Jean-Pierre Vilardaga, Irene Lin, and Robert A. Nissenson

Transcription

1 Analysis of Parathyroid Hormone (PTH)/Secretin Receptor Chimeras Differentiates the Role of Functional Domains in the PTH/ PTH-Related Peptide (PTHrP) Receptor on Hormone Binding and Receptor Activation Jean-Pierre Vilardaga, Irene Lin, and Robert A. Nissenson Endocrine Research Unit Veterans Affairs Medical Center and Departments of Medicine and Physiology University of California San Francisco, California The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r s N-terminal region, and the carboxy-core domain (transmembrane segments extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (camp induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60 100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues ) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues and particularly residues of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r s N-terminal region: one site in the amino-terminal half (residues 1 62) (site 1) that participates in high-affinity /01/$3.00/0 Molecular Endocrinology 15(7): Copyright 2001 by The Endocrine Society Printed in U.S.A PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues ) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the firstand third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r s helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation. (Molecular Endocrinology 15: , 2001) INTRODUCTION PTH plays a fundamental role in the regulation of calcium homeostasis in bone and kidney (1). PTH actions are

2 Structure-Function of PTH/PTHrP Receptor 1187 mediated by two related G protein-coupled receptors (GPCRs) that have been classified according to their different affinities for PTH-related peptide (PTHrP): the PTH1r (PTH/PTHrP receptor) that responds equally well to PTH and PTHrP; and the PTH2 receptor that responds to PTH (2, 3). These receptors belong to the class II GPCR family, which includes receptors for peptide hormones such as secretin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), glucagon, calcitonin, CRF, GLP-1, and several others (List of members of GPCR s class II is found in MSF.html). Agonist occupancy of these receptors leads to activation of adenylyl cyclase (via G s ), and in several cases [including the PTH1 receptor (PTH1r)] PI-specific phospholipase C (via G q ). The members of class II GPCRs share no sequence homology with members of the other classes of GPCRs (e.g. class I: rhodopsin-like receptors, peptide and glycoprotein hormone receptors; class III: metabotropic glutamate and Ca 2 -receptors; class IV: pheromone-like receptors, and class V: camp receptors). Class II GPCRs are characterized by 1) a large amino-terminal extracellular domain ( amino acids) that includes four strictly conserved cysteine residues (Fig. 1) that may participate in disulfide bridges (4); 2) the absence of the amino acid sequence DRY (or ERW) in the aminoterminal part of the second intracellular loop (highly conserved in class I GPCRs); 3) a distinctive distribution of transmembrane proline residues; and 4) the presence of a unique set of conserved transmembrane residues. The mechanisms of hormone binding and signal transduction for class II GPCRs are poorly understood. Structure-function studies of these receptors based on mutagenesis and construction of chimeric receptors point to an important role of the receptor s aminoterminal extracellular domain (N terminus) for agonist binding (5 10) and ligand discrimination (11, 12). An interaction between PTH(1 34) (a synthetic PTH fragment with the same pharmacological properties as PTH) and the PTH1r s N terminus has recently been supported by studies using photoaffinity cross-linking (13, 14), deletion mutagenesis (15), chimeric receptors (16, 17), and mutagenesis (18). Our previous studies involving the two chimeric receptors NPTH/SECr (the N terminus of the PTH1r attached to the remainder of the SECr) and NSEC/PTHr (the reciprocal chimeric receptor) support the view that the N-terminal extracellular domain as well as the Fig. 1. Amino Acid Sequence and Predicted Topological Structure of the Opossum PTH1r Arrow, Predicted cleavage site of the signal peptide. Gray circles, Conserved residues with the rat secretin receptor. Y, theoretical sites of N-glycosylation. [ ], boundaries of exon 2.

3 MOL ENDO 2001 Vol. 15 No carboxy-core of the PTH1r are both critical for PTH(1 34) binding and signal transduction (17). To further define the structural basis of the interaction between PTH(1 34) and the PTH1r s N terminus and carboxy core domains, we performed a detailed pharmacological analysis of 27 SEC/PTH1r chimeras. This approach was feasible because of the high sequence identity (60%) within the transmembrane (TM) segments of the secretin and PTH1 receptors (Fig. 1), and because of the mutual absence of cross-recognition of these hormones with each other s receptor. The results indicate that 1) two distinct PTH(1 34) binding sites are present within the receptor s N terminus: the first site (site 1) resides on the amino-terminal half of the PTH1r s N terminus and is implicated in high affinity binding of PTH, while a second site (site 2) of lower affinity is located on the carboxy-terminal half and is necessary to promote receptor signaling. Moreover, the data suggest that site 1 acts in concert with site 2 for optimal function of the receptor (hormone binding and camp response); 2) that the first- and third extracellular loops of the PTH1r are critical for highaffinity PTH(1 34) binding and signal transduction; 3) helix 5 in combination with the extracellular domains (N-termini and loops) regulates PTH1r affinity for PTH; 4) effective signaling in response to PTH requires the presence of the PTH1r s TM3 and TM6. RESULTS Binding and Functional Properties of Wild-Type PTH1 and SECrs Stably Expressed in HEK-293 Cell Line Stable HEK-293 cell lines expressing the PTH1 or secretin receptor were tested for their ability to bind 125 I- PTHrP(1 34) or 125 I-secretin and for their capacity to respond to PTH and secretin with respect to stimulation of adenylyl cyclase. In both cell lines, Scatchard analysis revealed the presence of a single population of highaffinity binding sites for their respective agonists. The PTH1r cell line expressed receptors per cell, with a dissociation constant for PTH(1 34) of K i 5.6 nm; the secretin receptor cell line expressed receptors per cell, with a K i value for secretin of 3.2 nm (data not shown). As expected, 125 I-secretin