Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor

Ligand binding to the PTH1 receptor is described by a “two-site” model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3–34) and PTH(1–14): PTH(1–14), which binds predominantly to the J domain, only partially inhibited binding of125I-PTH(3–34); and PTH(3–34), shown to bind predominantly to the N domain, only partially inhibited PTH(1–14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of125I-PTH(3–34) with 30 μm guanosine 5′-3-O-(thio)triphosphate (GTPγS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1–36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1–34) affinity for RG; residue 5 of MAP-PTHrP(1–36) was a determinant of R/RG binding selectivity, and PTH(1–14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3–34) binding was GTPγS-insensitive. Finally, several observations suggest the receptor conformation is more “closed” at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1–14) and PTH(3–34). At RG PTH(1–14) better occluded binding of 125I-PTH(3–34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states.


I-PTH(3-34) with 30 M guanosine 5-3-O-(thio)triphosphate (GTP␥S) present, and binding to RG was measured by displacement of 125 I-[MAP]
PTHrP(1-36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and PTH(1-14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3-34) binding was GTP␥S-insensitive. Finally, several observations suggest the receptor conformation is more "closed" at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1-14) and PTH . At RG PTH(1-14) better occluded binding of 125 I-PTH(3-34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states.
The parathyroid hormone 1 (PTH1) 1 receptor is a cell-surface signal transducer for PTH and PTH-related protein (PTHrP). PTH plays a central role in calcium homeostasis; the hormone acts on target cells in bone (osteoblasts) and kidney (renal tubule cells) to increase blood calcium levels (1). PTHrP is an autocrine factor, believed to be involved in the maintenance of numerous tissues, and an important developmental regulator, controlling breast, pancreas, skin, and bone development (2,3). PTH and PTHrP are involved in the etiology and treatment of disease. PTH, when administered intermittently, acts as a bone anabolic agent potentially useful for the treatment of osteoporosis (4). PTHrP is overproduced by certain tumors, leading to hypercalcemia through activation of the PTH1 receptor (5). The intracellular signaling pathways activated by PTH and PTHrP via the PTH1 receptor include stimulation of adenylyl cyclase, increases of intracellular calcium, and activation of phospholipase C and phospholipase D (6 -10).
Owing to its important physiological, pathophysiological, and therapeutic roles, the molecular mechanisms of PTH1 receptor function have been studied extensively (11). The receptor belongs to the type II family of G-protein-coupled receptors (GPCRs), which respond to peptide ligands of intermediate size such as secretin, glucagon, calcitonin, corticotropin-releasing hormone, and vasoactive intestinal polypeptide. The receptor can be divided into two functional domains; the large extracellular N-terminal domain (N domain) has been proposed to provide most of the binding energy for receptor-ligand interaction (12,13), and the remaining juxtamembrane region of the receptor (J domain) is a determinant of receptor activation and second messenger generation (12)(13)(14)(15). Likewise the ligand (PTH or PTHrP) can be divided into two binding regions; the 15-34 portion is a determinant of receptor binding affinity (12,16,17), and the 1-14 portion is a determinant of receptor activation for stimulation of cAMP production (12, 18 -21). (The cAMP-stimulating activity and high affinity binding of PTH and PTHrP are retained within an N-terminal fragment of 34 residues (22).) These observations suggested a "two-site" mode of receptor-ligand interaction (Fig. 1), in which the C-terminal portion of the ligand interacts with the N domain of the receptor (N interaction), and the N-terminal ligand region binds to the J domain of the receptor (J interaction) (11)(12)(13)19). This model has also been demonstrated for other type II GPCRs * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (23)(24)(25)(26). For the PTH1 receptor, this low resolution molecular model is supported by a large number of receptor manipulation and photochemical cross-linking studies, which have also suggested points of contact and/or proximity between specific amino acid side chains of the ligand and the receptor (14, 15, 27, 28, 30 -32). These observations have been combined with ligand structure data (33,34) and computer models of the receptor to provide atomic resolution structural models of certain regions of receptor-ligand interaction (28,33,35,36).
