preincubated for 30 min with 3-isobutyl-1-methylxanthine (1 mM) and subsequently exposed to various concentrations of the appropriate ligand (10-11 to 10-6 M). To determine desensitization of cAMP signaling, cells were treated with the indicated ligands (100 nM) for 30 min, followed by a 2-h washout at 37 °C. Residual cAMP accumulation was then measured for 15 min in the presence of 3-isobutyl-1-methylxanthine (1 mM). In all cases, reactions were stopped with 1.2 M trichloroacetic acid, and cAMP was isolated by the two-column chromatographic method (11). Transients of intracellular calcium stimulated by PTHrP-(1-36), PTHrP-(2-36), and Bpa1-PTHrP-(1-36) were assessed spectroscopically in Fura-2-loaded HEK-293 cells stably transfected with human PTH1Rc (clone C-21) as described (12).

Determination of total inositol phosphate accumulation was carried out essentially as described by John et al. (13). Briefly, HEK-293 cells transiently expressing either wild-type PTH1Rc or PTH1Rc(H223R) were incubated overnight with myo-[3H]inositol in Dulbecco's minimal essential medium and 0.1% bovine serum albumin. The medium was changed to medium containing 20 mM LiCl for 15 min, and cells were stimulated with 1 µM PTHrP-(1-36), PTHrP-(2-36), or Bpa1-PTHrP-(1-36) for 1 h at 37 °C. After aspiration of the medium, cells were lysed with 20 mM formic acid for 1 h on ice, and the neutralized supernatants were chromatographed on Dowex 1-X8 as described (13).

Fluorescence Microscopy-- Cellular distribution of fluorescent ligands, receptor-green fluorescent protein (GFP) conjugate (PTH1Rc-GFP), and beta -arrestin-2-GFP (beta -arr2-GFP) was assessed by real-time fluorescence microscopy as previously described (7). Briefly, transfected cells grown on glass coverslips were rinsed with phosphate-buffered saline and mounted in an open-air, temperature-controlled block on a Nikon Diaphot 300® epifluorescence microscope. beta -arr2-GFP or PTH1Rc-GFP distribution was first analyzed in the absence of ligand by monitoring the cells maintained in phosphate-buffered saline and 0.1% bovine serum albumin at 37 °C for up to 30 min. The effects of ligands on beta -arr2-GFP or PTH1Rc-GFP localization were evaluated by subsequently adding the various ligands (100-1000 nM) in the same buffer and by monitoring redistribution of the green fluorescence over time. Dual fluorescence microscopy to colocalize beta -arr2-GFP or PTH1Rc-GFP and rhodamine-labeled ligands was performed by preincubating cells with ice-cold phosphate-buffered saline and 1% bovine serum albumin (blocking of nonspecific binding), followed by incubation with the fluorescent ligand for 10 min on ice or at room temperature. The unbound ligand was then removed by carefully rinsing the coverslips with phosphate-buffered saline, and the cells were warmed to 37 °C for microscopy monitoring. Images of the distribution of beta -arr2-GFP or PTH1Rc-GFP and rhodamine-labeled ligands were acquired sequentially, i.e. within a 10-s interval and in the same cellular plan, using fluorescein and rhodamine filters, respectively. Overlay images were generated using Image-Pro Plus® software (Media Cybernetics, Silver Spring, MD).

Molecular Modeling-- The molecular dynamics simulations of the ligand-receptor complexes were carried out following published procedures (14, 15). The starting structure, based on extensive NMR characterization of the ligands and extra- and intracellular portions of PTH1Rc, also includes ligand-receptor contact points derived from photoaffinity labeling experiments (16-23). The simulations utilized a three-layer (water/decane/water) simulation cell, allowing for long simulations while maintaining the overall biphasic hydrophobic/hydrophilic character as well as the molecular motions of the long alkyl chains found in membranes (24). All simulations were performed using the GROMACS package (25). Analysis of motions of the transmembrane (TM) helices was carried out using the microdomain procedure described by Weinstein et al. (26), in which the receptor is defined as a set of domains. The rotation and translation of each domain during the simulation were then mapped upon the torques or forces exerted on the domain, clearly illustrating how interdomain interactions influence the motion of the domain. The structural consequences of the modified ligands, even if involving many sequential interactions, can be unambiguously identified.

