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J. Biol. Chem., Vol. 275, Issue 35, 27274-27283, September 1, 2000

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Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor

N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY^*

Sam R. J. Hoare, Janet A. Clark, and Ted B. Usdin

From the Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, Bethesda, Maryland 20892

Received for publication, May 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tuberoinfundibular peptide of 39 residues (TIP39) and the parathyroid hormone-2 (PTH2) receptor form part of an extended family of related signaling molecules that includes the PTH1 receptor, which responds to PTH and PTH-related protein. TIP39 does not appreciably activate the PTH1 receptor, but in this study it is shown to bind the receptor with moderate affinity (59 nM). In this study, we investigated the molecular determinants of both ligand and receptor for the PTH2 receptor selectivity of TIP39 and quantitatively evaluated the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. A chimeric receptor composed of the N-terminal extracellular domain of the PTH1 receptor and the remainder (juxtamembrane domain) of the PTH2 receptor (P2-NP1) was fully activated by TIP39 (E_max = 98% of the rPTH-(1-34), E_max, EC₅₀ = 2.0 nM). This receptor chimera bound TIP39 with an equivalent affinity to the wild-type PTH2 receptor (2.3 and 2.0 nM, respectively). The reciprocal chimeric receptor (P1-NP2) was not activated by TIP39 and bound the ligand with an affinity equivalent to that of the PTH1 receptor. Thus, the juxtamembrane receptor domain specifies the signaling and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. Removing six N-terminal residues of TIP39 eliminated activation of the PTH2 receptor and reduced binding affinity 70-fold. In contrast, this truncation increased affinity for the PTH1 receptor 10-fold, reversing the PTH2/PTH1 receptor binding selectivity and resulting in a high affinity interaction of TIP-(7-39) with the PTH1 receptor (6 nM). These findings can be explained by a strong interaction between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH2 receptor, with the corresponding domain of the PTH1 receptor acting as a selectivity barrier against high affinity binding of TIP39. As a result, TIP-(7-39) is a highly potent, selective antagonist for the PTH1 receptor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tuberoinfundibular peptide of 39 residues (TIP39)¹ is a recently discovered neuropeptide that was purified from bovine hypothalamus on the basis of its ability to activate the PTH2 receptor (1). TIP39 is a good candidate for the PTH2 receptor's endogenous ligand. It strongly activates the human, rat, and zebrafish² PTH2 receptors (1). PTH also strongly activates the human PTH2 receptor (2), but it is only a weak partial agonist for the rat (3) and zebrafish² receptors. The physiological roles of TIP39 and the PTH2 receptor are currently being investigated. The PTH2 receptor is most abundant in the nervous system. Its expression is relatively high in the hypothalamus, where nerve terminals in the median eminence and cell bodies in the periventricular nucleus have particularly high receptor levels, suggesting a role in the modulation of pituitary function (1). PTH2 receptor concentration in the superficial lamina of the spinal cord dorsal horn suggests a role in the modulation of pain perception (1). In the periphery, the receptor is expressed by discrete cells in a number of tissues including pancreatic islet somatostatin cells, heart and vascular muscle cells, and cells within bronchioles and vasculature in the lung (4).

The PTH2 receptor and TIP39 form a part of an extended family of related receptors and ligands (1). The human PTH2 receptor shares 51% amino acid sequence identity with the human PTH1 receptor. The PTH1 receptor mediates the principal actions of PTH (elevation of blood calcium levels) and PTHrP (a locally acting autocrine/paracrine factor and developmental regulator) (5, 6). Both PTH receptors belong to the type II family of G-protein-coupled receptors that respond to peptide modulators, including calcitonin, glucagon, secretin, and vasoactive intestinal polypeptide. The similarity identified for PTH receptors extends to their ligands (Fig. 1). Five residues are identical when the sequences of TIP39, PTH, and PTHrP are aligned. TIP39 is somewhat more similar to PTH. Seven of the 19 C-terminal amino acids are identical between bovine TIP39 and PTH from most species. The PTH2 and PTH1 receptors, together with their ligands, have presumably evolved to selectively mediate different physiological functions. In this regard, the PTH1 receptor mediates the responses to PTH and PTHrP (6) but does not respond to TIP39 (1), whereas the PTH2 receptor responds to TIP39 and perhaps PTH but not to PTHrP (1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence alignment of bovine TIP39 with the N-terminal sequence of bPTH and human PTHrP (hPTHrP). Residues common to all three sequences are boxed. Residues common to only bTIP39 and bPTH are enclosed by the dashed box, and additional residues common to only bPTH and PTHrP are underlined.

The molecular basis of PTHrP selectivity for the PTH1 receptor over the PTH2 receptor has been studied extensively (7-11). For the PTH1 receptor, these and other selectivity studies (12-16) have been used to propose models of the molecular basis of receptor-ligand interaction (17, 18). The principal biological activities of PTH and PTHrP are retained in N-terminal fragments of approximately 34 residues. The data are consistent with a "two-site" model; amino acid residues in the N-terminal extracellular domain of the PTH1 receptor interact with the C-terminal region of the bioactive fragments of PTH and PTHrP. In the second interaction, the N-terminal portion of PTH and PTHrP interacts with the juxtamembrane domain of the PTH1 receptor, leading to receptor activation. This model has been proposed for other type II G-protein-coupled receptors (19-22). Receptor-ligand cross-linking studies have confirmed this binding orientation for ligand binding to the PTH1 receptor (17, 23, 24) and also for PTH binding to the human PTH2 receptor (25).

The receptor interactions of TIP39 have not previously been examined, beyond the initial observation of selective activation of the PTH2 receptor. We have now begun investigating the molecular basis of TIP39 selectivity for the PTH2 receptor over the PTH1 receptor. Previous studies of PTH receptors and other type II G-protein-coupled receptors have demonstrated the involvement of particular domains or amino acid residues in binding or activation but have not taken into account the effects of receptor-G-protein coupling in evaluating their contribution to ligand binding affinity. In this study, we have considered the nature of receptor states in evaluating the contributions of binding determinants to receptor selectivity. This has enabled us to quantitatively evaluate the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. Coupled with the conformational characterization of TIP39 presented in the accompanying paper (41), these data provide structural insight into the the molecular interactions of this new receptor-ligand system.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Peptides-- The following peptides were purchased from Bachem (Torrance, CA) or Peninsula Laboratories (Belmont, CA): rPTH-(1-34), [Nle^8,21,Tyr³⁴]rPTH-(1-34) amide, [Nle^8,18,Tyr³⁴]bPTH-(3-34) amide, PTHrP-(1-34), and human glucagon-(1-29). bTIP39 was obtained from AnaSpec Inc. (San Jose, CA) or Biomolecules Midwest (Waterloo, IL). The letters "r" and "b" designate the peptide sequence as rat and bovine, respectively. The peptides were dissolved in 10 mM acetic acid, with the concentration calculated using the peptide content and weight provided by the supplier. Aliquots were stored at 80 °C and used once. N-terminally truncated TIP39 analogues were purchased from Biomolecules Midwest, purified by HPLC, and quantified using the copper bicinchoninic acid method (Pierce) with TIP39 as the standard. ¹²⁵I-cAMP was obtained from NEN Life Science Products, and Na¹²⁵I (2000 Ci/mmol) was from ICN Biomedicals (Costa Mesa, CA). [3-¹²⁵I-iodotyrosyl¹⁰]glucagon (2000 Ci/mmol) was from Amersham Pharmacia Biotech. Lactose peroxidase was obtained from Sigma. Cell culture supplies were obtained from Life Technologies, Inc. except for Dulbecco's modified Eagle's medium, which was from Mediatech (Herndon, VA).