did not bind to the PTH1r and secretin did not displace 125 I-PTHrP(1 34) binding. Likewise, PTHrP(1 34) tracer did not bind to the secretin receptor, and PTH(1 34) did not displace 125 I-secretin binding. Ligand-induced camp production was tested in both cell lines. The camp concentration reached a maximum ( 300 pmol/10 5 cells/10 min) at an agonist concentration of 10 nm (data not shown). The EC 50 values for adenylyl cyclase activation by PTH(1 34) and secretin for their respective receptors are subnanomolar (EC nm). The disproportionately low EC 50 value as compared with their K i values may be due to an amplification process (presence of spare receptors) as previously demonstrated for the secretin receptor stably expressed in Chinese hamster ovary (CHO) cells (19). These data demonstrated that PTH(1 34) and secretin did not cross-react with each other s receptor. Construction and Expression of Chimeras Receptors in HEK-293 Cells We constructed four sets of chimeric receptors. Figures 2 and 3 provide a schematic diagram of the various chimeric receptors studied. Studies with the first set of chimeric receptors were carried out to map the regions of the N-terminal domain of the PTH1r that cooperate functionally with the receptor s carboxy core. To accomplish this, we divided the PTH1r s amino-terminal extracellular domain into three domains according to the position of the strictly conserved cysteine residues within the N-terminal region of class II GPCRs (i.e. Cys 105, 114, 128, and 145 for the PTH1r; Fig. 1): domain A (residues 1 106), domain B (residues ), and domain C (residues ) (Fig. 4). Each domain was introduced, either singly or in combination, in place of the corresponding domain of the chimera NSEC/PTHr. The second and third set were designed to analyze the effect on receptor function (hormone binding and camp response) of progressive replacement of the sequence of the secretin receptor within the chimera NPTH/SECr with that of the PTH1r. In set 2, PTH1r sequence was progressively introduced from the cytoplasmic end of TM1 through the third extracellular loop (3e). In set 3, PTH1r sequence was progressively introduced in the opposite direction, from the carboxy-terminal tail through the first extracellular loop (1e). The fourth set was designed to distinguish the relative contribution to ligand binding and signaling of the entire extracellular domain (N-terminal region extracellular loops) vs. the TM segments. The binding affinity for PTH(1 34) and camp response of the chimeric receptors was analyzed in stably transfected HEK-293 cells. Table 1 summarizes the PTH(1 34) binding and signaling properties of the PTH1 and hybrid receptors. The expression levels of the chimeric receptors were generally similar to that of the PTH1r (Table 1). None of the chimeric receptors was able to bind or respond to secretin (data not shown). Properties of Chimeras NPTH/SECr and NSEC/ PTHr Stably Expressed in the HEK-293 Cell Line We have previously characterized chimeras NPTH/ SECr and NSEC/PTHr transiently expressed in COS-7A cells (17). Here we describe the pharmacological properties of these two chimeras stably expressed in HEK-293 cells. Chimera NPTH/SECr did not bind 125 I-PTHrP(1 34) and displayed a very low displacement of 125 I-secretin by secretin (IC nm, data not shown); camp responses to secretin or PTH(1 34) were not detectable. This defect in hormone binding was not attributable to impaired surface

4 Structure-Function of PTH/PTHrP Receptor 1189 Fig. 2. Schematic Structure of Chimeric Receptors, Set 1 A, Sequence comparison of the amino-terminal extracellular domains of the opossum PTH1r (PTH) and the rat SECr (SEC). The highly conserved residues with the receptors of class II are bold. B, Schematic representation of the amino-terminal extracellular domain of the PTH1r and SECr (black circles, conserved cysteine residues. Y, potential sites of N-glycosylation. Dotted lines delineate domains A, B, and C). C, Schematic diagram of the wild-type and set 1 of chimeric receptors (black and white portions represent PTH1r and secretin receptor sequences, respectively). expression of the chimera which was 75% that of the wild-type PTH1r (Table 1). The reciprocal hybrid receptor NSEC/PTHr failed to display an adenylyl response to secretin (data not shown) and displayed a very low camp response to a high concentration of PTH(1 34) (Table 1). This chimeric receptor showed weak displacement of 125 I-PTHrP(1 34) by PTH(1 34) (IC 50 1,000 nm) and was unable to bind secretin. The surface expression of NSEC/PTHr could not be determined with our available anti-pth receptor antibody due to the absence in this chimera of the PTH1r s N-terminal epitope (see Materials and Methods). From these data, it is unclear whether the loss of function seen in the N-terminal receptor chimeras was due to 1) loss of direct hormone contact sites in the receptors N-terminal domains; 2) changes in the overall conformation of the receptors; or 3) in the case of NSEC/ PTHr, possibly reduced levels of receptor expression. However, we recently found that hybrid PTH-secretin ligands are capable of fully activating NSEC/PTHr and NPTH/SECr, indicating that the chimeric receptors maintain a functional conformation, and that NSEC/ PTHr is properly expressed at the plasma membrane (Nissenson, R. A., and J. P. Vilardaga, manuscript in preparation). These results indicate that the PTH1r s N-terminal domain and carboxy core (TM domain and adjacent loop) both influence PTH(1 34) binding and receptor activation, but neither domain is by itself sufficient to provide binding or camp responses to PTH(1 34). Thus the extracellular N-terminal domain and the carboxy-core domain of the receptor must cooperate in complexing the hormone and in initiating signal transduction. Role of the N-Terminal Extracellular Domain of the PTH1r in Signal Transduction Compared with NSEC/PTHr, chimera A showed a strong increase in binding affinity for PTH(1 34), with a potency close to that observed for the wild-type