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

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Position 1-modified PTHrP Analogs-- Residues 1 and 2 in PTHrP are essential for activation of receptor-mediated signaling (Ref. 27 and references therein). Substitution of residue 2 (Val) in PTHrP with Bpa results in a ligand, [Bpa2,Ile5,Arg11,13]PTHrP-(1-36), with antagonist/inverse agonist properties (16). In contrast, modification of residue 1 (Ala) in PTHrP-(1-36) by either deletion or substitution with Bpa (yielding PTHrP-(2-36) and Bpa1-PTHrP-(1-36), respectively) did not significantly alter the affinity for PTH1Rc or the efficacy in acutely stimulating the Gs/adenylyl cyclase signaling pathway in HEK-293 cells stably expressing human PTH1Rc (C-21 cells) (Table I). However, although PTH-(1-34) and PTHrP-(1-36) stimulated rapid and robust transient increases in intracellular calcium in C-21 cells, both PTHrP-(2-36) and Bpa1-PTHrP-(1-36) lost their ability to stimulate Gq/phospholipase C, as evidenced by both the absence of intracellular calcium transients upon stimulation of PTH1Rc in C-21 cells (Table I) as well as the lack of stimulation of phosphatidylinositol hydrolysis (see Fig. 3C).

                              
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Table I
Characterization of affinity and signaling properties of position 1-modified PTHrP analogs in HEK-293/C-21 cells
All data are means ± S.E. of three to four independent experiments performed in triplicates.

Cellular Distribution of Ligands, PTH1Rc, and beta -Arrestin-2-- Using beta -arr2-GFP (28), the translocation of beta -arrestin-2 from the cytosol to the cell membrane and its colocalization with agonist-occupied receptors were monitored in C-21 cells. In contrast to native PTHrP-(1-36) and PTH-(1-34) (6, 7), neither Bpa1-PTHrP-(1-36) nor PTHrP-(2-36) induced detectable beta -arr2-GFP mobilization (Fig. 1). Furthermore, a rhodamine-labeled Bpa1-PTHrP-(1-36) analog that bound to PTH1Rc on the cell membrane did not colocalize with beta -arr2-GFP on the cell surface or intracellularly (Fig. 2A). Rapid receptor internalization did not occur in response to the modified PTHrP analogs in cells expressing PTH1Rc-GFP (data not shown) (29), and Bpa1-PTHrP-(1-36)-receptor complexes were not detectable intracellularly (Fig. 2B). These results, distinct from those obtained with the full-length agonists PTH-(1-34) and PTHrP-(1-36) (Fig. 2, A and B) (6, 7), indicate that the Gs-selective agonists PTHrP-(2-36) and Bpa1-PTHrP-(1-36) are unable to stimulate translocation of beta -arrestin-2 to the cell membrane, beta -arrestin-2 interaction with agonist-occupied PTH1Rc, and subsequent ligand-PTH1Rc complex endocytosis.


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  		Fig. 1.   Fluorescence microscopy monitoring of beta -arrestin-2 mobilization in response to PTHrP analogs. HEK-293/C-21 cells stably expressing human PTH1Rc were transiently transfected with beta -arr2-GFP (0.3 µg of DNA/35-mm glass coverslip) as previously described (6, 7). In the absence of stimulation, beta -arr2-GFP was distributed throughout the cytoplasm (panel a). Stimulation for up to 40 min at 37 °C of the cells shown in panel a with 100 nM PTHrP-(2-36) did not mobilize beta -arr2-GFP (panel b). A similar lack of beta -arr2-GFP mobilization was observed with 100 nM Bpa1-PTHrP-(1-36) (panel c). In contrast, 100 nM PTHrP-(1-36) recruited beta -arr2-GFP to the cell membrane within 5 min (panel d). Each panel is representative of eight independent experiments yielding similar results.