Preparation of Radioligands-- The radioligands ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34) and ¹²⁵I-[Nle^8,18,Tyr³⁴]bPTH-(3-34) were prepared using chloramine T as catalyst and the di-iodinated peptide (4000 Ci/mmol) purified by HPLC, as described previously (9). ¹²⁵I-TIP39 was prepared using the lactose-peroxidase method (27). TIP39 (5 µg in 5 µl of reaction buffer (0.1 M sodium acetate buffer, pH 6.5)) was dispensed into a siliconized microcentrifuge tube, followed by the sequential addition of 0.5 mCi of Na¹²⁵I, 5 µl of 20 µg/ml lactose peroxidase in reaction buffer, and 45 µl of reaction buffer. After mixing, 10 µl of 0.001% H₂O₂ was added. After 20 min at room temperature, the reaction was terminated by the addition of 0.5 ml of reaction buffer supplemented with 0.1% sodium azide. After a further 5 min, 0.5 ml of reaction buffer supplemented with 1 M NaCl, 0.1% bovine serum albumin, and 1% potassium iodide was added. The radioligand was then desalted using a C18 cartridge and purified by high pressure liquid chromatography. The radioactive peak fractions corresponded with a single peak of UV absorbance.

Plasmid Constructions-- The PTH2/PTH1 receptor chimeras have been described previously (9). Chimeric receptors and their parent wild-type receptors contain a sequence encoding a 12-residue hemagglutinin tag inserted at the 3'-end of the coding sequence. The chimeric receptors were constructed by exchanging residues 215-594 of the PTH1 receptor with residues 172-550 of the PTH2 receptor. Amino acids 62-106 (encoded by exon E2 of the PTH1 receptor gene) were removed from the PTH1 receptor used for construction of these chimeras to facilitate comparisons with the PTH2 receptor, which lacks a homologous sequence (9). TIP39 displayed an indistinguishable activation and ligand binding profile for the exon-deleted and full-length forms of the PTH1 receptor (data not shown). A slightly different chimeric receptor nomenclature was used in this study compared with the study of Clark et al. (9). P1-NP2 is the same construct as P_rP-NP₂, and P2-NP1 corresponds to P₂-NP_rP.

Chimeric PTH2/glucagon receptors were constructed by exchanging the N-terminal extracellular domain between the hemagglutinin-tagged PTH2 receptor in pcDNA1/Amp and the human glucagon receptor in pCI.neo (28). A BstZ17I restriction site was engineered into the human glucagon receptor sequence by converting Cys⁴³⁵ to thymidine, using the GeneEditor Site-directed Mutagenesis System (Promega, Madison, WI) according to the manufacturer's protocol, allowing the first 443 base pairs of the coding sequence of the PTH2 receptor and the first 434 base pairs of the glucagon receptor to be exchanged as BstZ17I/XbaI fragments.

Cell Culture and Transient Receptor Expression in COS-7 Cells-- COS-7 cells were grown as described previously (9). For cAMP accumulation assays, COS-7 cells were transfected as described previously (9) except that transfections were performed in 10-cm tissue culture dishes using 10 µg of plasmid DNA. The cells were transferred following trypsinization to 96-well plates at a density of 50,000 cells/well the following day. Cells were used for cAMP accumulation assays 3 days after transfection. For preparation of transfected COS-7 cell membranes, confluent 15-cm tissue culture plates were transfected with 30-100 µg of DNA, and cells were harvested 3 days after transfection. HEK293 cells stably expressing the human PTH2 or PTH1 receptors were grown as previously (29) and transferred to polyornithine-coated 96-well tissue culture plates 1 day prior to assay.

Measurement of Ligand-stimulated cAMP Accumulation-- Ligand-stimulated accumulation of cAMP was measured as described previously (3), using a radioimmunoassay to quantify cAMP (9).

Isolation of Cell Membranes-- P2 membrane preparations from HEK293 cells expressing the human PTH2 and PTH1 receptors were isolated as described previously (30). COS-7 cell membranes were prepared using a modified procedure. Cells were washed with 10 ml of PBS/plate and mechanically dislodged in 10 ml of 4 mM EDTA in phosphate-buffered saline. Cells were centrifuged at 1000 × g for 10 min, and the cell pellet was suspended in lysis buffer (10 mM Tris, 2 mM EDTA, 6 mM MgCl₂, and 100 µM (4-(2-aminoethyl))-benzenesulfonylfluoride, pH 7.5) using 32 ml of lysis buffer for five confluent 15-cm plates of cells. After 1 h at 4 °C, 8 ml of 1.25 M sucrose was added, and cells were immediately homogenized by 50 strokes with a Dounce homogenizer. The homogenate was then centrifuged at 1000 × g for 10 min to remove unbroken cells and larger debris. Cell membranes were collected by centrifugation, quantified, and stored as described previously (30).

Radioligand Binding Assays-- In these assays, the binding of a range of concentrations of an unlabeled ligand was measured by displacement of radioligand binding. Three methods were employed. An assay employing centrifugation to separate bound and free radioligand was used to accurately measure ligand binding parameters (30). A higher through-put method employing rapid filtration was used to generate comparative ligand binding data (30). Whole-cell binding assays (9) were used to measure radioligand binding to chimeric PTH2/glucagon receptors, since this assay provides the highest total binding/nonspecific binding ratio, important for detecting lower affinity binding of radioligands. In all these assays, a very low concentration of radioligand was used so that the IC₅₀ closely approximates the ligand affinity.