5 MOL ENDO 2001 Vol. 15 No Fig. 3. Schematic Structure of Chimeric Receptors, Sets 2 4 A, Restriction map of the wild-type PTH1r and SECr cdnas. The locations of the restriction sites used for the construction of chimeric receptors are indicated above and below the structure of secretin- and PTH1rs, respectively. The location of the seven-transmembrane segments are represented in black boxes; B, Schematic diagram of the wild-type and sets 2, 3, and 4 of chimeric receptors. Black and white portions represent PTH1r and SECr sequences, respectively. PTH1r (K i 9.2 vs. 5.6 nm). Despite this gain in hormone binding affinity, chimera A was totally unresponsive to PTH(1 34) in terms of stimulation of intracellular camp production. This was not due to decreased levels of expression of chimera A relative to the wildtype PTH1r (Table 1). Thus, the signaling defect of chimera A may be due to improper positioning of PTH within the PTH1r. This suggests that structural determinants in the domain BC of the PTH1r are required to properly position the hormone in the receptor. To determine whether domain BC as an effect on the signaling properties of PTH1r, the pharmacological properties of chimera receptors B, C, and BC were analyzed. Although chimera C did not display a gain of affinity for the binding of PTH(1 34) (IC nm), it did display a marked increase in camp responses to high concentrations (10 5 M) of PTH(1 34). In a receptor-agonist system with equal magnitude for the biological response, relative potencies (EC 50 ) should reflect relative binding affinities (K i ) (20). For chimera C the maximal extent of PTH stimulation of camp formation was the same as in cells expressing PTH1r. Thus, the high EC 50 ( 3, 500 nm) value for PTH(1 34) observed for chimera C suggests that PTH1r domain C could be part of an interaction of low affinity (IC 50 3, 500 nm) with the hormone, sufficient to promote a full signaling response. Domain C appears to be involved in the activation of the PTH1r. Data obtained with chimera B are not informative because of the absence of hormone binding and the low camp response toward PTH. Simultaneous substitution of domains B and C of the PTH1r (chimera BC) strongly increased the potency of PTH(1 34) for increasing cellular camp levels (EC nm), as well as conferring a maximal camp response similar to that of the wild-type PTH1r. As expected from the functional (camp) response, the binding affinity was also increased (K i 30.7 nm). Therefore, domain BC of the PTH1r appears to be part of a PTH(1 34) binding site that is involved in signal transduction. Thus, the conformation conferred by domain BC appears to be involved in transducing the signal from the N-terminal extracellular domain to the carboxy core domain of the receptor. When we compared the properties of wild-type PTH1r and chimera BC, we observed that the increase

6 Structure-Function of PTH/PTHrP Receptor 1191 Fig. 4. Functional Properties of Chimeric Receptors, Set 1 A, Inhibition of 125 I-PTHrP(1 34) binding by PTH(1 34); and B, dose-response curves for adenylyl cyclase activation for the PTH1r and chimeric receptors stably expressed in HEK-293 cells. The structure of the chimeric receptors scanning the PTH1r s amino-terminal extracellular domain for their involvement in receptor function is represented. Black and white portions represent PTH1r and secretin receptor sequences, respectively: wild-type PTH1r (F), NSEC/PTHr ( ), chimera A ( ), chimera B (X), chimera C( ), chimera AB ( ), chimera AC (ƒ), chimera BC (E). in the K i value (6-fold) observed for the chimera BC as compared with the PTH1r did not reflect the increase in the EC 50 (200-fold) for PTH-stimulated camp production. This discrepancy could reflect 1) a decrease in cell surface receptor expression (i.e. the roughly similar value for K i and EC 50 suggest the absence of spare receptors in cells expressing the chimera BC) and/or 2) a contribution of the binding site in domain A to signal transduction in the wild-type PTH1r. To test whether the interaction of PTH(1 34) with domain A could act in concert with additional hormone interaction sites in domains B or C, we constructed and expressed chimeras AB and AC. The apparent binding affinity of PTH(1 34) to chimera AB (K i 12.5 nm) and to chimera AC (K i 15.6 nm) was markedly increased as compared with chimeras B and C, respectively, and was similar to the affinity of PTH(1 34) for chimera A (K i 9.2 nm). Although introduction of domain A increased the binding affinity of chimeras B and C, it did not enhance their signaling properties. Altogether, these data not only confirm the importance of the PTH1r s N-terminal domain for hormone binding, but also indicate its critical contribution to signal transduction. Our data identify two domains (domains A and BC) of the N-terminal extracellular domain of PTH1r that differentially affect PTH binding and receptor activation. It appears that the N-terminal domain participates in at least two interactions with the hormone: one interaction with domain A (site 1) that is not required for full efficacy of the camp response; and an additional interaction with domain BC (site 2) that is necessary to promote the signal transduction process. Contact of the hormone with sites 1 and 2 appears to be required for optimal functionality of the receptor. Differential Role of the Extracellular Loop 1 and Extracellular Loop 3 of PTH1r in Hormone Binding and Receptor Activation Chimeric receptors (CRs) that contain the N-terminal sequence and the first one, three, or four TM segments including the connecting loops of the PTH1r (chimeric receptors CR1, CR2, and CR3) showed no specific 125 I-PTHrP(1 34) binding and no camp responses to PTH(1 34), even though these chimeric receptors displayed cell surface expression that was % that of the wild-type PTH1r (Table 1). Further substitution of the sequence extending from the N terminus through TM6 of the PTH1r resulted in a chimera (CR4) that displayed high-affinity PTH binding (K i 12 nm vs. 5.6 nm for the wild-type PTH1r) (Fig. 5A). CR4 also initiated PTH-stimulated camp production (maximal response 75% of the wild-type receptor), but the potency of PTH was markedly reduced (EC nm vs. 0.1 nm for the wild-type receptor) (Fig. 5B). Additional replacement of the third extracellular loop (chimera CR5) resulted in a remarkable 400-fold increase in the potency of PTH(1 34) in stimulating camp production (EC nm), with only a 2.5-fold increase in apparent binding affinity for PTH(1 34) (Fig. 5, A and B, and Table 1). These results indicate that the third extracellular loop of the PTH1r plays an important role in the process of receptor activation after agonist binding. The fact that CR5 is virtually identical to the wild-type PTH1r with respect to ligand binding affinity and signaling indicates that PTH1r-specific residues in the region extending from TM7 to the C terminus do not play a critical role in promoting high-affinity PTH binding or receptor signaling to adenylyl cyclase.