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  		Fig. 2.   Cellular colocalization of ligand, PTH1Rc, and arrestin-2 in response to PTHrP analogs. A, HEK-293 cells transiently coexpressing human PTH1Rc and beta -arr2-GFP were incubated with 50 nM Rho-Bpa1-PTHrP-(1-36) (panels a-c) or Rho-PTH-(1-34) (panels d-f). After 15 min at 37 °C, Rho-Bpa1-PTHrP-(1-36) appeared bound on the cell surface without evidence of internalization (panel a); beta -arr2-GFP was not recruited to the cell surface (panel b); and therefore, the two fluorescences did not colocalize on the overlay image (panel c). In contrast, after stimulation with Rho-PTH-(1-34), both ligand (panel d) and beta -arr2-GFP (panel e) colocalized intracellularly (panel f). B, HEK-293 cells transiently expressing human PTH1Rc-GFP were incubated with 50 nM Rho-Bpa1-PTHrP-(1-36) (panels a-c) or Rho-PTH-(1-34) (panels d-f). After 15 min at 37 °C, Rho-Bpa1-PTHrP-(1-36) was linearly distributed on the cell surface (panel a); PTH1Rc-GFP was mostly localized on the cell surface (panel b); and therefore, both ligand and receptor colocalized (in orange-yellow in the overlay image) (panel c). In contrast, in response to Rho-PTH-(1-34), both ligand (panel d) and receptor (panel e) were internalized and colocalized in the cytoplasm (panel e). Each panel is representative of three to seven independent experiments yielding similar results.

Radioligand Distribution-- To quantify and confirm these observations, the distribution of radioiodinated PTH-(1-34), PTHrP-(2-36), and Bpa1-PTHrP-(1-36) was monitored (Fig. 3A); 125I-PTH-(1-34) was rapidly (t1/2 = 10 min) and efficiently internalized in C-21 cells, whereas internalization of 125I-Bpa1-PTHrP-(1-36) and 125I-PTHrP-(2-36) was slower (t1/2 = 30 min) and only partial. In addition, the dissociation of receptor-bound 125I-Bpa1-PTHrP-(1-36) and 125I-PTHrP-(2-36) (14 ± 1 and 16 ± 2% at 60 min, respectively) was lower than that of 125I-PTH-(1-34) (26 ± 6% at 60 min), in agreement with the proposed "pseudo-irreversible" binding of agonist to receptor-G protein complexes (30). As a consequence, 1 h after extensive washout, both Bpa1-PTHrP-(1-36) and PTHrP-(2-36) remained mostly associated with PTH1Rc on the cell membrane (47 ± 5 and 61 ± 3% of total bound ligand, respectively), whereas only 10 ± 1% of PTH-(1-34) was still associated with membrane receptors.