In the centrifugation assay, cell membranes (45-50 µg), radioligand (100,000-300,000 cpm), and unlabeled ligand were incubated in a final volume of 1 ml of assay buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 3 mM MgSO₄, pH 7.5, supplemented with 0.3% nonfat dried milk powder, 100 µM (4-(2-aminoethyl))-benzenesulfonylfluoride, and 1 µg/ml bacitracin) for 2 h at 21 °C. Membranes were collected at 18,000 × g, the surface of the pellet was gently washed, and the radioactivity was counted as described previously (30). For the PTH1 receptor, ¹²⁵I-[Nle^8,18,Tyr³⁴]bPTH-(3-34) was used as radioligand at a final concentration of approximately 20-32 pM. The PTH2 receptor was labeled with ¹²⁵I-TIP39 (24-52 pM, assuming monoiodination of TIP39 using the lactose peroxidase method) and ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34) (14-28 pM). Less than 20% of the total radioligand added was bound within the membrane pellet. For ¹²⁵I-TIP39 binding to the PTH2 receptor in HEK293 membranes, this requirement necessitated the use of 15 µg of membrane protein from transfected cells, made up to 45 µg with membranes from nontransfected HEK293 cells. (Greater than 50% of the total radioligand was bound if all of the membrane in the incubation was from transfected cells).

In the filtration assay 5-10 µg of membrane protein, 50,000-100,000 cpm of radioligand (56-112 pM for ¹²⁵I-TIP39 and 28-56 pM for ¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34)), and unlabeled ligand were incubated for 2 h at 21 °C. Membranes were harvested as described (30). Total binding was less than 15% of the total amount of radioactivity added. The whole-cell binding assay was performed as described previously (9).

Data Analysis-- Concentration dependence data for ligand-stimulated cAMP accumulation and displacement of radioligand binding were analyzed using the following four-parameter logistic equation using Prism 2.01 (GraphPad Software Inc., San Diego, CA),

(Eq. 1)

where X represents the logarithm of the ligand concentration and n_H represents the pseudo-Hill slope. For cAMP accumulation, y represents the amount of cAMP produced at a given peptide concentration, min is the cAMP level in the absence of ligand, and max is the maximum level produced. For inhibition of radioligand binding, y is the cpm bound at a given unlabeled ligand concentration, min is nonspecific binding (measured in the presence of a high concentration of the unlabeled version of the radiolabeled ligand), and max is total binding (measured in the absence of unlabeled ligand). Statistical comparison of multiple means was performed initially by single factor analysis of variance followed by post hoc analysis with the Newman-Keuls test. Statistical comparison of two means was performed using a two-tailed Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding of TIP39 and rPTH-(1-34) to PTH2 and PTH1 Receptors-- In HEK293 cells, the stably expressed human PTH2 receptor (293PTH2 receptor) is potently activated by TIP39 (EC₅₀ = 0.44 nM) and by rPTH-(1-34) (EC₅₀ = 58 pM, E_max = 85% of the response to TIP39), whereas PTHrP-(1-34) is much less potent (Fig. 2A). The human PTH1 receptor stably expressed in HEK293 cells (293PTH1 receptor) is potently activated by rPTH-(1-34) and PTHrP-(1-34) (EC₅₀ values of 0.50 and 0.44 nM, respectively) but is not appreciably activated by TIP39 (Fig. 3A). TIP39 therefore selectively activates the PTH2 receptor in HEK293 cells. This activation profile closely resembles that of the receptors transiently expressed in COS-7 cells (1). It is possible that TIP39 binds to the PTH1 receptor but fails to activate it. It is also not clear how closely related are the concentration dependences of TIP39 activation and binding. We therefore measured the binding of TIP39 to PTH1 and PTH2 receptors. The binding assays were performed in the absence and presence of 10 µM GTPS to determine whether ligand binding was sensitive to receptor-G-protein (R-G) coupling.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Comparison of human PTH2 receptor activation and binding by TIP39 and rPTH-(1-34). The 293PTH2 receptor was used for these experiments. A, stimulation of cAMP production in intact cells by TIP39 (), rPTH-(1-34) (), and PTHrP-(1-34) (). B, inhibition of 125I-TIP39 binding to isolated cell membranes by TIP39. C, inhibition of 125I-TIP39 binding to cell membranes by [Nle8,21,Tyr34]rPTH-(1-34). Binding of the ligands was measured in the absence () and presence () of 10 µM GTPS, using the centrifugation binding assay described under "Experimental Procedures." Specific 125I-TIP39 binding was defined as the difference between total binding (no unlabeled ligand present) and nonspecific binding (the lower plateau of the binding curve for B and 125I-TIP39 binding measured in the presence of 1.00 µM unlabeled TIP39 for C).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Comparison of PTH1 receptor activation and binding by TIP39 and rPTH-(1-34). The 293PTH1 receptor was used for these experiments. A, stimulation of cAMP production in intact cells by TIP39 (), rPTH-(1-34) () and PTHrP-(1-34) (). B, inhibition of 125I-[Nle8,21,Tyr34]rPTH-(1-34) binding to isolated cell membranes by TIP39 in the absence () and presence () of 10 µM GTPS, using the centrifugation binding assay described under "Experimental Procedures." Specific [Nle8,18,Tyr34]bPTH-(3-34) binding was defined as the difference between total binding (no unlabeled ligand present) and nonspecific binding (measured in the presence of 300 nM unlabeled [Nle8,18,Tyr34]bPTH-(3-34)).

¹²⁵I-[Nle^8,21,Tyr³⁴]rPTH-(1-34) (¹²⁵I-rPTH-(1-34)) has been used previously as a radioligand for the PTH2 receptor (9, 10), but we found that the signal-to-noise ratio was low in membrane binding assays (typically 3.5- and 2-fold in the absence and presence of GTPS, respectively). Since TIP39 is a potent agonist for the PTH2 receptor, we evaluated it as a radioligand. TIP39 contains a tyrosine residue at position 29 that can be radioiodinated as well as a methionine residue at position 30 that can potentially be oxidized during iodination. TIP39 labeled in a chloramine-T catalyzed reaction did not bind detectably to the PTH2 receptor. ¹²⁵I-TIP39 prepared in a lactose peroxidase-catalyzed reaction bound to the PTH2 receptor in HEK293 membranes with a considerably higher signal-to-noise ratio than ¹²⁵I-rPTH-(1-34) (20- and 15-fold in the absence and presence of GTPS, respectively), and no specific binding was detected in membranes prepared from nontransfected HEK293 cells. Unlabeled TIP39 displaced ¹²⁵I-TIP39 binding to the PTH2 receptor with high potency (IC₅₀ = 0.59 nM, Fig. 2B, Table I). The presence of 10 µM GTPS produced a parallel 4.7-fold rightward shift of the binding curve, suggesting that TIP39 binds with higher affinity to the R-G complex than to the uncoupled receptor (Fig. 2B). The pseudo-Hill slope for TIP39 was significantly less than unity (Table I). No increase of the slope was observed by doubling or trebling the 2-h incubation time (data not shown). At the PTH1 receptor, TIP39 completely inhibited ¹²⁵I-[Nle^8,18,Tyr³⁴]bPTH-(3-34) binding with a moderate affinity of 59 nM, the binding curve described by a pseudo-Hill slope of unity (Fig. 3B, Table I). Binding was insensitive to GTPS, indicating that the ligand binds with indistinguishable affinity to the R-G and R states of the receptor (Fig. 3B, Table I). TIP39 therefore binds selectively to the PTH2 receptor over the PTH1 receptor. The peptide is a high affinity agonist of the PTH2 receptor and a moderate affinity antagonist of the PTH1 receptor. Binding selectivity was maintained in the presence of GTPS (Table I), indicating that this selectivity results from a stronger interaction with the PTH2 receptor and is not simply a result of receptor-G-protein coupling enhancing ligand affinity for this receptor.