Receptor-ligand interaction models for the PTH1 receptor have not taken into account the dynamic nature of receptor conformation. Conformational change is central to the ability of a GPCR to transduce the extracellular signal of ligand binding across the plasma membrane to the intracellular signal of G-protein activation (37,38). As a result, evaluating the effect of this receptor conformational change is essential for understanding ligand binding and signal transduction mechanisms (37,38). These mechanisms have been examined extensively for GPCRs of the type I family, such as the ␤ 2 -adrenergic receptor, leading to the development of the ternary complex model and its extended variants (37)(38)(39). These models describe the reciprocal effects of G-protein (G) and agonist on their binding to the receptor (R). For type II GPCRs a great deal is known regarding the orientation of ligand binding, but very little is known regarding how the two-site binding mechanism is affected by the conformational changes in the receptor that result from R-G interaction. In this study we evaluated the effect of R-G interaction on the two-site binding mechanism for the PTH1 receptor. EXPERIMENTAL  MAP is a model amphipathic ␣-helix (Glu-Leu-Leu-Gln-Lys-Leu-Leu-Glu-Lys-Leu) substituted for residues 22-31 of PTHrP, a modification originally described for RS-66271 (40). The MAP-containing analogues used in this study differ from the originally described RS-66271 by extension of the C terminus to residue 36 and by substitution of position 36 with a tyrosine residue to enable radioiodination. The letters "b," "r," and "h" designate the peptide sequence as bovine, rat, or human, respectively. The peptides were dissolved in 10 mM acetic acid, with the peptide concentration calculated using the peptide content and weight provided by the supplier. Aliquots were stored at Ϫ80°C and used once. 125    were prepared using chloramine T as catalyst, and the mono-iodinated peptide (2000 Ci/mmol) was purified by high pressure liquid chromatography, as described previously (41).
Cell Culture, Transfection, and Isolation of Cell Membranes-HEK293 cells stably expressing the human PTH1 receptor were grown in G418-containing media as described previously (42). For assays of cAMP accumulation cells were transferred to polyornithine-coated 96well tissue culture plates 1 day prior to assay (25,000 cells/well). For preparation of cell membranes cells were grown in 15-cm tissue culture plates. For transfection COS-7 cells were grown to confluence, as described previously (41), in 15-cm tissue culture plates. Cells were transfected using the DEAE-dextran method as described previously (41) with 25 g of a plasmid (pCDNA.1) encoding the human PTH1 receptor (6), in the absence or presence of varying amounts of a plasmid encoding a G-protein ␣-subunit. (In the cotransfection of receptor and G-protein the DNA was mixed before addition of DEAE-dextran.) The G-proteins used were wild-type G␣ s in pCDNA.I/Amp and a mutant G-protein, G␣ s (␣ 3 /␤ 5 ), in which five residues in rat G␣ s were substituted for residues in the corresponding positions of rat G␣ i2 (N271K/K274D/R280K/ T284D/I285T), also in pCDNA.I/Amp (43). The mutations decrease the ability of the G-protein to activate adenylyl cyclase but increase the affinity of receptor for G-protein (43). For the experiment in Fig. 3B, COS-7 cells were transfected in 24-well plates with 200 g of pCDNA.1 plasmid DNA encoding the human PTH1 receptor or a human PTH1 receptor from which residues 24 -181 had been removed (PTH1⌬NT (21)). Previously described methods were used to isolate cell membranes from HEK293 cells and COS-7 cells (44,45). Membrane protein was quantified using the copper bicinchoninic acid method (Pierce) with bovine serum albumin as the standard.
Measurement of cAMP Accumulation-Previously described methods were used to measure cAMP accumulation in HEK293 cells expressing the PTH1 receptor (42) and in COS-7 cells expressing the PTH1 or PTH1⌬NT receptors (21  ) to membranes prepared from HEK293 cells or COS-7 cells expressing the PTH1 receptor (44). To each well of a polypropylene v-bottomed 96-well plate (Nunc, Naperville, IL), we sequentially added 25 l of buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 3 mM MgSO 4 , pH 7.5, supplemented with 0.3% nonfat dried milk powder, 100 M (4-(2-aminoethyl))benzenesulfonyl fluoride, and 1 g/ml bacitracin), 25 l of radioligand, 25 l of unlabeled ligand, and 50 l containing 3-10 g of membranes for HEK293 cell membranes or 10 -20 g for COS-7 cell membranes. The mixture was incubated for 3 h at 21°C prior to separation of bound and free radioligand by transfer to a Multiscreen filtration plate (MAHVN45, Millipore, Bedford, MA) on a vacuum manifold. The filters were washed three times with 150 l of buffer without supplements. Filters were pre-treated for 20 min on ice with buffer supplemented with 0.3% nonfat dried milk. Filters were removed, and radioactivity was determined in a Wallac 1470 Wizard gamma counter.