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  		Fig. 3.   A, internalization of radiolabeled PTH-(1-34), Bpa1-PTHrP-(1-36), and PTHrP-(2-36) in HEK-293/C-21 cells. HEK-293/C-21 cells stably expressing PTH1Rc were incubated for 2 h at 4 °C with the radiolabeled agonist 125I-PTH-(1-34) (black-square), 125I-Bpa1-PTHrP-(1-36) (black-triangle), or 125I-PTHrP-(2-36) (); washed; and incubated in fetal bovine serum-supplemented Dulbecco's minimal essential medium for the indicated times. The percentage of specific radioligand internalization was evaluated as reported (6, 7). Each point represents the mean percentage ± S.E. of total cell-associated ligand from triplicate determinations. Similar results were obtained in three additional experiments. Average values of all experiments for internalization at 60 min were as follows: PTH-(1-34), 71 ± 9% of the total cell-associated radioligand; Bpa1-PTHrP-(1-36), 37 ± 6% (p < 0.001 compared with PTH-(1-34)); and PTHrP-(2-36), 25 ± 7% (p < 0.001 compared with PTH-(1-34)). B, sustained adenylyl cyclase activity by position 1-modified PTHrP analogs is inhibited by the PTH1Rc(H223R) mutant. HEK-293 cells transiently expressing either wild-type PTH1Rc (white and thinly hatched bars) or the constitutively active PTH1Rc(H223R) mutant (black and thickly hatched bars) (0.4 µg of DNA/well in 24-well plates) were treated with the indicated ligands (100 nM) for 30 min, followed by a 2-h washout at 37 °C. Residual and re-challenged cAMP accumulation was then measured for 30 min in the presence of 3-isobutyl-1-methylxanthine (1 mM) without (white and black bars) and with re-exposure (hatched bars) to PTHrP or its modified analogs, respectively. In cells expressing wild-type PTH1Rc, residual cAMP accumulation after preincubation with Bpa1-PTHrP-(1-36) and PTHrP-(2-36) was significantly higher than after preincubation with PTHrP-(1-34). Residual cAMP accumulation after preincubation with Bpa1-PTHrP-(1-36) and PTHrP-(2-36) was lower (p < 0.001) in cells expressing PTH1Rc(H223R) than in cells expressing wild-type PTH1Rc. Also, in cells expressing PTH1Rc(H223R), the response to a re-challenge with the agonist (p < 0.01 compared with residual cAMP levels) was similar to that in cells not previously treated with the ligand. The maximal cAMP accumulation in these cells, evaluated by stimulation of adenylyl cyclase with forskolin (10 µM), was 24,027 ± 947 cpm/well. Results are presented as means ± S.E. from three independent experiments performed in triplicate. C, inositol phosphate (IP) generation by PTHrP-(1-34), Bpa1-PTHrP-(1-36), and PTHrP-(2-36) in HEK-293 cells expressing wild-type PTH1Rc or PTH1Rc(H223R). HEK-293 cells transiently expressing either wild-type PTH1Rc (white bars) or the PTH1Rc(H223R) mutant (gray bars) (0.4 µg of DNA/well in 24-well plates) were loaded with myo-[3H]inositol and treated with the indicated ligands (1 µM) for 60 min at 37 °C in the presence of 20 mM LiCl. In cells expressing wild-type PTH1Rc, only PTHrP-(1-34) induced generation of inositol phosphates (p < 0.001 versus basal activity). Cells expressing the PTH1Rc(H223R) mutant were unresponsive to all ligands. Results are expressed as means ± S.E. from two independent experiments performed in triplicate. Basal values were subtracted from the values obtained in the presence of the hormone. D, rescue of Bpa1-PTHrP-(1-36) internalization by PTH1Rc(H223R). HEK-293 cells cotransfected with the PTH1Rc(H223R) mutant and beta -arr2-GFP were incubated for 10 min at room temperature with Rho-Bpa1-PTHrP-(1-36) (50 nM). In these cells, beta -arr2-GFP was constitutively recruited to the cell membrane (panel a). The ligand at first appeared to be linearly bound on the cell surface (panel b) and colocalized with beta -arr2-GFP (in yellow in the overlay image) (panel c). After 15 min at 37 °C, both beta -arr2-GFP (panel d) and the fluorescent ligand (panel e) were internalized and colocalized in the cytoplasm (panel f).

PTH1Rc Desensitization and Resensitization of cAMP Signaling-- Because receptor interaction with beta -arrestin-2 is an essential early step in rapid desensitization of Gs/cAMP signaling by PTH1Rc (7), we hypothesized that Bpa1-PTHrP-(1-36) and PTHrP-(2-36) would induce sustained activation of adenylyl cyclase. Indeed, exposure of HEK cells expressing PTH1Rc either stably (C-21) or transiently to Bpa1-PTHrP-(1-36) or PTHrP-(2-36) for 30 min, followed by a 2-h washout, resulted in a 5-7-fold increase in residual levels of cAMP accumulation at the end of this period compared with cells similarly exposed to PTHrP-(1-36) or PTH-(1-34) (Fig. 3B and Table I). Moreover, cells pretreated with PTHrP-(1-36) and PTH-(1-34) were fully responsive to restimulation with these agonists after 2 h (+883 and +796% above residual levels, respectively), indicating that complete receptor resensitization had occurred (Fig. 3B and Table I). In cells pretreated with Bpa1-PTHrP-(1-36) or PTHrP-(2-36), restimulation with the same ligands resulted in small increases in cAMP accumulation above the residual levels (13.5 and 36%, respectively) (Fig. 3B and Table I). Time course experiments indicated that significant differences in residual and restimulated cAMP levels between PTHrP-(1-36) and its position 1-modified analogs were detectable after 30 min and persisted for up to 4 h after exposure to the agonists (data not shown).