View this table: [in this window] [in a new window]   Table I Binding of TIP39 and rPTH-(1-34) to PTH2 and PTH1 receptors in HEK293 membranes Binding of unlabeled TIP39 and [Nle8,18,Tyr34]rPTH-(1-34) (rPTH-(1-34)) was measured by displacement of radioligand binding using the centrifugation method described under "Experimental Procedures." Displacement data were fitted to a four-parameter logistic equation to obtain estimates of logIC50 and nH (pseudo-Hill slope). Maximum inhibition of specific binding was calculated as (maximum  minimum)/(maximum  nonspecific binding) × 100, where nonspecific binding was measured as described in the legends to Figs. 2 and 3. ND, not determined. Differences between two parameter values were tested statistically using a two-tailed Student's t test as follows: a, p = 0.020; b, p = 0.91; c, p = 0.033; d, p = 0.58; e, p = 0.57; f, p = 0.74; g, p = 0.13; h, p = 0.12; i, p = 1.0. Of the tests performed, the only significant differences were found between the IC50 values for TIP39 binding in the absence versus the presence of GTPS and between the IC50 values for rPTH-(1-34) binding in the absence versus the presence of GTPS.

The PTH2 receptor is activated with high potency by PTH as well as TIP39. We compared the binding of [Nle^8,21,Tyr³⁴]rPTH-(1-34) (rPTH-(1-34)) with that of TIP39. rPTH-(1-34) displaced ¹²⁵I-TIP39 binding to the PTH2 receptor with high affinity (IC₅₀ = 1.1 nM; Fig. 2C, Table I). GTPS produced a slight (1.5-fold) but statistically significant increase of IC₅₀ for rPTH-(1-34) but did not alter the slope (Table I). The human PTH2 receptor therefore binds two distinct peptide ligands with high affinity. Since the receptor bound ¹²⁵I-rPTH-(1-34), we next addressed whether the ligand binding parameters of unlabeled ligands were dependent upon the radioligand used. With ¹²⁵I-rPTH-(1-34) as the radioligand, the binding parameters of TIP39 and unlabeled rPTH-(1-34) were not significantly different from the values obtained using ¹²⁵I-TIP39 as the tracer (Table I). Both unlabeled ligands completely inhibited binding of both radioligands (Table I), consistent with identical or overlapping binding sites for TIP39 and rPTH-(1-34).

Activation of Chimeric PTH2/PTH1 receptors by TIP39 and rPTH-(1-34)-- Chimeric PTH2/PTH1 receptors were used to examine the molecular determinants of the receptor that specify the PTH2 receptor signaling selectivity of TIP39. These receptors were constructed by exchanging between the human PTH2 and PTH1 receptors a region comprising transmembrane domains 2-7 and the intervening loops (including the first intracellular loop) and the C-terminal tail, collectively referred to as the juxtamembrane domain. Chimeric and wild-type receptors were expressed in COS-7 cells, and ligand-stimulated cAMP accumulation was measured.

The four receptors studied (chimeric and wild type) produced an equivalent maximal accumulation of cAMP in response to rPTH-(1-34) and were activated with an equivalent potency (EC₅₀) by this ligand (Fig. 4, Table II). As described previously (1), the PTH2 receptor was fully and potently activated by TIP39 in COS-7 cells, whereas the ligand produced no detectable response at the PTH1 receptor (Fig. 4, Table II). A chimeric receptor made up of the juxtamembrane region of the PTH2 receptor and N-terminal extracellular domain of the PTH1 receptor (P2-NP1) was also fully activated by TIP39 (Fig. 4C, Table II); the maximal cAMP accumulation was 98% of that for rPTH-(1-34) at the same receptor. TIP39 activated this receptor with high potency (EC₅₀ = 2.0 nM), slightly lower than the potency of this ligand at the wild-type PTH2 receptor (EC₅₀ = 0.42 nM). The reciprocal chimera P1-NP2 containing the juxtamembrane domain of the PTH1 receptor was not detectably activated by TIP39 (Fig. 4D). These findings indicate that the juxtamembrane receptor region specifies the PTH2/PTH1 receptor signaling selectivity of TIP39.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Activation of chimeric PTH2/PTH1 receptors and wild-type PTH receptors by TIP39 and [Nle8,21,Tyr34]rPTH-(1-34). Wild-type and chimeric receptors were expressed in COS-7 cells. Ligand-stimulated cAMP was measured as described under "Experimental Procedures" (, TIP39; , [Nle8,21,Tyr34]rPTH-(1-34)). A, PTH2 receptor; B, PTH1 receptor. C, chimeric receptor composed of N-terminal domain and first transmembrane domain of the PTH1 receptor fused to the remainder of the PTH2 receptor (P2-NP1). D, chimeric receptor composed of the N-terminal domain and first transmembrane domain of the PTH2 receptor fused to the remainder of the PTH1 receptor (P1-NP2).

View this table: [in this window] [in a new window]   Table II Parameters of rPTH-(1-34) and TIP39 stimulation of cAMP production in COS-7 cells transiently expressing chimeric PTH2/PTH1 receptors and wild-type PTH receptors Adenylyl cyclase-stimulating activity of the ligands was measured, and the data were analyzed as described under "Experimental Procedures." *, values measured at 320 nM TIP39. Single-factor analysis of variance indicated that the logEC50 values of rPTH-(1-34) for the four receptors were not significantly different (p = 0.77) (a) and that the Emax values were not significantly different (p = 0.80) (b). c, P22-NP1 TIP39 logEC50 value significantly different from P2 value (p < 0.01).