The amount of radioligand added varied from 70 to 220 pM for 125 I-PTH  and from 90 to 220 pM for 125 I-[MAP]PTHrP . For HEK293 cell membranes the total amount of PTH1 receptor present in the assay varied from 20 to 66 pM, whereas for COS-7 membranes the level varied from 90 to 180 pM. Total binding was less than 15% of the number of counts added in all cases. Nonspecific binding was measured by inclusion of a large excess of the unlabeled analogue of the radioligand (300 nM for PTH     in the equilibration phase of the assay. Data Analysis-Radioligand binding data were analyzed by nonlinear regression using Prism 2.01 (GraphPad Software Inc. San Diego, CA). Radioligand saturation of the PTH1 receptor was analyzed using a single affinity state saturation equation. Displacement of radioligand binding to the PTH1 receptor was analyzed using a single affinity state binding model (Equation 1) or a two affinity state model (Equation 2), where Y is the total counts/min bound in the presence of the competing ligand; NSB is nonspecific binding; SB is specific binding in the absence of the competing ligand, and X is the logarithm of the unlabeled ligand concentration.
Statistical comparison of multiple means was performed by single factor analysis of variance. Statistical comparison of two means was performed using a two-tailed Student's t test. Unless otherwise stated, data points in figures are presented as the mean Ϯ S.E. of triplicate measurements, and in some cases the error bars are enclosed within the symbol. , a Novel Radioligand Selective for the RG State of the PTH1 Receptor-To evaluate the effects of receptor-G-protein coupling on the mechanism of ligand binding to the PTH1 receptor, assays were developed for measuring the equilibrium binding affinity of ligands for the uncoupled receptor (R) and the receptor-Gprotein complex (RG). For the R state, the binding affinity of unlabeled ligands was measured by displacement of the antagonist radioligand 125 I-[Nle 8,18 ,Tyr 34 ]bPTH . Binding was measured in the presence of a high concentration (30 M) of GTP␥S, which binds to the G-protein and uncouples it from the receptor (47). (Binding of 125 I-PTH(3-34) is insensitive to GTP␥S (48).) For the RG state a radioligand was required that bound selectively to this receptor conformation. We previously showed that the PTHrP analogue RS-66271 (40) (11,12). The Cterminal portion of the ligand interacts with the large extracellular N-terminal domain (N domain) of the receptor, and the N-terminal region of the ligand interacts with the juxtamembrane domain (J domain) comprising the transmembrane ␣-helices and interconnecting loops. It is assumed that ligand (L) first interacts with the N domain (defined by the equilibrium association constant K N ) and then subsequently interacts with the J domain (defined by the isomerization constant K NJ ). This assumption of a sequential mechanism is based upon the much lower binding affinity of ligands for the J domain compared with the N domain. PTH(1-34) displays Ͼ1000-fold lower potency at a receptor from which the N-terminal domain has been removed (21). As a result, the equilibrium fraction of receptors with ligand bound only to the J domain (RL J ) is very small compared with the fraction with ligand bound to the N domain or bound to N and J domains (RL N and RL NJ , respectively). For this reason RL J has been excluded from the model. The equation describing equilibrium binding of L to R is shown in Equation 4,

Characterization of 125 I-[MAP]PTHrP
where K L ϭ K N (1 ϩ K NJ ) and [R tot ] is the total concentration of receptors. The equation is mathematically identical to a single affinity state binding isotherm, in which K L is the macro-affinity constant for receptor-ligand interaction. The K i value measured in the experiments in Fig. 4 provide a measure of 1/K L . For the 1-34 ligand fragment, the contribution of the micro-affinity constants K N and K NJ to the value of K L cannot be determined by measurement of equilibrium binding of L to R. To assess ligand binding to the N domain and J domain, ligands were used that interact almost exclusively with each of these domains (PTH(3-34) and PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), respectively, see text for details).   