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Molecular Endocrinology, doi:10.1210/me.2003-0275
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Molecular Endocrinology 17 (12): 2647-2658
Copyright © 2003 by The Endocrine Society

Identification of a Contact Site for Residue 19 of Parathyroid Hormone (PTH) and PTH-Related Protein Analogs in Transmembrane Domain Two of the Type 1 PTH Receptor

Robert C. Gensure, Naoto Shimizu, Janet Tsang and Thomas J. Gardella

Endocrine Unit (R.C.G., N.S., J.T., T.J.G.) and Pediatric Endocrine Unit (R.C.G.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, 50 Blossom Street, WEL 501, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent functional studies have suggested that position 19 in PTH interacts with the portion of the PTH-1 receptor (P1R) that contains the extracellular loops and seven transmembrance helices (TMs) (the J domain). We tested this hypothesis using the photoaffinity cross-linking approach. A PTHrP(1–36) analog and a conformationally constrained PTH(1–21) analog, each containing para-benzoyl-L-phenylalanine (Bpa) at position 19, each cross-linked efficiently to the P1R expressed in COS-7 cells, and digestive mapping analysis localized the cross-linked site to the interval (Leu232-Lys240) at the extracellular end of TM2. Point mutation analysis identified Ala234, Val235, and Lys240 as determinants of cross-linking efficiency, and the Lys240->Ala mutation selectively impaired the binding of PTH(1–21) and PTH(1–19) analogs, relative to that of PTH(1–15) analogs. The findings support the hypothesis that residue 19 of the receptor-bound ligand contacts, or is close to, the P1R J domain—specifically, Lys240 at the extracellular end of TM2. The findings also support a molecular model in which the 1–21 region of PTH binds to the extracellular face of the P1R J domain as an {alpha}-helix.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE TYPE-1 PTH receptor (P1R) is a class-2 G protein-coupled receptor (GPCR) that mediates the calcium-homeostatic actions of PTH and the developmental actions of PTHrP (1, 2). The first 34 amino acids of PTH and PTHrP contain sufficient information for full biological activity on the P1R (1, 2). Structure-activity relationship studies on PTH(1–34) and PTHrP(1–34) have indicated that the ligand determinants of receptor-binding affinity and receptor activation reside approximately in the carboxyl-terminal and amino-terminal portions of the molecule, respectively (2, 3). As for each class-2 GPCR that binds a peptide hormone, the P1R contains a relatively large (~170 amino acid) amino-terminal extracellular (N) domain that contributes importantly to ligand-binding affinity. Mutational and photoaffinity cross-linking studies suggest that the N domain of the P1R provides the principal docking site for the C-terminal binding domain of the ligand (3, 4, 5, 6) and that the juxtamembrane (J) region of the P1R, which contains the seven transmembrane helices (TMs) and the connecting loops, provides the principal interaction sites for the amino-terminal signaling portion of the ligand (3, 7, 8, 9, 10, 11). A two-site mode of ligand interaction has therefore been suggested for PTH and the P1R, and this general mechanism may be used by other class-2 GPCRs that bind peptide hormones, including the receptors for calcitonin (3, 12), glucagon and glucagon-like peptide 1 (13), vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide (14, 15), and corticotropin-releasing factor (CRF) (16, 17, 18). As for each of the class-2 GPCRs, the specific ligand/receptor interaction sites in the P1R are still poorly defined.

In the current models of the PTH·P1R complex (4, 19, 20), it has been assumed that residues in the ligand C terminal of position 13 (9) interact with the P1R N domain. In our functional studies on the PTH/P1R interaction mechanism, we have shown that modified N-terminal PTH analogs, such as [Aib1, 3,Gln10, Har11, Ala12,Trp14]PTH(1–14) [herein denoted as [M3]PTH(1–14)] bind to and activate equally well the intact P1R and a P1R mutant (P1R-delNt) that is missing most (residues 24–181) of the N domain (11, 21, 22, 23, 24). These observations suggest that residues 1–14 of PTH do not utilize the N domain of the P1R, as defined by the deletion in P1R-delNt, for binding interactions or to induce receptor activation. Extending such PTH fragments to residue 20 provides a 10- to 100-fold improvement in binding affinity and signaling potency on both the intact P1R and on P1R-delNt; these findings suggest that residues in the 15–20 region of PTH interact with the receptor’s J domain (25). We also found that the substitution of Glu19->Arg in modified PTH(1–20) analogs results in similar improvements in potency on P1R-delNt, a result that led us to the hypothesis that residue 19 of the ligand interacts with the P1R J domain (25). On the other hand, Piserchio et al. (26) recently showed by nuclear magnetic resonance (NMR) methods that the Glu19->Arg substitution in [Ala1,3,12,Gln10,Har11,Trp14,Arg19]PTH(1–20) [herein denoted as [M]PTH(1–20)], stabilizes the {alpha}-helical structure in the C-terminal region of the peptide and results in a peptide that is nearly completely {alpha}-helical. Residue 19 of the ligand could, therefore, contribute to affinity/potency by providing a direct receptor contact, by stabilizing a bioactive ligand conformation, or both.