Binding of TIP39 and rPTH-(1-34) to Chimeric PTH2/PTH1 Receptors-- We examined the molecular determinants of the receptor specifying TIP39's binding selectivity for the PTH2 receptor using the chimeric PTH2/PTH1 receptors described above. rPTH-(1-34) bound with high affinity to wild-type and chimeric PTH receptors in COS-7 membranes (Fig. 5, Table III), suggesting that the conformation of the chimeric receptors was not greatly disrupted compared with that of the wild-type receptors. The P2-NP1 chimera, comprised of the juxtamembrane domain of the PTH2 receptor and N-terminal region of the PTH1 receptor, bound TIP39 with high potency (IC₅₀ = 2.3 nM) that was not significantly different from the TIP39 IC₅₀ at the wild-type PTH2 receptor (2.0 nM) (Fig. 5, Table III). The reciprocal chimeric receptor (P1-NP2, containing the juxtamembrane domain of the PTH1 receptor) bound TIP39 with low affinity (IC₅₀ = 280 nM), comparable with the ligand's affinity for the wild-type PTH1 receptor (160 nM) (Fig. 5, Table III). Therefore, the juxtamembrane domain of the PTH2 receptor specifies the PTH2 receptor binding selectivity of TIP39 as well as specifying the signaling selectivity (Fig. 4, Table II). For the PTH2 and P2-NP1 receptors, the pseudo-Hill slope value was less than 1 whereas the value for the PTH1 and P1-NP2 receptors was approximately unity. TIP39 completely inhibited binding of ¹²⁵I-rPTH-(1-34) to the PTH1 and P1-NP2 receptors, and rPTH-(1-34) completely displaced ¹²⁵I-TIP39 binding to the PTH2 and P2-NP1 receptors (Table III).

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Binding of TIP39 and [Nle8,21,Tyr34]rPTH-(1-34) to chimeric PTH2/PTH1 receptors and wild-type PTH receptors. A, PTH2 receptor. B, PTH1 receptor. C, P2-NP1 receptor. D, P1-NP2 receptor. (See Fig. 4 legend for description of the receptor chimeras.) Membranes were prepared from COS-7 cells transfected with receptor cDNAs. Binding of unlabeled TIP39 () and [Nle8,21,Tyr34]rPTH-(1-34) () was measured by displacement of radioligand binding using the filtration binding assay described under "Experimental Procedures." 125I-[Nle8,21,Tyr34]rPTH-(1-34) was used to label PTH1 and P1-NP2 receptors and 125I-TIP39 for PTH2 and P2-NP1 receptors. Specific binding was defined as the difference between total binding (no unlabeled ligand present) and nonspecific binding (the lower plateau of the binding curve for homologous displacement assay or binding measured in the presence of a 1.00 µM concentration of the unlabeled analogue of the radioligand for heterologous displacement assays).

View this table: [in this window] [in a new window]   Table III Parameters for rPTH-(1-34) and TIP39 displacement of radioligand binding to chimeric PTH2/PTH1 receptors and wild-type PTH receptors Radioligand binding to membranes isolated from COS-7 cells transiently expressing the receptors was measured as described under "Experimental Procedures." Data were fitted to a four-parameter logistic equation to obtain estimates of IC50, Hill slope, and asymptotic maximum and minimum. Maximal inhibition values were calculated as (maximum  minimum)/(maximum  nonspecific binding) × 100, where nonspecific binding was measured in parallel using a 1.00 µM concentration of the unlabeled analogue of the radioligand. a, significantly different from the TIP39 values for P1 and P1-NP2 (p < 0.001). b, TIP39 values for P2 and P2-NP1 are not significantly different (p > 0.05). c, significantly different from the TIP39 values for P1 and P1-NP2 (p < 0.005). d, TIP39 values for P2 and P2-NP1 are not significantly different (p > 0.05).

The receptor states identified in these binding assays were evaluated using GTPS to promote receptor/G-protein dissociation (31). GTPS (10 µM) reduced radioligand binding by 53 ± 4%, 5 ± 2%, 69 ± 6%, and 63 ± 1% at the PTH2, PTH1, P2-NP1, and P1-NP2 receptors, respectively. Thus, for the chimeric receptors and the PTH2 receptor, the predominant state identified in these assays was the receptor-G-protein complex. The state of the PTH1 receptor identified in the assay cannot be defined unambiguously. However, the R-G coupling status of this receptor is not relevant to the evaluation of ligand binding selectivity given that TIP39 does not detectably discriminate the R-G complex from the uncoupled receptor (Fig. 3B, Table I). These considerations suggest that the juxtamembrane domain specifies TIP39's PTH2/PTH1 receptor binding selectivity under conditions in which the receptor-G-protein complex is the receptor state predominantly detected in the binding assay. The signal/noise ratio did not permit critical evaluation of the binding selectivity of the chimeric receptors in the presence of GTPS. However, in whole cell binding assays, in which the receptor is probably predominantly uncoupled from G-protein (26), the juxtamembrane domain again specified the PTH2/PTH1 receptor binding selectivity of TIP39 (IC₅₀ values for the PTH2, PTH1, P2-NP1, and P1-NP2 receptors of 3.3, 415, 5.4, and 1600 nM, respectively; graphical data not shown).

Effect of N-terminal Truncation of TIP39 on Stimulation of cAMP Production and Ligand Binding at PTH2 and PTH1 Receptors-- The above evaluation of chimeric PTH2/PTH1 receptors indicated that the juxtamembrane region of the receptor specified both the signaling selectivity and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. In studies of the orientation of ligand binding to type II G-protein-coupled receptors, the N-terminal region of the ligand has been shown to interact with the juxtamembrane domain of the receptor, leading to receptor activation and second messenger generation (8, 12, 13, 21). We therefore tested whether the N-terminal region of TIP39 was required for receptor activation by measuring the effects of removing residues from its N terminus on ligand-stimulated adenylyl cyclase activity. We also measured the extent to which the N-terminal region of TIP39 specifies the selective binding of the ligand to the PTH2 receptor by measuring the binding affinity of N-terminally truncated ligands for the PTH2 and PTH1 receptors.

At the 293PTH2 receptor, deletion of one, two, or four residues from the N terminus of TIP39 reduced the potency for stimulation of cAMP accumulation but did not affect the maximal ligand-stimulated adenylyl cyclase activity (Fig. 6A). Deletion of six N-terminal residues, producing TIP (7-39), resulted in the loss of detectable ligand-stimulated cAMP accumulation (Fig. 6A). The N-terminal region of TIP39 is therefore a determinant of PTH2 receptor activation. None of the truncated TIP39 analogues detectably activated the PTH1 receptor (Fig. 6B).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of N-terminal truncation of TIP39 on ligand-stimulated cAMP accumulation at PTH2 and PTH1 receptors. Adenylyl cyclase activity was measured in 293PTH2 cells (A) and 293PTH1 cells (B) as described under "Experimental Procedures" for TIP39 (), TIP-(2-39) (), TIP-(3-39) (), TIP-(5-39) (), TIP-(7-39) (), and rPTH-(1-34) ().