binding, but the total receptor level (labeled with 125 I-PTH(3-34)) was considerably reduced (Fig. 2D). This reduction of receptor expression probably resulted from cAMP-dependent down-regulation of the receptor; overexpression of G␣ s resulted in an increase of the basal cAMP level in COS-7 cells (from 0.84 Ϯ 0.04 to 2.28 Ϯ 0.01 pmol/well). In an attempt to minimize this reduction of receptor expression, we used a mutant G-protein (G␣ s (␣ 3 /␤ 5 ) (43)) that is impaired in adenylyl cyclase activation. This Gprotein mutant also displays higher receptor binding affinity, which stabilizes high affinity binding of agonist ligands. Overexpression of G␣ s (␣ 3 /␤ 5  The sites recognized by this radioligand (B max ϭ 180 Ϯ 50 fmol/mg) represented only a fraction of the sites recognized by 125 I-PTH(3-34) (820 Ϯ 90 fmol/mg), indicating that only a fraction (22%) of the total receptor population is coupled to G-protein in HEK293PTH1 membranes (Fig. 2B). Radioligand association experiments demonstrated a slow rate of association of 125 I-[MAP]PTHrP(1-36) (t1 ⁄2 of 1 h). Dissociation of the radioligand was complex (Fig. 2C). Three phases were detected in the absence of GTP␥S as follows: a very rapid phase (t1 ⁄2 Ͻ 10 s, representing 18 Ϯ 2% of specific binding), a more slowly dissociating phase (t1 ⁄2 ϭ 47 Ϯ 1 min, 50 Ϯ 1% of binding), and a pseudo-irreversible phase (32 Ϯ 2% of binding). 30 M GTP␥S removed most of the pseudo-irreversible component (now representing 4 Ϯ 1% of specific binding), reduced the fraction of binding and t1 ⁄2 of the slower phase (30 Ϯ 0%, 29 Ϯ 1 min), and increased the amount of binding dissociating at the very rapid rate (to 66 Ϯ 1%). These data suggest that GTP␥S converts the   to membranes prepared from COS-7 cells expressing the PTH1 receptor alone or coexpressed with G-protein ␣-subunits. COS-7 cells in 15-cm tissue culture plates were transfected with 25 g of plasmid DNA encoding the PTH1 receptor, with or without 100 g of plasmid encoding wild-type rat G␣ s or the mutant G-protein G␣ s (␣ 3 /␤ 5 ) (43), as described under "Experimental Procedures." Radioligand binding to 10 g of membrane protein was measured for each condition. The experiment was preformed twice with similar results, using membranes from different transfections in the two experiments. slow and pseudo-irreversible phases to the rapidly dissociating phase.
These findings indicate that the large majority of sites labeled by 125 I-[MAP]PTHrP(1-36) represent the RG complex. However the radioligand is not completely selective for the RG state. We note that a small fraction (21%) of the binding is not displaced by GTP␥S and that a similar fraction (18%) dissociates very rapidly. These components likely represent a low affinity state, since a low affinity state was detected in homologous-displacement experiments (Fig. 4D). (This low affinity state was not detected in the radioligand saturation experiments because the maximum concentration of radioligand that could be used was limited (to about 10 nM), due to the decrease of the specific binding:nonspecific binding ratio with increasing radioligand concentration.) This low affinity state might represent the uncoupled receptor or a GTP␥S-insensitive form of the RG complex. In support of the former, the affinity of the low affinity component in 125 I-[MAP]PTHrP(1-36) displacement assays was not significantly different from that for the R state, measured by displacement of 125 I-PTH  binding in the presence of GTP␥S (Table I). Association of the radioligands with the low affinity R state might not be directly detectable by measurement of equilibrium binding. This low affinity state may arise instead by spontaneous dissociation of G-protein from the RG complex.
Comparison of Ligand Binding Affinity for R and RG States of the PTH1 Receptor-The effect of RG coupling on ligand binding to the PTH1 receptor was determined by measuring the affinity of unlabeled ligands for the R and RG states of the receptor in HEK293PTH1 membranes. Ligand binding affinity for the uncoupled receptor was measured by displacement of 125 I-PTH  in the presence of 30 M GTP␥S. (This concentration of GTP␥S was near-saturating for removal of the RG state. It reduced specific binding of 125 I-[MAP]PTHrP  binding by 77%, close to the lower plateau value of the displacement curve in Fig. 2A of 78%.) Ligand binding to the RG state was measured by displacement of 125 I-[MAP]PTHrP  in the absence of exogenous guanine nucleotides. It is important to note that this assay provides only an approximate measurement of ligand affinity for RG, since a fraction of specific 125 I-[MAP]PTHrP  binding is insensitive to guanine nucleotides ( Fig. 2A), and a fraction of binding is pseudo-irreversible (Fig. 2C).