In the current study we used the photoaffinity cross-linking approach to test the hypothesis that residue 19 of the receptor-bound ligand (PTH or PTHrP) directly contacts the P1R J domain. We used a PTHrP(1–36) analog, as well as a modified PTH(1–21) analog, each of which contained the photo-labile amino acid, para-benzoyl-L-phenylalanine (Bpa), at position 19. We cross-linked these ligands to the wild-type (WT) P1R or mutant P1Rs expressed in COS-7 cells and mapped the cross-linking site by digestive methods. The results of the studies provide support for our starting hypothesis, as they reveal that each ligand cross-links to a site within the extracellular portion of the receptor’s second TM domain. We incorporated the new findings, along with previously published cross-linking information and the proposed helical structure of PTH(1–21) (20, 26), into a new model of the PTH·P1R-J domain complex, which now includes residues in the 14–19 region of the ligand.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To explore possible receptor interaction sites for residue 19 in the ligand, we first synthesized and pharmacologically characterized the photo-labile analog Bpa19-PTHrP(1–36) (Table 1Go). The analog functioned as a fully potent agonist for cAMP production in P1R-expressing HKRK-B7 cells (EC50 = 1.1 ± 0.2 nM; response maximum = 431± 35 pmol/well), and bound to these cells with adequately high affinity [IC50 = 75 ± 7 nM, as compared with 15 ± 1 nM for PTHrP(5–36) analog, as determined in competition assays performed with 125I-PTHrP(5–36) tracer radioligand]. The radioiodinated Bpa19-PTHrP(1–36) analog cross-linked efficiently to the P1R expressed in COS cells, and no cross-linking was observed in the presence of excess unlabeled PTH(1–34) (Fig. 1Go).


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  		Table 1. Peptide Primary Structures

 


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  		Fig. 1. Cross-Linking of Bpa19-PTHrP(1–36) to the WT P1R

COS-7 cells transiently transfected with the WT P1R were incubated with 125I-Bpa19-PTHrP(1–36), either in the absence (lane 1) or presence of excess (1 x 10-6 M) PTH(1–34) (lane 2), and photoaffinity cross-linking was induced by UV irradiation. Cell lysates were analyzed by SDS-PAGE/autoradiography.

 
The cross-linked complex, Bpa19-PTHrP(1–36)·P1R, was gel-purified and digested with cyanogen bromide (CNBr) to cleave the peptide chain C terminal of methionine residues (absent in the ligand and occurring at 13 positions in the P1R, Fig. 2Go). Tricine-SDS-PAGE analysis of the digested complex resulted in the appearance of a band of approximately 10 kDa (Fig. 3AGo). The mobility of this band is consistent with a cross-link to any of six predicted CNBr/P1R digestion fragments: Thr33-Met63, Ile190-Met224, Leu232-Met312, Leu446-Met499, Val500-Met555, and Ala556-Met593 (Figs. 2Go and 3Go). Importantly, the data ruled out the possibility that cross-linking occurred to the CNBr fragment Glu64-Met189, as this P1R fragment is glycosylated and migrates above the 46-kDa size marker (4). The majority of the receptor’s amino-terminal extracellular domain, therefore, could be disregarded as a possible cross-linking site for Bpa19-PTHrP(1–36).



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  		Fig. 2. Schematic of the Human P1R

The intact P1R (lacking residues 1–22 of the signal sequence) is shown with the amino terminus at the top and the carboxyl terminus at the bottom. Shown are the locations of the methionine residues (green), lysine residues (blue), and potential sites for Asn-linked glycosylation (red). The cross-link interval for Bpa19-PTHrP(1–36) is also shown (gray). Position numbers are indicated for selected residues.

 


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  		Fig. 3. CNBr and LysC Digestion of Complexes Formed between Bpa19-PTHrP (1–36) and P1R

A, Gel-purified complexes of 125I-Bpa19-PTHrP(1–36) bound to the P1R were treated with 70% formic acid (F. A.) in the absence (lane 1) or presence (lane 2) of CNBr for 24 h. B, Purified complexes were incubated for 24 h without cleavage reagent as control (lanes 1), with LysC (lane 2), or with LysC followed by PNGase F (lane 3). The samples were analyzed by Tricine/SDS-PAGE/autoradiography, with equal amounts of radioactivity loaded into each lane. The positions of the size markers (in kilodaltons) are indicated; the 2.35- and 3.5-kDa size markers comigrated.