In radioligand binding assays, deletion of one, two, four, and six residues from TIP39 results in a progressive reduction of the ligand binding potency for the 293PTH2 receptor (Fig. 7A, Table IV). TIP-(7-39), which does not activate the PTH2 receptor, binds with 70-fold lower affinity to the PTH2 receptor than full-length TIP39 (Fig. 7A, Table IV). (GTPS (10 µM) reduced binding of ¹²⁵I-TIP39 to the PTH2 receptor by 62 ± 2%, indicating that the radioligand detects predominantly the receptor-G-protein complex of the PTH2 receptor in these assays.) In contrast, at the PTH1 receptor TIP-(7-39) binds with a 5.6-fold higher affinity than TIP39 (Fig. 7B, Table IV). The N-terminal region of TIP39 is therefore a determinant of TIP39's selective binding to the PTH2 receptor under conditions in which the G-protein-coupled receptor state is predominantly detected in the binding assay.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of N-terminal truncation of TIP39 on ligand binding to PTH2 and PTH1 receptors. Binding of unlabeled ligands was measured by displacement of radioligand binding to 293PTH2 membranes (A) and 293PTH1 membranes (B) using the filtration binding assay described under "Experimental Procedures." Under the conditions of the assay, the receptor-G-protein complex is the predominant receptor state detected. , TIP39; , TIP-(2-39); , TIP-(3-39); , TIP-(5-39); , TIP-(7-39). 125I-TIP39 was the radioligand for the PTH2 receptor, and 125I-[Nle8,21,Tyr34]rPTH-(1-34) was the radioligand for the PTH1 receptor. Specific binding was defined as in Fig. 5. In the curve-fitting analysis for TIP-(3-39), TIP-(5-39), and TIP-(7-39) at the PTH2 receptor, nonspecific binding was fixed at the binding measured in the presence of 1.00 µM TIP39.

View this table: [in this window] [in a new window]   Table IV Binding properties of N-terminally truncated TIP39 analogues at PTH2 and PTH1 receptors in HEK293 cell membranes Binding of truncated TIP39 analogues was measured by displacement of 125I-TIP39 binding to 293PTH2 membranes and 125I-[Nle8,21,Tyr34]rPTH-(1-34) binding to 293PTH1 membranes using the filtration binding assay described under "Experimental Procedures." Under the conditions of the assay, the receptor-G-protein complex is the predominant receptor state detected. Values were calculated as described for Table III. ND, a maximal inhibition value was not calculated for these experiments because the lower plateau was fixed at nonspecific binding in the curve-fitting analysis. a, significantly different from TIP39 logIC50 for the PTH2 receptor (p < 0.01). b, significantly different from TIP39 logIC50 for the PTH1 receptor (p < 0.005).

The effect of ligand truncation on receptor binding affinity was also measured at the G-protein-uncoupled receptor by measuring ligand binding in the presence of 10 µM GTPS. Under these conditions, TIP-(7-39) bound to the PTH2 receptor with a 32-fold lower binding potency than full-length TIP39 (Fig. 8A). In contrast, TIP-(7-39) bound with 12-fold higher affinity to the PTH1 receptor (Fig. 8B). The N-terminal region of TIP39 is therefore a determinant of PTH2/PTH1 receptor binding selectivity at the G-protein-uncoupled receptor state.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effect of N-terminal truncation of TIP39 on ligand binding to PTH2 and PTH1 receptors in the presence of 10 µM GTPS. Binding of TIP39 () and TIP-(7-39) () was measured by displacement of 125I-TIP39 binding to 293PTH2 membranes (A) and 125I-[Nle8,21,Tyr34]rPTH-(1-34) binding to 293PTH1 membranes (B) using the centrifugation binding assay described under "Experimental Procedures." This assay measures the affinity of ligands for the free receptor, uncoupled from G-protein. Specific binding was defined as in Fig. 5. The mean TIP-(7-39) logIC50 values for the PTH2 and PTH1 receptors were as follows (IC50 values in parentheses): 7.01 ± 0.04 (98 nM) and 8.30 ± 0.05 (5.0 nM), respectively.

In summary, removal of six residues from the N terminus of TIP39 reduces receptor binding affinity at the PTH2 receptor but increases binding affinity at the PTH1 receptor. As a result, the truncation reverses the PTH2/PTH1 receptor binding selectivity of TIP39, such that TIP-(7-39) is a selective, high affinity (<10 nM) antagonist of the PTH1 receptor and a weak antagonist of the PTH2 receptor.

Binding of TIP39 to Chimeric PTH2/Glucagon Receptors-- The PTH2/PTH1 receptor selectivity studies above suggest that the juxtamembrane region of the PTH2 receptor contributes strongly to the binding affinity of TIP39. However, the PTH1 receptor binds TIP39 with a moderate affinity and so does not provide a null background in which to measure the contribution of binding interactions to the overall affinity of the ligand. In particular, the PTH2/PTH1 selectivity experiments have not addressed the role of the N-terminal extracellular domain in the binding of TIP39. It is possible that the N-terminal region contributes to the interaction of TIP39 with both PTH2 and PTH1 receptors, but this interaction may not be detected because the selectivity experiments only address the molecular determinants of the difference of ligand affinity between the two receptors.

In order to more directly examine the molecular basis of TIP39 recognition by the PTH2 receptor, we measured TIP39 binding to chimeric PTH2/glucagon receptors. The human glucagon receptor expressed in COS-7 cells did not detectably bind TIP39 at ligand concentrations up to 1 µM (Fig. 9B). This receptor was not detectably activated by TIP39, but glucagon-(1-29) stimulated cAMP accumulation (data not shown), with a logEC₅₀ of 8.85 ± 0.02 (EC₅₀ = 1.4 nM) and an E_max of 4.3 ± 0.2 pmol/well (comparable with the E_max for TIP39 activation of the PTH2 receptor; see Table II). A chimeric receptor comprising the N-terminal domain of the PTH2 receptor and juxtamembrane region of the glucagon receptor (G-NP2) bound ¹²⁵I-TIP39 when expressed in COS-7 cells (Fig. 9C). Unlabeled TIP39 displaced this binding with a logIC₅₀ of 6.74 ± 0.42 (IC₅₀ = 182 nM; Fig. 9C). The TIP39 affinity of the G-NP2 receptor was 55-fold lower than that of the PTH2 receptor in COS-7 cells (Fig. 9A; logIC₅₀ = 8.48 ± 0.42, IC₅₀ = 3.3 nM). These data indicate that the N-terminal domain of the PTH2 receptor does contribute to TIP39 binding. The reciprocal chimeric receptor (P2-NG) detectably bound ¹²⁵I-glucagon but not ¹²⁵I-TIP39. Glucagon-(1-29) displaced binding of ¹²⁵I-glucagon to the P2-NG receptor (logIC₅₀ = 6.75 ± 0.14; Fig. 9D), whereas TIP39 did not inhibit binding of the radioligand to this receptor (Fig. 9D). The P2-NG receptor was weakly activated by TIP39 (EC₅₀ > 1 µM) but not by glucagon-(1-29), and the G-NP2 receptor was weakly activated by glucagon-(1-29) (EC₅₀ > 1 µM) but not by TIP39.