7 MOL ENDO 2001 Vol. 15 No Table 1. Functional Properties of the PTH1r and Chimeric Receptors Stably Expressed in HEK-293 Cells Receptors Surface Expression (% PTH1r) PTH(1-34)-Stimulated camp Accumulation 125 I-PTHrP(1-34) Binding max a (pmol/well) EC b 50 (nm) K i (PTH1-34) (nm) B c max (receptors 10 6 /cell) PTH1R N-PTH/SECr No specific binding detectable N-SEC/PTHr ND ,000 ND Chimera A Chimera B ND ,000 1,000 ND Chimera C ND , ,000 ND Chimera AB , Chimera AC , Chimera BC ND CR ,000 ND CR ,000 ND CR ,000 ND CR CR CR ,000 ND CR CR CR CR CR CR CR ,000 ND CR CR CR ,000 ND CR CR CR Surface expression was assessed by anti-ok1 antibody binding as described. a Basal was 2 3 pmol/well; max was determined at [PTH(1 34)] 10 6 M. b :EC 50 was not measurable. c ND, Not done because tracer binding capacity was too low to obtain accurate B max values. Compared with chimera NPTH/SECr, chimera CR6 did not show a gain in receptor function (Table 1). Further replacement of TMs 5 and 6 (as well as the connecting third intracellular loop) resulted in a chimera (CR7) that was able to bind PTH(1 34) with an apparent affinity approximately 10-fold lower than that of the PTH1r (Table 1). Chimera CR7 also responded to PTH(1 34) with respect to camp production, although the potency of PTH was low (EC nm) and the maximal response to PTH was only 43% that of the wild-type PTH1r (Table 1). These data support the view that, in the presence of PTH1r s N-terminal domain, the region spanning from TM5 through 3e contributes significantly to hormone binding and responsiveness. However, the reduced activity of chimeras CR7 relative to that of the wild-type PTH1r indicates that other regions with the PTH1r s carboxy core are required for 1) direct interaction with the hormone and/or 2) to maintain the overall architecture of the PTH1r required for high-affinity hormone binding. Indeed, possible incompatibilities between TM helices could lead to improperly folded cell surface receptors. Additional replacement of the second extracellular loop (chimera CR8), and the second intracellular loop (2i) TM4 (chimera CR9) did not significantly change the binding affinity or the doseresponse profile for PTH, compared with CR7 (Table 1). Further introduction of TM3 (chimera CR10) slightly enhanced the binding affinity for PTH (by 1.5-fold; K i 27 nm) and increased the potency of PTH(1 34) for promoting camp production by about 30-fold (EC nm) (Fig. 5B). As mentioned above, this gain in receptor function could reflect a direct interaction of the hormone with TM3, or could be secondary to conformation changes in the helix packing of the TMs between TMs 3, 4, 5, and 6. Extending the substitution through the first extracellular loop (chimera CR11) further increased ligand binding affinity (6-fold) and stimulation of the camp response (40-fold) (Fig. 5, A and B). Thus, the first extracellular loop is implicated in the high-affinity binding of PTH(1 34) and in receptor activation. The functional characteristics of the chimera CR11 were indistinguishable from those of the wildtype PTH1r, indicating the nonessential role of the divergent residues in TM1 and TM2 (Table 1 and Fig. 5).