Rescue of Signaling Regulation and Endocytosis by the PTH1Rc(H223R) Mutant-- To elucidate whether such critical differences between PTHrP and its analogs modified at position 1 were dependent on differences in ligand-induced receptor conformation or Gq-mediated signaling, we investigated whether regulation of cAMP signaling and cellular distribution of Bpa1-PTHrP-(1-36) with beta -arr2-GFP could be rescued by the PTH1Rc(H223R) mutant. Indeed, this natural mutant is Gq signaling-deficient both in COS-7 cells (31) and in HEK-293 cells, where no measurable generation of inositol phosphates in response to PTHrP-(1-34), Bpa1-PTHrP-(1-36), or PTHrP-(2-36) was detected (Fig. 3C). Furthermore, in HEK-293 cells, PTH1Rc(H223R) is constitutively associated with beta -arrestin-2 on the cell membrane and internalizes normally in response to PTH-(1-34) and PTHrP-(1-34) (7). In this system, brief exposure to Bpa1-PTHrP-(1-36) and PTHrP-(2-36), followed by a 2-h washout, did not result in cAMP levels higher than the basal activity, which is reflective of the constitutive activity of the receptor mutant (Fig. 3B). Moreover, 2 h after the first agonist stimulation, PTH1Rc(H223R)-expressing cells were fully responsive to a re-challenge with agonist (Fig. 3B), indicating that the elevated constitutive activity in these cells is not a limiting factor for the ligand-induced generation of cAMP. This was accompanied by internalization of 125I-Bpa1-PTHrP-(1-36) and 125I-PTHrP-(2-36) bound to PTH1Rc(H223R) (68 ± 3 and 54 ± 3%, respectively), which was similar to 78 ± 2% for 125I-PTH-(1-34). Additionally, rhodamine-labeled Bpa1-PTHrP-(1-36) colocalized with PTH1Rc(H223R)-associated beta -arr2-GFP on the cell membrane and then intracellularly (Fig. 3D), as was previously seen with rhodamine-labeled PTH-(1-34) (7). These experiments show that the absence of activation of the Gq/phospholipase C signaling pathway is not responsible for lack of rapid cAMP desensitization of wild-type PTH1Rc in response to PTHrP analogs. Rather, these data indicate that interaction of the receptor with beta -arrestin-2 is sufficient for the regulation of Gs/cAMP signaling.

Molecular Modeling of Ligand-PTH1Rc Complexes-- To further explore the structural details underlying these effects, we examined the conformational changes induced by binding of native PTHrP-(1-36) and its position 1-modified analogs to human PTH1Rc using molecular dynamics simulations. The starting ligand-receptor structure, based on extensive NMR characterization of the ligands and extra- and intracellular portions of PTH1Rc, also includes ligand-receptor contact points identified by photoaffinity labeling experiments (16-23). Among these, of particular significance is the interaction of position 1 of Bpa1-PTHrP-(1-36) with Met425 at the extracellular end of TM6 of PTH1Rc (16).

Analysis of simulations with PTHrP-(2-36) and Bpa1-PTHrP-(1-36) indicated significant differences from simulations with the native hormone, PTHrP-(1-36) (Fig. 4), in particular with regard to the orientation of TM5 and TM6. The root-mean-square deviation for TM5-TM6 was 3.5 Å between the wild-type ligand and Bpa1-PTHrP-(1-36). A much smaller value of 1.5 Å was obtained for the remaining helices (TM1-TM4 and TM7). This resulted in major effects on the structure of the third intracellular loop (IC3), particularly the N-terminal helix (Thr387-Arg396) of the intracellular loop. With PTHrP-(1-36), this helix was found to be parallel to the membrane surface, leading to the exposure of the charged residues, Lys388, Arg390, and Glu391. In contrast, with the Gs-selective modified ligands, the helix-turn-helix structure of the third loop, as observed in our NMR studies (23, 32), was maintained. In all of the simulations, smaller effects on the orientation of the C-terminal portion of IC3 were observed. These changes, mostly evident with Lys405, were identical to those observed for PTH1Rc mutants that constitutively activate adenylyl cyclase (14). The results for PTHrP-(2-36) were very similar to those for Bpa1-PTHrP-(1-36), suggesting that the first residue of PTHrP is actively involved in the induction of the receptor conformation recognized by beta -arrestin-2.