View larger version (38K): [in this window] [in a new window]   Fig. 9.   Binding of TIP39 to chimeric PTH2/glucagon receptors and PTH2 and glucagon receptors. A hemagglutinin-tagged PTH2 receptor (P2) (A), the human glucagon receptor (G) (B), a chimeric receptor comprising the N-terminal extracellular domain of the PTH2 receptor and the juxtamembrane region of the glucagon receptor (G-NP2) (C), and the reciprocal chimera (P2-NG) (D) were expressed in COS-7 cells. Binding of TIP39 () or human glucagon-(1-29) () was measured by displacement of radioligand binding (125I-TIP39 for P2 and G-NP2 and 125I-glucagon for G and P2-NG) using intact cells in 96-well plates, as described under "Experimental Procedures." The total 125I-TIP39 in A and C was 70,000 cpm, the total 125I-glucagon in B was 14,000 cpm, and the total 125I-glucagon in D was 47,000 cpm. Data points are mean ± S.E. of triplicate measurements. The experiments were performed three times with similar results, except for the glucagon receptor for which the experiment was performed twice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TIP39 is a good candidate for an endogenous ligand for the PTH2 receptor (1). This peptide ligand has previously been shown to selectively activate the PTH2 receptor and not the closely related PTH1 receptor. In this study, we found that TIP39 also displays binding selectivity for the PTH2 receptor. We have now identified molecular determinants of TIP39 and the PTH receptors that specify the PTH2 receptor selectivity of TIP39 and evaluated the contribution of these regions to the binding affinity of TIP39. The principal findings of this study are as follows. 1) The juxtamembrane domain of the receptor is a determinant of TIP39's signaling selectivity. 2) The N-terminal region of the ligand is required for PTH2 receptor activation. 3) Ligand binding selectivity is specified by the juxtamembrane domain of the receptor. 4) The N-terminal region of TIP39 is a determinant of ligand binding selectivity; removal of 6 residues from the ligand reverses the PTH2/PTH1 receptor selectivity such that TIP-(7-39) is a selective high affinity antagonist for the PTH1 receptor. 5) A chimeric receptor containing the N-terminal domain of the PTH2 receptor (G-NP2) bound TIP39 (with 55-fold lower affinity than the wild-type receptor), demonstrating that the N-terminal extracellular domain of the PTH2 receptor contributes to TIP39 binding. These findings are consistent with a model in which TIP39 binds with moderate affinity to the N-terminal extracellular domain of the PTH2 receptor, while an interaction between the juxtamembrane domain and N-terminal region of the ligand strongly stabilizes the receptor-ligand complex.

The molecular basis of TIP39's selective activation of the PTH2 receptor was investigated using chimeric PTH2/PTH1 receptors and truncated TIP39 analogues. The juxtamembrane domain was identified as the principal determinant of signaling selectivity (Fig. 4, Table II). The N-terminal region of TIP39 is required for activation of the PTH2 receptor; TIP-(7-39) did not detectably activate the receptor (Fig. 6A). These findings are consistent with a model in which the N-terminal region of the ligand interacts with the juxtamembrane domain of the receptor leading to receptor activation and second messenger generation. Such an interaction has been proposed for ligand activation for each type II G-protein-coupled receptor for which there are relevant studies (8, 9, 11, 12, 16, 21, 22, 33, 34), including interaction of the human PTH2 receptor with PTH (19, 25). In the evaluation of chimeric PTH2/PTH1 receptors, we found that the juxtamembrane domain did not just support full efficacy (E_max) of TIP39; the P2-NP1 chimera was activated by TIP39 with a potency (logEC₅₀) almost as high as that for the wild-type PTH2 receptor (Fig. 4, Table II). This finding suggested that the juxtamembrane domain of the PTH2 receptor may also contribute to the ligand binding selectivity of TIP39. Measurement of TIP39 binding to chimeric and wild-type receptors confirmed this hypothesis; the juxtamembrane region of the receptor specified the binding selectivity (Fig. 5, Table III).

The data from PTH2/PTH1 chimeric receptor experiments can be used to formulate binding models for TIP39 interaction with the PTH2 and PTH1 receptors. The findings of this study are consistent with the previously proposed two-site model for ligand interaction with type II G-protein coupled receptors; the juxtamembrane domain of the PTH2 receptor is required for activation (Fig. 6), and the N-terminal extracellular domain contributes to binding of TIP39, as demonstrated by the G-NP2 PTH2/glucagon receptor chimera (Fig. 9). A description of the model parameters is given in the legend to Fig. 10. Within the framework of this model, two types of interaction could contribute to the PTH2/PTH1 receptor binding selectivity of TIP39. 1) The juxtamembrane domain of the PTH2 receptor may interact with high affinity with the N-terminal region of TIP39 (resulting in a high value of K_J and/or K_NJ; Fig. 10). 2) The juxtamembrane domain of the PTH1 receptor may be incompatible with the N-terminal region of TIP39, such that this receptor domain acts as an affinity barrier preventing high affinity binding of TIP39 to the PTH1 receptor, reducing the overall affinity of the ligand (low value of K_J and/or K_NJ). We addressed these possibilities using the TIP39 analogues that had been truncated at the N terminus.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Two-site model of receptor-ligand interaction. In this model, two sites on the ligand (A) interact with two corresponding sites on the receptor (R). The C-terminal region of the ligand interacts with the N-terminal extracellular domain of the receptor (forming RAN, defined by the equilibrium association constant KN), and the N-terminal region of the ligand interacts with the receptor juxtamembrane domain (forming RAJ, defined by KJ). Following binding to either of these domains, ligand binding can be stabilized by interaction with the second domain of the receptor; the stabilization arising from interaction with the juxtamembrane region following binding to the N-terminal domain is defined as KNJ, and the reciprocal interaction is defined as KJN. The macroaffinity constant of the ligand, KA, is equal to (KN + KJ + KNKNJ). The stability constant KNJ can limit the macroaffinity of the ligand to (KN + KJ) as the value of KNJ approaches 0. For example, if KN is 109 M1 and KJ is 108 M1, then KNJ values of 0.01, 1, and 100 will give KA values of 1.11 × 109 M1, 2.10×109 M1, and 1.011×1011 M1, respectively (0.900 nM, 0.476 nM, and 9.89 pM when expressed as a equilibrium dissociation constants, respectively). This model is further elaborated in Ref. 39.