All agonist ligands tested (Fig. 3A) bound with significantly higher affinity to RG than R ( Fig. 4 and Table I). The extent of the preference for the RG complex over the R state varied from 12-fold for PTH(1-34) to 160-fold for PTHrP . In contrast, antagonist ligands did not appreciably discriminate RG from R ( Fig. 4B and Table I); PTH(7-34) did not significantly discriminate RG from R, and PTH(3-34) bound with a slightly higher affinity (2-fold) to the uncoupled receptor than the RG complex (Table I) Fig. 2A). Therefore K i(high) ( Table I) (1-36).) The 1-2 Region of PTH(1-34) Is a Determinant of R/RG Selectivity at the PTH1 Receptor-By assuming that the twosite model (Fig. 1, Equation 4) is appropriate for analysis of these ligand binding data (see below), the considerations above indicate that the macro-affinity (K N (1 ϩ K NJ )) for agonist ligands was higher at the RG complex than the R state. We next determined the extent to which this increase resulted from an increase of ligand affinity for the N domain (K N ) and/or for bound with 11-fold higher affinity to RG (displacement of 125 I-[MAP]PTHrP(1-36)) than to R (displacement of 125 I-PTH  in the presence of GTP␥S) (Fig. 4A and Table I). In contrast, PTH  bound with a similar affinity to the RG and R states of the PTH1 receptor ( Fig. 4B and Table I (50,51).) The same substitution increases the affinity of PTHrP for the PTH1 receptor (50,51). In this study, His 5 in [MAP]PTHrP(1-36) was replaced with Ile. The substitution modified the R/RG selectivity; [MAP]PTHrP(1-36) bound with 97-fold higher affinity to RG than R (Fig. 4D and Table I). The selectivity was reduced to 17-fold for [Ile 5 ,MAP]PTHrP(1-36) (Fig. 4E and Table I). Position 5 is therefore a determinant of R/RG selectivity for [MAP]PTHrP .
The replacement of His 5 by Ile also greatly increased the affinity of [MAP]PTHrP(1-36) for the uncoupled receptor (by 160-fold (Fig. 4, D and E, and Table I)), a larger effect than that previously observed for PTHrP (7- fold (50, 51)). The molecular basis of this affinity-enhancing effect is not known. We investigated the extent to which this effect was preserved at a receptor from which most of the N-terminal domain had been removed (PTH1⌬NT, residues 24 -181 removed (21)).
[MAP]PTHrP(1-36) failed to stimulate cAMP accumulation in COS-7 cells transfected with the PTH1⌬NT receptor (Fig. 3B). Substitution of His 5 with Ile restored the ability of the ligand to stimulate cAMP accumulation via the PTH1⌬NT receptor (Fig.  3B). This finding indicates that the enhancing effect of the substitution is preserved at a receptor from which the N domain has been largely removed, suggesting direct and/or indirect effects of residue 5 on ligand interaction with the J domain of the receptor.

PTH(3-34) Binds Predominantly to the N Domain of the Receptor and Does Not Appreciably Discriminate R and RG
States of the PTH1 Receptor-A direct method with which to address the R/RG selectivity of the N interaction would be to measure R/RG selectivity of a ligand that only interacts with the N domain of the receptor. PTH(3-34) is a candidate ligand; circumstantial evidence suggests that this ligand may bind predominantly to the N domain. This domain of the receptor is a determinant of the selectivity of N-terminally truncated analogues of PTH(1-34) for the human PTH1 receptor over the rat PTH1 receptor (13). Dissociation of 125 I-PTH(3-34) from the   binding, and by inhibition of 125 I-PTH  binding in the presence of GTP␥S Inhibition of radioligand binding to the PTH1 receptor in HEK293 cell membranes was measured as described under "Experimental Procedures." Data were fitted to equations assuming inhibition of radioligand binding to one (Equation 1) or two affinity states (Equation 2), and the best fit was determined using a partial F test. Displacement of 125 I-[MAP]PTHrP(1-36) binding provides a measurement of ligand binding to the G-protein-coupled state of the PTH1 receptor, whereas inhibition of 125 I-bPTH  binding in the presence of GTP␥S provides a measurement of ligand binding affinity for the uncoupled receptor. Statistical significance of the difference between pK i(GTP␥S) and pK i(high) was tested by Student's t test. NA, not applicable.  h Statistical significance was also tested for the difference between pK i(GTP␥S) and pK i(low) as 0.28.