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  		Fig. 4.   Molecular model of the complex between PTH1Rc and Bpa1-PTHrP-(1-36). A, the ligand is depicted in gray, with the backbone displayed as a ribbon. The receptor is depicted as a ribbon, which is colored dark (N terminus-TM1 and TM4-second extracellular loop-TM5) and light (TM2-first extracellular loop-TM3 and TM6-third extracellular loop (EC3)-TM7) green. The molecular dynamics simulations of the ligand-receptor complexes were carried out following published procedures (14, 15). B, shown is the interaction of Bpa of Bpa1-PTHrP-(1-36) with Met425 (shown in Corey-Pauling-Koltun spheres) of PTH1Rc (16). The color scheme is the same as described for A. C, shown is the structure of PTH1Rc resulting from simulations of the ligand-receptor complex with PTHrP-(1-36) (light green) and Bpa1-PTHrP-(1-36) (dark green).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to characterize the structural and functional consequences of modifications affecting the first amino acid residue in PTHrP and thereby to provide new insights into the PTH1R conformational states associated with coupling to G proteins and desensitization of intracellular signaling. Our data clearly show that modifications of residue 1 in the biologically active N-terminal fragment of PTHrP result in ligands (i.e. PTHrP-(2-36) and Bpa1-PTHrP-(1-36)) that, although maintaining high affinity and efficacy in stimulating cAMP production, are unable to stimulate the Gq/phospholipase C signaling pathway. Additionally, in contrast to native PTHrP and PTH (6-9), the modified PTHrP analogs do not stimulate beta -arrestin-2 translocation to the cell membrane and its association with agonist-activated PTH1Rc, functionally resulting in impaired desensitization of cAMP signaling. The observation that regulation of signaling by PTHrP-(2-36) and Bpa1-PTHrP-(1-36) and ligand-receptor endocytosis were rescued by the mutant receptor PTH1Rc(H223R), which is defective in Gq-mediated signaling (31) but is constitutively associated with beta -arrestin-2 (7), clearly indicates that the signaling selectivity of these analogs per se is not the cause of the impaired desensitization. Rather, when combined with the findings that receptor phosphorylation is not necessary for beta -arrestin-2 translocation (7, 8) and with the previous characterization of the molecular motions of the PTH1Rc(H223R) mutant showing that the conformation of the third intracellular loop of the unbound receptor mutant is very similar to the one of cognate agonist-bound PTH1Rc (14), these experiments suggest that the known interaction between residue 1 of PTHrP and the extracellular end of transmembrane helix VI of PTH1Rc (16) mediates the conformational changes leading to the high affinity state for beta -arrestin-2. In structural terms, interaction of Bpa1-PTHrP-(1-36) and PTHrP-(2-36) with PTH1Rc results in stabilization of a non-desensitized G protein-coupled conformation. In contrast, complexes with native PTHrP-(1-36) allow for a distinct conformational state in which interactions with beta -arrestin-2 are favored over G protein coupling (i.e. the desensitized state).

Analysis of the conformational changes within PTH1Rc upon interaction with either PTHrP-(1-36) or the non-desensitizing analogs was carried out by molecular modeling of ligand-receptor complexes in a simulation cell that mimics the biphasic amphipathic character of cell membranes (24). The starting ligand-receptor structure, based on experimental characterization of the ligands and relevant portions of PTH1Rc, also includes several ligand-receptor contact points identified by biochemical methodologies (16-23). Among these, particularly important is the interaction of position 1 of Bpa1-PTHrP-(1-36) with Met425 at the extracellular end of TM6 of PTH1Rc (16). The molecular dynamics simulations indicate that the major differences in the receptor structure upon interaction with either PTHrP-(1-36) or the non-desensitizing position 1-modified analogs arise from the distinct interaction of the modified residues with TM5 and TM6, resulting in altered conformation of IC3. Interestingly, residues in the N-terminal helix of IC3, particularly Val384 and Leu385, have been previously shown to be involved in Gq coupling (33), consistent with the observations that the position 1-modified analogs lose their ability to elicit intracellular calcium transients and phosphatidylinositol hydrolysis. Given the importance of IC3 in coupling to both G proteins and arrestins (33-38), it is not surprising that major differences in the structures of active and desensitized ligand-receptor complexes are found within this domain. This mechanism of receptor function is in good agreement with mutagenesis studies showing the importance of relative movements between TM3 and TM6 of PTH1Rc for Gs and Gq activation (9, 39). These studies, in which receptor stabilization was achieved by engineered Zn2+-binding sites, also suggested that G protein activation and PTH1Rc phosphorylation and internalization require different conformational switches (9).