8 Structure-Function of PTH/PTHrP Receptor 1193 Fig. 5. Role of Exoloops 1 and 3 in Receptor Function A, Inhibition of 125 I-PTHrP(1 34) binding by PTH(1 34) and B, dose-response curves for adenylyl cyclase activation for the PTH1r and chimeric receptors stably expressed in HEK-293 cells. PTH1r (F), CR4 ( ), CR5 (E), CR10 ( ), CR11(ƒ). Substitution of the extracellular loop 1 in CR10 (resulting in CR11) and of the extracellular loop 3 in CR4 (resulting in CR5) show disparate effects on PTH binding and camp inducibility. Indeed, as compared with the first extracellular loop substitution (CR10 vs. CR11: K i decreases 6-fold and EC 50 decreases 40-fold), the third extracellular loop substitution (CR4 vs. CR5: K i decreases 2.5-fold and EC50 decreases 420-fold) shows a 10-fold better PTH potency for camp induction (Fig. 5B). On the other hand, the first exoloop substitution shows a 2.4-fold better affinity for PTH binding (Fig. 5A). These disparate effects underscore the differential roles of exoloop 1 and exoloop 3 of PTH1r on PTH binding and receptor activation. Helices 3, 5, and 6 of PTH1r Are Important for High-Affinity Hormone Binding and Receptor Activation Substitution of TMs 1, 2, 4, and 7 resulted in a receptor (chimera CR12) with properties similar to those of the wild-type PTH1r (Table 1 and Fig. 6). Introduction of the secretin receptor s second extracellular loop into chimera CR12 had no influence on the binding and signaling properties of the resulting receptor (data not shown). These results support the nonessential role of the divergent residues in TMs 1, 2, 4, and 7 as well as the second extracellular loop (2e) for PTH1r function, and suggest that, in the presence of the N-terminal domain, the first and the third extracellular loops, as well as TMs 3, 5, and 6 of the PTH1r define the principal determinants of high affinity PTH(1 34) binding and receptor activation. To distinguish the relative contribution of the extracellular region and the TM domains in PTH1r function and to approach the specificity of the interhelical interaction between TMs 3, 5, and 6, a series of chimeric receptors containing the entire PTH1r s extracellular domain in combination with various PTH1r TM segments (TMs 3, 5, and 6) was constructed (set 4 in Fig. 3) and stably expressed in HEK-293 cells. Substitution of the secretin receptor s entire extracellular domain with that from the PTH1r resulted in a chimera (CR13) that displays a very weak PTH(1 34) affinity binding (K i 1000 nm) or signaling (Fig. 6A), even though this chimera was expressed at the cell surface with a density 60% that of the wild-type PTH1r (Table 1). This is in contrast with the results with chimera CR12 and indicates that the pharmacologically inactive chimeric receptor CR13 could be rescued by introduction of TMs 3, 5, and 6 (Fig. 6). None of the chimeras in which only a single TM was substituted (CR14: introduction of TM3 into CR13; CR15: introduction of TM5 into CR13; and CR16: introduction of TM6 into CR13) were able to rescue the pharmacologically inactive CR13 receptor (Fig. 6, A and B). However, CR15 displayed the most important gain in PTH(1 34) binding affinity. Indeed this chimera showed a K i value close to that observed for the wild-type PTH1r (K i 9.5 vs. 5.6 nm) and an equivalent B max value. Chimera CR14 displayed reduced binding affinity (K i 50 nm), and chimera CR16 showed little if any specific PTH binding. Functionally, chimeras CR15 and CR16 were totally unresponsive to PTH(1 34) stimulation; this was unexpected for chimera CR15 in view of its ability to bind PTH(1 34) with high affinity. In contrast, chimera CR14 was able to respond modestly to PTH(1 34) (maximal camp response 41% that of the wild-type PTH1r; EC nm) (Table 1 and Fig. 6B). These results indicate that PTH was recognized when TM3 or TM5 of PTH1r was incorporated into CR13. It is important to note that these data do not show that either TM 3 or 5

9 MOL ENDO 2001 Vol. 15 No Fig. 6. Functional Properties of Chimeric Receptors, Set 4 A and C, Inhibition of 125 I-PTHrP(1 34) binding by PTH(1 34) and B and D, dose-response curves for adenylyl cyclase activation for the PTH1r and chimeric receptors stably expressed in HEK-293 cells. PTH1r (F), CR12 ( ), CR13 (Œ), CR14 ( ), CR15 (E), CR16 (X), CR17 ( ), CR18 (f), CR19 ( ). makes a direct contact with PTH, but suggest that the conformation conferred by TM5 and TM3 plays a role in PTH binding and signal transduction, respectively. Helices 3 and 6 of PTHR1 Receptor Are Determinant for Signal Transduction To further explore the helix specificity by which introduction of TMs 3, 5, and 6 can restore the function lost in CR13, three additional chimeric receptors (CR17: introduction of TMs 5 and 6; CR18: introduction of TMs 3 and 5; CR19: introduction of TMs 3 and 6) were constructed and characterized. Compared with CR13, CRs 17, 18, and 19 displayed a remarkable increase in PTH binding and camp induction. However, they show distinct differences in camp induction profiles (Fig. 6D). CR19 with the lower PTH affinity binding (Ki 45 nm vs. 8.9 nm and 11.9 nm for CR17 and 18, respectively) has the strongest effect in the camp response toward PTH (EC nm vs. 140 nm and 94 nm for CR17 and CR18, respectively). These data support an important role for TM3 and TM6 in the process of signal transduction in the PTH1r. These data indicate that, in the presence of the PTH1r s extracellular domains, specific molecular interactions between TMs 3, 5, and 6 play a decisive role to govern the overall function of the PTH1r. Structural elements in TM5 appear to contribute to high affinity PTH binding, and determinants in TM3 and TM6 contribute to a lesser extent to PTH binding but are essential for effective signal transduction. DISCUSSION The N-Terminal Domain The critical contribution of the N-terminal extracellular domain for hormone binding has now been well documented for a number of class II GPCRs including the receptors for secretin, VIP, PACAP, calcitonin, glucagon, and GHRH (5 18, 21, 22). The present study confirmed this finding for the PTH1r and further defined the role of the N-terminal domain in the hormone binding and receptor activation. To accomplish this, we constructed a series of six secretin/pth chimeric receptors (chimeras A, B, C, AB, AC, and BC) designed to delineate the ligand binding domain in the PTH1r s N-terminal region. Our strategy was based on