i Statistical significance was also tested for the difference between pK i(GTP␥S) and pK i(low) as 0.21.
j Statistical significance was also tested for the difference between pK i(GTP␥S) and pK i(low) as 0.35. PTH1 receptor is mono-exponential, consistent with a single site interaction (in contrast to the complex dissociation of 125 I-PTH(1-34) which suggests more than one site of receptorligand interaction (48)). In this study we determined the extent to which PTH  binds to the J domain in a direct and quantitative fashion, by measuring the effect of blocking this domain on the binding affinity of the ligand. For this purpose we used [Ala 3,10,12 , Arg 11 ]rPTH(1-14) (PTH (1-14), which binds predominantly if not exclusively to the J domain (see above).
The fitted values are given in the legend to Fig. 5. The affinity of 125 I-PTH(3-34) for PTH(1-14)-occupied receptor was only slightly lower than the affinity of 125 I-PTH(3-34) for the nonoccupied receptor (3.9 and 3.1 nM, respectively). Occupancy of the J domain by PTH(1-14) therefore minimally affects the binding affinity of 125 I-PTH  for the PTH1 receptor, strongly suggesting that almost all the binding energy of this ligand is supplied by the N interaction (Fig. 1). PTH  does not appreciably discriminate the R and RG states of the receptor (Fig. 4B), indicating that the N interaction is insensitive to receptor-G-protein coupling.
Assessment of Ligand Conformation at the R and RG States of the PTH1 Receptor-The observation of higher affinity binding of agonist ligands to the RG versus the R state of the receptor implies a different conformation of the receptor when coupled to G-protein. Other ligand binding data in this study provide circumstantial evidence for a model of how the conformation of the receptor differs between the R and RG states. At the uncoupled receptor, PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH  bind almost independently of each other (Fig. 5A), suggesting that the receptor conformation is "open" enough to allow access of both ligands to their binding sites on the receptor. PTH(1-14) more effectively blocks 125 I-PTH  binding in the absence versus the presence of GTP␥S; the negative cooperativity between the binding of 125 I-PTH  and PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) is significantly greater in the absence of GTP␥S (Fig. 5A). Although this difference is small, only a small fraction of the receptor population is coupled to G-protein (22%), suggesting a considerable reduction of PTH(3-34) binding affinity resulting from PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) occupancy at the RG state. This suggests that the conformation of the receptor is more "closed," preventing simultaneous access of both ligands to their binding sites on the receptor. Further circumstantial evidence for a more closed conformation at the RG state is provided by the observation of pseudoirreversible binding of agonist radioligands to the RG state ( Fig. 2C (48)), suggesting that the ligand is trapped within the RG complex. We tested this hypothesis more directly by increasing the fraction of PTH1 receptors in the RG state and by testing the effect of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) on the binding of 125 I-PTH . COS-7 cells were transfected with either the PTH1 receptor alone or cotransfected with the PTH1 receptor and the mutant G-protein G␣ s (␣ 3 /␤ 5 ). Coexpression with G-protein substantially increased the fraction of PTH1 receptors in the RG state. In membranes prepared from COS-7 cells expressing the PTH1 receptor alone, a high affinity state for [MAP]PTHrP(1-36) could not be detected in a 125 I-PTH(3-34) displacement assay (Fig. 8A). In membranes containing the receptor and G␣ s (␣ 3/5 ), the high affinity state (IC 50 ϭ 270 pM) represented 40 Ϯ 4% of the 125 I-PTH  binding displaced by [MAP]PTHrP(1-36) (Fig. 8A). The additional high affinity binding produced by expression of the mutant G-protein was not entirely sensitive to GTP␥S; the nucleotide did not completely remove the high ). A high affinity state of binding was detected for the PTH1 receptor coexpressed with G␣ s (␣ 3 /␤ 5 ), both in the absence and presence of GTP␥S, the two affinity state fit providing an improvement (p ϭ 0.016 and 0.013, respectively). The experiment was performed three times for one transfection and once for a second transfection with similar results. B, inhibition of 125 I-PTH  binding to the PTH1 receptor by [Ala 3,10,12 ,Arg 11 ]rPTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), for the receptor expressed alone or coexpressed with G␣ s (␣ 3 /␤ 5 ). A single affinity state binding curve describes the displacement for the PTH1 receptor coexpressed with the G-protein in the absence of GTP␥S (Equation 1). We were unable to reliably fit data from the other experimental conditions to this equation. The experiment was performed twice for one transfection and once for a second