At the PTH2 receptor, TIP-(7-39) does not detectably activate the PTH2 receptor, so it may not appreciably interact with the juxtamembrane domain of the receptor. The postulated weakening of this interaction reduces binding affinity 36- and 70-fold at the R and R-G states, respectively (Figs. 7 and 8), suggesting that an interaction of the N-terminal region of TIP39 with the juxtamembrane domain of the PTH2 receptor contributes strongly to the overall binding energy of the ligand. This binding energy could result from a high value of K_J and/or K_NJ. We estimate the value of K_N for the R state within this model to be at most 10⁷ M¹ (at least 100 nM as a dissociation constant), since the affinity of TIP39 for the G-NP2 receptor in whole cells is 182 nM and the affinity of TIP (7-39) for the G-protein-uncoupled receptor is 98 nM. The lack of detectable TIP39 binding to the P2-NG receptor chimera suggests that K_J may not contribute the additional binding energy, implying that the high affinity interaction of TIP39 results from strong stabilization by interaction with the juxtamembrane region subsequent to interaction with the N-terminal domain (high K_NJ). However, a structural incompatibility between the PTH2 and glucagon receptors cannot be excluded as an explanation for the lack of TIP39 binding to the P2-NG receptor, so the prediction of a high K_NJ value requires further investigation. At the PTH1 receptor, TIP-(7-39) binds with 12-fold higher affinity than full-length TIP39 (Fig. 8). This suggests that an incompatibility between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH1 receptor impedes receptor-ligand interaction (low value of K_J and/or K_NJ), lowering the macroaffinity of the ligand. Removal of this affinity barrier increases K_J and/or K_NJ such that TIP-(7-39) binds with higher affinity to the PTH1 receptor. (A similar model has been proposed to account for selective ligand binding to other type II G-protein-coupled receptors (11, 16)). These postulated binding mechanisms are consistent with a structural model of TIP39 interaction with PTH2 and PTH1 receptors (see accompanying paper (41)). While we were able to use the model in Fig. 10 to investigate the microaffinity constants of receptor-ligand interaction, the model does not take into account potential structural modifications of the ligand arising from the N-terminal truncation. Such modifications could complicate the interpretation presented above if they affected binding of TIP39 with other regions of the receptor. This possibility requires further investigation.

The molecular determinants of TIP39's binding selectivity differ from those identified in selectivity studies of the PTH1 receptor. In almost every study of the PTH1 receptor, binding selectivity of PTH and PTHrP is specified by the N-terminal domain of the receptor and the C-terminal region of the ligand (9, 10, 12, 14). The juxtamembrane region of the PTH1 receptor does not appear to contribute greatly to the macroaffinity of the ligand at the R state; in the presence of GTPS, bPTH-(3-34), which does not appreciably stimulate cAMP accumulation at the PTH1 receptor, binds with similar affinity to the full agonist bPTH-(1-34) (36). In addition, most mutations in the juxtamembrane region of the PTH1 receptor that impair signaling produce only modest reductions of binding affinity (15, 37, 38). In contrast, the data in this study indicate that the juxtamembrane domain of the PTH2 receptor contributes strongly to the macroaffinity of TIP39; the PTH2/PTH1 receptor binding selectivity is specified by this receptor domain, and TIP-(7-39) binds with 32-fold lower affinity than full-length TIP39 at the uncoupled receptor (and with 70- fold lower affinity in assays where the R-G state was predominantly detected).

We observed that the binding of TIP39 to the PTH2 receptor was described by a pseudo-Hill slope of less than unity. A similarly shallow binding curve was observed in the presence of 10 µM GTPS and in assays where the incubation time was doubled or tripled, indicating, respectively, that the shallow slope did not result from R-G coupling or from incomplete equilibration. The two-site model proposed for type II G-protein-coupled receptors should yield a ligand binding curve with a Hill slope of unity at equilibrium (36, 39). Slopes deviating from unity have been commonly observed for the binding of glucagon to its receptor (reviewed in Ref. 40) and have been noted for the binding of ligands to the PTH1 receptor (19). To the best of our knowledge, there is no consensus on the mechanism that produces this effect. Hill slopes of <1 are possible within an extended version of the two-site model in which two molecules of ligand (L) can bind simultaneously to a single receptor (forming RL₂) (39). The shallow slope results from negative cooperativity in the binding of the second ligand molecule to the receptor. Hill slopes of <1 are also possible within a model that assumes dimerization of the ligand; binding of ligand to the receptor is inhibited by the binding of ligand to itself (in the formation of the ligand dimer L₂). Importantly, TIP39 binds with high affinity in the formation of RL in both of these models, the lower affinity shoulder of the binding curve arising from negative cooperativity or ligand dimerization. These hypothetical models require more direct examination.

Since TIP39, PTH, and PTHrP are related, discrete structural modifications of these peptides may provide useful ligands with altered binding and/or activation specificity (10). In this study, removing six residues from TIP39 changed it into a high affinity, selective antagonist for the PTH1 receptor. PTH1 receptor antagonists have been proposed as possible treatments for hypercalcemia, which results from elevation of the serum level of PTH or PTHrP (32, 35). TIP-(7-39) is a novel antagonist that is structurally distinct from truncated analogues of PTH and PTHrP that have been investigated previously as potential anti-hypercalcemic agents (32, 35).

In conclusion, we have identified determinants of TIP39's selectivity for the PTH2 receptor and evaluated their contributions to ligand binding affinity. In contrast to the closely related PTH1 receptor, the juxtamembrane domain of the PTH2 receptor and the N-terminal region of TIP39 are determinants of ligand binding selectivity. The binding of TIP39 to the PTH2 receptor is consistent with the two-site model of ligand interaction proposed for the PTH1 receptor, but with the juxtamembrane domain of the receptor contributing more strongly to the macroaffinity of the ligand.

	FOOTNOTES

^* 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 all correspondence should be addressed: Rm. 3D06, Bldg. 36, 36 Convent Dr. MSC4094, NIH, Bethesda, MD 20892-4094. Tel: 301-402-4161; Fax: 301-435-5465; E-mail: usdin@codon.nih.gov.

Published, JBC Papers in Press, June 14, 2000, DOI 10.1074/jbc.M003910200

² Hoare, S. R. J., Rubin, D. A., Jüppner, H., and Usdin, T. B. (2000) Endocrinology, in press.

	ABBREVIATIONS

The abbreviations used are: TIP39, tuberoinfundibular peptide of 39 residues; bTIP39, bovine TIP39; PTH, parathyroid hormone; rPTH, rat PTH; bPTH, bovine PTH; PTHrP, parathyroid hormone-related protein; G-protein, guanine nucleotide-binding regulatory protein; R-G, receptor-G-protein; GTPS, guanosine 5'-3-O-(thio)triphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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