10 Structure-Function of PTH/PTHrP Receptor 1195 the ability to restore PTH binding and signaling that had been lost in the chimera NSEC/PTHr (see Results). The binding to chimeras A (K i 9.2 nm) and BC (K i 30.7 nm) demonstrated that the unfavorable interaction of PTH(1 34) with the chimera NSEC/PTHr (K i 1000 nm) can be overcome by replacing domain A or BC of the secretin receptor with the corresponding domain of the PTH1r (residues for domain A; and residues for domain BC). This suggests the existence of two distinct PTH(1 34) binding sites in the PTH1r s N-terminal domain: one in domain A (site 1) displays high affinity for PTH, and a second in domain BC (site 2) displays slightly lower affinity ( 3.5-fold). Despite displaying high-affinity for PTH(1 34), chimera A was not able to induce camp accumulation, indicating a complete loss of the transduction process associated with PTH(1 34) stimulation. This absence of functional activity was not attributable to a decrease in cell surface receptor expression. Rather it was caused by conformational incompatibilities, possibly involving the N terminus and the carboxy-core domain of the receptor, that interfere with a proper positioning of PTH. The remarkable gain of PTH-stimulated camp accumulation with chimera BC, as compared with NSEC/PTHr, indicates that hormone interaction with site 2 (PTH1r s domain BC) was essential to promote receptor activation. However, chimera BC did not fully reproduce the binding and signaling properties of the wild-type PTH1r (K i 30.7 vs. 5.6 nm; EC vs. 0.1 nm), suggesting that hormone interaction with site 1 (domain A) may optimize the function of the receptor by lowering the K i and EC 50 values. We speculate that the interaction of PTH(1 34) with site 1 (domain A) is not requisite for the recognition of PTH by domain BC, but could facilitate the association of PTH with site 2 (domain BC), thus allowing initiation of the signal transduction process. Our study revealed that full stimulation of the camp response was achieved only when the PTH1r s domain C was present in the chimeras (chimeras C, AC, and BC), suggesting that the conformation conferred by domain C was required for maximal PTH-stimulated camp production. That chimera C, in the absence of high-affinity PTH(1 34) binding, was able to stimulate a full camp response at a high hormone concentration (10 5 M) suggests that domain C of the PTH1r could be part of a low-affinity binding site for PTH(1 34) that is sufficient to promote receptor activation. These results are in agreement with a recent photoaffinity cross-linking study demonstrating close proximity between PTH(1 34) and amino acids in domain C of the human PTH1r (14). Therefore, our results indicate that the receptor s N-terminal domain not only has a fundamental role in PTH(1 34) binding, but also makes a critical contribution to the process of signal transduction. Since the N-terminal domain of a GPCR cannot interact with G proteins on the cytoplasmic face of the plasma membrane, it is not clear how this region of the receptor can control the signal transduction process. The current model for activation of GPCRs is that receptor occupancy by an agonist results in the stabilization or induction of the active receptor conformation that is competent to activate the cognate G protein (23). According to this model, we speculate that domain BC of the PTH1 receptor (site 2) could be of a structural domain of the receptor that stabilizes or induces the active receptor conformation after PTH binding. In our recent study using PTH1/PTH2 chimeric receptors, we proposed a possible two-site model for PTH(1 34)- PTH1r interaction (2), in which the combined hormone interaction with the receptor s N-terminal domain and the receptor s carboxy core are required for receptor activation. The results of the present study allow us to extend this model: as chimera A displays PTH binding affinity 3.5-fold higher than the chimera BC, we propose that the initial contact between PTH and the receptor could occur with receptor elements in domain A as well as elements in the carboxy core. Indeed, cross-linking studies have identified an interaction between residue 23 in PTH and the extreme amino terminus of the PTH1R (14). This binding event facilitates an energetically less favorable interaction between PTH and elements in domain BC and in the carboxy core. The latter interaction initiates the conformational switch that results in signal transduction. Although our data clearly suggest that the receptor N-terminal domain plays a role in receptor activation, previous reports (16, 24) might deemphasize the importance of the N-terminal domain in the activation process. Indeed, the substitution of the N termini of PTH1- and of GHRH receptors by the corresponding N termini of other class II receptors [as the calcitonin receptor and the VIP receptor] have resulted in chimeras N-Cal/PTHr and N-VIP/GHRHr that fully stimulate adenylyl cyclase in response to high concentrations ( 1 M) of PTH and GHRH, respectively. These data contrast with the very low camp response to PTH of our chimera N-SEC/PTHr. Alignment of the N-terminal regions between the secretin-, PTH-, and calcitonin receptors indicates a significant higher amino acid sequence identity between corresponding domain C of PTH- and calcitonin receptor (36%) vs. corresponding domain C of PTH- and SEC receptor (16%). Because our data suggest that domain C of PTHR is sufficient to promote receptor activation, a possible explanation for the differences between the signaling response of chimeras N-Cal/PTHr and N-SEC/PTHr, might be that some of the determinants required for PTH binding are present in the N-terminal domain (corresponding to domain C) of the calcitonin receptor and sufficient to promote full activation. The Carboxy Core Domain The very low PTH binding affinity and lack of functional response to PTH displayed by the chimera NPTH/ SECr (IC nm) demonstrate that domains within the PTH1r s carboxy core must complement the receptor s N-terminal region with respect to hormone