transfection with similar results. affinity state (Fig. 8A). (The mechanism underlying this effect, which has also been observed for the ␤ 2 -adrenergic receptor (43), is not presently understood.) PTH(1-14) did not appreciably inhibit binding of 125 I-PTH  to the PTH1 receptor expressed alone; significant inhibition was observed at the highest concentration tested (100 M), but we were unable to reliably fit the data to a single affinity state binding model (Equation 1) or the simultaneous binding model (Equation 3). In contrast, PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) inhibited 125 I-PTH  binding to membranes containing the PTH1 receptor and G␣ s (␣ 3 /␤ 5 ), with a maximal inhibition of 48% (Fig. 8B) and with an affinity (190 nM) similar to that for the RG state measured in competition against 125 I-[MAP]PTHrP(1-36) (300 nM, Fig. 5C). Further evidence that this high affinity state represented the RG state was its removal by GTP␥S (Fig. 8B). These data suggest that PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) almost or completely inhibits the binding of 125 I-PTH  to the 40% of the receptor population in the RG state. This finding is in good agreement with the hypothesis of a more closed conformation of the RG state that prevents simultaneous binding of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH . DISCUSSION Previous studies employing modified receptors and ligands have indicated a two-site mode of ligand recognition by the PTH1 receptor. The C-terminal portion of the 1-34 fragment of PTH or PTHrP interacts with the extracellular N-terminal receptor region (N interaction), and the N-terminal portion of the ligand binds to the juxtamembrane domain of the receptor (J interaction) (11,12). In this study we examined the effect of different receptor conformational states arising from receptor-G-protein interaction on the molecular mechanism of ligand binding to the PTH1 receptor. The principle findings of this study are as follows. 1) Receptor-ligand interaction for the unmodified receptor is well described by the two-site model. Strong, direct evidence for this binding mode was provided by the novel observation of allosteric interactions between the binding of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH  to the receptor together with the inference of simultaneous receptor binding of these two ligands. 2) Agonist ligands bind with higher affinity to the RG state than to the uncoupled receptor, whereas antagonist ligands bind with similar affinity to these states. 3) The J interaction is stabilized by R-G coupling, whereas the N interaction is not appreciably affected by R-G interaction. 4) A more closed receptor conformation is suggested for the PTH1 receptor when coupled to G-protein. These findings are summarized in the model in Fig. 9.
The effect of R-G-coupling on ligand binding to the receptor was evaluated by comparing ligand affinity for the uncoupled receptor with the affinity for the RG complex. The former was measured by displacement of 125 I-PTH    , an analogue of the bone anabolic agent RS-66271 (40). The R/RG selectivity of this radioligand was demonstrated by the 78% reduction of binding produced by GTP␥S, which breaks down the RG complex (47), and by the increase of binding produced by coexpression of the PTH1 receptor with a mutant G␣ s G-protein, G␣ s (␣ 3 /␤ 5 ), that stabilizes the RG state ((43) Fig. 8). All agonist ligands bound with higher affinity to the RG complex than to the uncoupled receptor, whereas antagonist ligands bound with similar affinity to these states, confirming the findings of previous studies of the PTH1 receptor (48,53) and in agreement with a large number of studies of type I GPCRs (38). This finding suggests that agonists enhance G-protein activation by stabilizing the RG state. For other GPCRs, measurement of ligand binding to R and RG states for a range of agonists has been used to test models of ligand-receptor-G-protein interaction. In this study we obtained estimates of the difference of ligand affinity for R and RG states (K i(GTP␥S) /K i(high) ) and the fraction of 125 I-[MAP]PTHrP(1-36) binding displaced with high affinity by the unlabeled ligand (% (high) ). The binding data in this study cannot be accounted for by the simplest form of the ternary complex model, since there was no correlation between these two measurements (39). This discrepancy suggests additional states of the receptor (such as the active and inactive forms of the receptor proposed in the extended ternary complex model (38)) and/or additional states of the G-protein in complex with the receptor. With respect to the latter, the role of the G-protein ␤␥ dimer in agonist binding to the PTH1 receptor remains to be determined. For type I GPCRs the ␤␥ dimer does not detectably affect agonist binding alone but is required together with the ␣-subunit for the detection of the high affinity agonist binding ternary complex state (54,55).