The concept that dynamic conformational changes upon agonist binding result in distinct functional receptor states (i.e. the active and resting states) led to the development of the ternary complex model for G protein-coupled and G protein-uncoupled receptors (40). Recent models propose multiple conformational states for GPCRs (41-48) that are stabilized by different classes of ligands (i.e. agonists, partial agonists, antagonists, and inverse agonists) and/or receptor oligomerization and that mediate distinct molecular and cellular events. We now provide experimental evidence showing that, in the PTH/PTHrP system, structurally similar agonists can selectively stabilize either the active G protein-coupled or the desensitized beta -arrestin-coupled receptor conformation. The differences between the active and desensitized receptor conformations appear to be related, at least in part, to the induction of distinct structures of IC3 by the ligand via the interaction of residue 1 with the extracellular end of TM6. Collectively, this study and previous reports (7-9) also suggest that receptor occupancy by agonists, rather than covalent receptor modification (i.e. phosphorylation), may be sufficient for induction of a purely structural desensitized receptor state. Indeed, receptor desensitization is one of the major mechanisms in determining both the physiology and pharmacology of GPCRs (49, 50). The possibility of modulating such phenomena through the appropriate design of PTH- and PTHrP-derived agonists may have profound influence on the exploitation of pharmacological interventions on PTH1Rc function.

    ACKNOWLEDGEMENTS

We thank Dr. R. Lefkowitz (Duke University) and Drs. P. Friedman and A. Stewart (University of Pittsburgh) for critical comments in preparing this manuscript. We also thank Drs. M. Caron (Duke University), C. Silve (INSERM, Paris) and H. Jueppner (MGH, Boston) for kindly providing cDNAs for beta -arr2-GFP, PTH1Rc-GFP, and PTH1Rc(H223R), respectively.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK47940 (to M. R.).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.

§ To whom correspondence may be addressed: Div. of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, E1140 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412-648-7347; Fax: 412-648-3290; E-mail: biselloa@msx.dept-med.pitt.edu.

Dagger Dagger Supported by Professorship Grant Nb 632-62937 from the Swiss National Science Foundation. To whom correspondence may be addressed. Tel.: 41-22-382-9972; Fax: 41-22-382-9973; E-mail: serge.ferrari@medecine.unige.ch.

Published, JBC Papers in Press, July 9, 2002, DOI 10.1074/jbc.M202544200

    ABBREVIATIONS

The abbreviations used are: GPCRs, G protein-coupled receptors; PTH, parathyroid hormone; PTHrP, PTH-related protein; PTH1Rc, PTH type 1 receptor; Bpa, p-benzoylphenylalanine; Bpa1-PTHrP-(1-36), [Bpa1,Ile5,Arg11,13,Tyr36]PTHrP-(1-36)-NH2; Rho-Bpa1-PTHrP-(1-36), [Bpa1,Ile5,Arg11,Lys13(Nepsilon -5-carboxymethylrhodamine), Tyr36]PTHrP-(1-36)-NH2; Nle, norleucine; Nal, naphthylalanine; Rho- PTH-(1-34), [Nle8,18,Lys13(Nepsilon -5-carboxymethylrhodamine),L-2-Nal23, Arg26,27,Tyr34]PTH-(1-34)-NH2; HPLC, high pressure liquid chromatography; HEK, human embryonic kidney; GFP, green fluorescent protein; beta -arr2, beta -arrestin-2; TM, transmembrane domain; IC3, third intracellular loop.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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