11 MOL ENDO 2001 Vol. 15 No binding. To identify these domains, we generated chimeric receptors by progressively replacing SECr domains in the chimera NPTH/SECr with the corresponding domains of PTH1r (sets 1 and 2). Analysis of the PTH(1 34) binding and signaling properties of these chimeras led to the following conclusions: 1. The divergent residues (i.e. amino acids that differ between PTH1r and SECr) present in TM1, TM2, TM4, TM7, the second extracellular loop, and the carboxyterminal tail are not important for PTH1r function (hormone binding and camp response). Of course, this does not preclude a functional role for amino acids in these TM domains that are conserved between PTH1r and SECr. Indeed, previous studies (25 27) have demonstrated that conserved residues in TM2 (e.g. His 222, Ser 226, Arg 230, and Ser 233) and TM7 (e.g. Gln 445) of the PTH1r are determinants of receptor function (Fig. 1). 2. Analysis of chimeras CRs 4, 5, 10, and 11 indicates that the PTH1r s first- and third extracellular loop participates differentially in receptor activation as well as in high affinity PTH binding. 3. Critical role of TMs 3, 5, and 6 for the overall activation process of the PTH1r. Analysis of the third set of chimeric receptors, designed to distinguish the role of the extracellular domains from the TM 3, 5, and 6 segments on receptor function, suggests that the extracellular domain and these three TMs act cooperatively to confer high-affinity binding of PTH. In support of this, introduction of TMs 3, 5, and 6 into the inactive receptor CR13, which contains all of the extracellular domains of the PTH1r receptor, rescue PTH1r s functional characteristics (see data for CR12). The present study cannot determine whether bound PTH is in direct contact with these TM domains of the receptor, or if the gain of function of CR12 is secondary to allosteric effect between TMs 3, 5, 6, and the extracellular domains. However, recent cross-linking studies have demonstrated direct contact of PTH with amino acid residue Met425 on TM6 of the human PTH1r (corresponding to Met419 in the opossum PTH1r, Fig. 1) (28). Although chimera CR15 (introduction of TM5 into CR13) was able to bind PTH with relatively high affinity, it was unable to initiate activation of adenylyl cyclase. Thus, TM components in TMs 3 and 6 in addition to TM5 are required for effective signal transduction in the agonist-occupied receptor. Indeed, the improved ability for PTH to stimulate the adenylate cyclase in chimera CR19 (introduction of TMs 3 6 into CR13; EC nm) compared with chimera CR17 (introduction of TMs 5 6 into CR13; EC nm) and chimera CR18 (introduction of TMs 3 5 into CR13; EC nm) underline the importance of TMs 3 and 6 in the activation process of PTH1r. The importance of TM3 and TM6 in signal transduction has also been reported for a variety of class I GPCRs (29). Studies on rhodopsin have indicated that receptor activation involves the rotation of TM6 and its movement away from TM3 at the cytoplasmic side of the plasma membrane (30), and evidence for a similar conformational shift in the PTH1R has recently been reported (31). In conclusion, by using a gain-of-function chimeragenesis strategy, we have delineated PTH1r s region that differentially influence PTH binding and receptor activation. PTH binding site is formed by residues scattered throughout the receptor s N terminus and first and third extracellular loops. Although the present results do not establish whether PTH is in contact with TMs 3, 5, and 6, our data support the view that helix interactions between these TM domains are critical for maintaining the PTH1r conformation required for highaffinity PTH binding. We present a composite diagram in Fig. 7 that indicates the regions of the PTH1r involved in maintaining high-affinity PTH binding (black box), and those required to promote receptor signaling (striped box). One may speculate on a possible mechanism of activation of the PTH1r in accordance with a two-site model: the N terminus [residues 1 104; deletion of exon 2, which is residues , has no influence on the function of PTH1r (15)]; exoloops 1 and 3 and TM5 constitute site 1 of the receptor accounting for an interaction of high affinity with PTH(1 Fig. 7. Regions of the PTH1r Critically Implicated in Hormone Binding and Signal Transduction Black section, Regions preferentially implicated in PTH binding; black heavy stripe sections, regions preferentially implicated in receptor activation. A two-site model is proposed for PTH1r activation. Initially, the hormone binds with high affinity to the N-terminal s region 1 62 and the extracellular loops (site 1). TM5 either constitutes an additional contact site in site 1 or is required to maintain the extracellular domain in a high-affinity conformation. After binding to site 1, PTH makes a second contact of lower affinity with the N- terminal s region , TM3 and TM6 (site 2), resulting in signal transduction. In this model, we indicate amino acid residues (white circles, and region ) that are suggested to make direct contact with PTH in cross-linking studies (13, 14, 34).