Since the macro-affinity of agonist ligands was increased by R-G interaction, we next investigated the extent to which the micro-affinity constants within the two-site binding mechanism (K N and/or K NJ ) were affected by R-G coupling. Modification of the N-terminal region of the ligand altered the RG/R binding selectivity as follows. 1) Removal of the two N-terminal residues from PTH(1-34) resulted in the loss of RG/R selectivity. 2) Replacement of His 5 of [MAP]PTHrP(1-36) with Ile reduced the RG/R selectivity from 97-to 17-fold, demonstrating a previously unknown role for residue 5 in the control of RG/R binding selectivity. These findings suggest that the affinity of ligand interaction with the J domain is increased by RG interaction, a hypothesis strongly supported by the detection of selective binding of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) to the RG state. In contrast ligand binding to the N domain is not appreciably sensitive to RG coupling; PTH(3-34) bound with similar affinity to the R and RG states of the receptor. Therefore, agonists enhance R-G interaction through ligand interaction with the J domain (K NJ ) and not through ligand binding to the N domain (K N ) (Fig. 9).
An increase of ligand affinity for RG compared with R indicates different receptor conformations at the G-protein-coupled and -uncoupled receptor states. At the uncoupled receptor, the allosteric binding data suggest than the conformation is open enough to permit the simultaneous binding of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH . For the RG state, three observations are consistent with the hypothesis of a more closed receptor conformation. 1) Agonist binding is pseudo-irreversible, suggesting that the ligand is trapped within the ligand-receptor-G-protein complex (48). 2) PTH(1-14) produces a greater reduction of the binding affinity of PTH  at the RG state, suggesting that simultaneous binding of one ligand better occludes binding of the second. 3) PTH  reduces the E max of PTH(1-14)-stimulated cAMP accumulation. This suggests a reduced ability of the PTH1 receptor to adopt an active conformation that couples to G-proteins when both PTH  and PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) are bound to the receptor. The terms open and closed are used operationally in this model. We offer no structural interpretation of the postulated open and closed states, since such an interpretation is beyond the scope of the present data. The hypothesis, currently based on indirect measurements of receptor conformation (ligand binding data), requires more direct examination.
Whereas this study has focused on the conformation of the receptor, the conformation of the ligand when bound to the receptor is largely unknown. For the free peptide, a recent crystal structure of PTH  indicates an extended helical conformation (33). NMR studies of PTH and PTHrP under secondary structure-inducing conditions are consistent with an ␣-helix in the N-and C-terminal portions linked by a region of variable structure (34,56). When bound to the receptor some studies suggest an extended ␣-helical ligand conformation (57), whereas other studies suggest tertiary interactions between the N-and C-terminal ligand domains (29). In this study, a comparison of the binding of PTH  and PTH(7-34) is difficult to reconcile with the extended conformation hypothesis. PTH(7-34) binds with much lower affinity to the PTH1 receptor than PTH . However, the weak effect of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) binding on the affinity of PTH  suggests that very little of the binding energy of PTH  is provided by receptor interactions involving the 3-14 portion of the ligand. As a result, the large loss of affinity resulting from deletion of resides 3-6 is difficult to explain by the loss of a strong direct interaction of this region of the ligand with the juxtamembrane region of the receptor. The effect could be accounted for by the loss of intramolecular stabilization interactions within the ligand, which result in the reduction of affinity of the 15-34 ligand region with the receptor.
In conclusion, for the first time we have extended the twosite model to take into account different conformations of the PTH1 receptor, the R and RG states. Agonist ligand interaction with the J domain of the PTH1 receptor discriminates the RG state from the R state suggesting that the J interaction increases receptor-G-protein interaction and enhances subsequent second messenger generation. Ligand binding to the N domain is insensitive to R-G interaction. Finally, the increase of agonist affinity for the RG state may result from a "closure" of the receptor conformation. Given the commonality of the low resolution binding mechanism for type II GPCRs, these findings may well be relevant to an understanding of the signal transduction mechanism of other members of this receptor family.