12 Structure-Function of PTH/PTHrP Receptor ). As discussed earlier, it is unclear whether TM5 is a direct site of contact with PTH or whether it influences binding affinity indirectly. High-affinity binding to site 1 facilitates the interaction of PTH with a second site of lower affinity, located in the N-terminal extracellular domain (residues ) and in TMs 3 and 6, to promote signal transduction. As the receptor s firstand third extracellular loops as well as TMs 3 and 6 appear to play an important role in signal transduction, we suspect that an interaction of PTH with the domains 1e TM3 and TM6 3e alters the arrangement of TM3 and TM6, resulting in the activation of the receptor. These results, together with evolving structural models, will provide new insights into the molecular basis of hormone-induced signal transduction for class II GPCRs. MATERIALS AND METHODS Materials Restriction endonucleases, DNA modifying enzymes (ligase, kinase, phosphatase alkaline, and polymerase), and all the products used in cell culture (culture medium, FCS, trypsin, penicillin, and streptomycin) were purchased from Life Technologies, Inc. (Gaithersburg, MD). Hygromycin was obtained from Roche Molecular Biochemicals (Indianapolis, IN). camp and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma (St. Louis, MO). Synthetic porcine secretin, bovine PTH(1 34), and human PTHrP(1 34) were purchased from Bachem (Torrance, CA). 125 I-secretin and 125 I-PTHrP(1 34) were prepared by the chloramine method, as previously described (17). All oligonucleotides used were synthesized and purified by the Biomolecular Resource Center of the University of California, San Francisco. The mouse monoclonal antibody (OK-1) against a peptide epitope (residues 69 to 82; Fig. 1) in the opossum PTH1r s amino-terminal extracellular domain was generated in collaboration with Dr. T. Anderson (Berkeley Antibody Co., Berkeley, CA). Construction of Chimeric Receptors and Nomenclature The opossum PTH1 and rat secretin receptor cdnas were used for the construction of the chimeric receptors (32, 33). The construction of the chimeric receptors NPTH/SECr and NSEC/PTHr was previously described (17). The cdnas for the N-terminal extracellular region of chimeras A [PTH1r(1 104)SECr(66 142)], B [SECr(1 66)PTH1r( )SECr( )], C [SECr(1 107)PTH1r( )], AB [PTH1r(1 145)- SECr( )], AC [PTH1r(1 104)SECr(66 107)PTH1r( )], and BC [SECr(1 66)PTH1r( )] (set 1 in Fig. 2) were engineered by a recombinant PCR technique using the Pfu DNA polymerase (Stratagene, La Jolla, CA), as previously described (34). The resulting amplified products were purified and digested with HindIII/AccI, and subcloned into the corresponding sites of the recombinant vector pbs/pthr, a derivative of pbluescript that contains the cdna of the opossum PTH1r. Chimeric receptors CR1 to CR11 (set 2 and 3 in Fig. 3) were constructed using preexisting restriction sites in the receptors cdnas (Fig. 3A) and by splicing restriction endonuclease fragments derived from the wild-type- or hybrid receptors cdnas with synthetic oligonucleotide adaptors ranging in size from 24 to 55 bases, as previously described (35). The cdna fragments were ligated into the vector pbluescript (Stratagene, La Jolla, CA) that had been linearized by digestion with HindIII and NotI. Chimeric receptor CR13 was constructed according to a scheme previously described (6). Chimeric receptors CR12 and CR14 to CR19 (set 4) were engineered by a recombinant PCR technique previously described (34), using the Pfu DNA polymerase (Stratagene) and the CR13 cdna as a DNA template. Proper construction of the hybrid cdnas was confirmed by DNA sequence analysis. HindIII/NotI fragments of the chimeric cdnas were then introduced into the respective sites in pcep4 (Invitrogen, San Diego, CA), a mammalian expression vector. Cell Culture and Transfection Stable cell lines that selectively express the PTH1r, the secretin receptor, or the chimeric receptors were established in the human embryonic kidney cell line 293-EBNA (HEK-293). These cells were grown in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin at 37 C in 5% CO 2 atmosphere. At 18 h before transfection, the cells were split into 75-cm 2 flasks at 30% confluency. Cells were transfected using the calcium phosphate precipitation method with 24 h incubation in the presence of 10 g of plasmid DNA per flask. Two days after transfection, selection of stably transfected cells was initiated by the addition of hygromycin (200 g/ml). Selection was generally complete after 3 4 weeks of hygromycin treatment. Stock cell lines were cultured in the continuous presence of hygromycin, except when subcultured for experiments in which case hygromycin was omitted. Competitive Radioligand Binding The transfected cells were split into six-well culture plates at a density of 10 6 cells per well. Twenty four to 48 h later, they were incubated with DHB (DMEM containing 20 mm HEPES and 0.1% BSA) for2hat4candthen incubated in 1 ml DHB with 100,000 cpm 125 I-hPTHrP(1 34)amide or 125 I-secretin ( 0.1 nm) and various concentrations of bpth(1 34) or porcine secretin at 4 C for 2 h. After extensive washes with ice-cold PBS, cells were lysed with 1 ml 0.8 N NaOH, and cell-bound radioactivity was determined by a -counter. Adenylyl Cyclase Activity Determination Intracellular camp accumulation was assessed in transfected cells split in 12-well plates and incubated for 10 min at room temperature in 0.5 ml DHB containing 1 mm IBMX and various concentrations of bpth(1 34) or porcine secretin. Cells were washed twice with ice-cold PBS, and camp was extracted with 1 ml of 100% ethanol and measured by RIA (36). Determination of Cell Surface Receptor Expression Transfected cells cultured in 12-well plates were incubated at room temperature for 2 h with 1 ml DMEM containing 5% heat-denatured FCS in the presence of 1.5 g/ml mouse monoclonal antibody OK-1. Cells were washed three times with PBS and incubated for 2 h at room temperature with 200,000 cpm/ml of 125 I-labeled goat anti-mouse Ig (Amersham Pharmacia Biotech). After three washes with ice-cold PBS, cells were lysed with 1 ml 0.8 N NaOH, and cell-bound radioactivity was determined. No specific binding was detected with mock-transfected HEK-293 cells. Specific antibody binding for the wild type PTH1r was 7.5%/well of added 125 I-labeled goat antimouse Ig. Specific binding was normalized to the cell number. Data Analysis Dose-response curves were analyzed by computer-assisted nonlinear regression GraphPad software (GraphPad Soft-