 
Digestion of the Bpa19-PTHrP(1–36)·P1R complex with endopeptidase LysC to cleave the peptide chains C terminal of lysine residues (occurring at position 13 in the ligand and at 27 positions in the P1R, Fig. 2Go) resulted in a diffuse band that migrated mostly above the 21-kDa size marker (Fig. 3BGo, lane 2). The mobility and diffuse appearance of this band suggested that the cross-linked LysC fragment was glycosylated. Only two of the predicted LysC-generated P1R fragments contain consensus sites for Asn-glycosylation: Gly142-Lys172 and Phe173-Lys240 (Fig. 2Go). The fragment Gly142-Lys172 does not overlap any of the six candidate CNBr-generated fragments. The fragment Phe173-Lys240, on the other hand, overlaps two of the CNBr-generated fragments: Ile190-Met224 and Leu232-Met312. The results, therefore, localize the Bpa19-PTHrP(1–36) cross-linking site to the LysC fragment Phe173-Lys240. Treatment of the LysC-digested conjugate with peptide N-glycosidase (PNGase) F resulted in a large proportion of apparently aggregated material that failed to enter the gel, along with a new distinct band, the mobility of which corresponded to a fragment of approximately 12 kDa (Fig. 3BGo, lane 3). The size of this band correlates more closely with the predicted size of the unglycosylated Bpa19-PTHrP(14–36)·(Phe173-Lys240) conjugate (~11 kDa) than it does with the predicted size of the unglycosylated Bpa19-PTHrP(14–36)·(Gly142-Lys172) conjugate (~6 kDa). The results of the PNGase F treatment, therefore, confirm that Bpa19-PTHrP(1–36) cross-linked to the LysC-fragment (Phe173-Lys240).

To further narrow the cross-link interval, we investigated which of the two possible CNBr-generated fragments, Ile190-Met224 vs. Leu232-Met312, contains the cross-linking site for Bpa19-PTHrP(1–36) We employed for this analysis several P1R mutants (8), each of which was altered at one of the relevant methionine residues (i.e. those positions at 189, 224, 231, and 312; Fig. 2Go). The ligand-binding and cAMP-signaling properties of these mutant receptors, as evaluated with PTH(1–34), were indistinguishable from those of the WT P1R (8). Digestion of the Bpa19-PTHrP(1–36)·P1R-M189V and Bpa19-PTHrP(1–36)·P1RM224L complexes with CNBr resulted, in each case, in an approximately 10-kDa fragment that was identical in size with that obtained with the WT P1R (data not shown). Digestion of the Bpa19-PTHrP(1–36)·P1R-M312V complex, however, resulted in a fragment of approximately 24 kDa, which was consistent with the addition of the 100-residue segment Ala313-Met414 to the Leu232-Met312 fragment (Fig. 4AGo, lane 1). These results, therefore, indicate that Bpa19-PTHrP(1–36) cross-linked to a site within the Leu232-Met312 fragment of the P1R. This interpretation is supported by the finding that CNBr digestion of the Bpa19-PTHrP(1–36)·P1R-M231I conjugate yielded a band that was only slightly larger than the 10-kDa fragment obtained with the WT P1R (Fig. 4BGo), consistent with the addition of the expected seven-residue segment His225-Met231 to the Leu232-Met312 fragment. The overlap between the CNBr-generated fragment, Leu232-Met312, and the LysC-generated fragment, Phe173-Lys240, narrows the cross-link interval to the nine-amino-acid segment Leu232-Lys240, which comprises the extracellular portion of TM2 (Fig. 2Go).



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  		Fig. 4. CNBr Digestion of Complexes Formed between Bpa19-PTHrP(1–36) and P1R Mutants Substituted at Methionine Residues

Gel-purified complexes formed between 125I-Bpa19-PTHrP(1–36) and P1R-M312V (panel A, lane 1), P1R-WT (panel A, lane 2, and panel B, lanes 1 and 3), or P1R-M231I (panel B, lane 2) were treated with CNBr for 24 h, and the resulting digested samples were analyzed by Tricine/SDS-PAGE/autoradiography with equal amounts of radioactivity loaded in each lane. The positions of the size markers (in kilodaltons) are indicated.

 
We then used point mutation analysis to analyze the role of the individual residues within the Leu232-Lys240 interval in the cross-linking efficiency of Bpa19-PTHrP(1–36). The mutant P1Rs generated for this analysis were each well expressed on the cell surface, as judged by their capacities to bind the N domain-selective radioligand 125I-PTHrP(5–36) (Table 2Go). With the exceptions of P1R-R233A, each mutant bound radioiodinated Bpa19-PTHrP(1–36) at a level comparable to that observed for P1R-WT (Fig. 5AGo). The low binding of P1R-R233A (~15% of P1R-WT) is consistent with previous findings that showed that mutations at this Arg impair binding of PTH(1–34) and PTHrP(1–34) analogs (27, 28). For the other mutant P1Rs, equal amounts of receptor-bound (covalently and noncovalently) radioactivity were applied to SDS-PAGE gels, and relative effects on cross-linking efficiency were assessed by comparing the intensities of the bands migrating at the position of the intact ligand·receptor complex. As shown in Fig. 5AGo, cross-linking to P1R-A234V, P1R-V235A, and P1R-K240A was reduced, relative to the cross-linking observed with P1R-WT and the other mutants tested (excluding P1R-R233A). These results identify Ala234, Val235, and Lys240A as receptor determinants of Bpa19-PTHrP(1–36) cross-linking efficiency.


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  		Table 2. Radioligand-Binding To WT and Mutant P1Rs

 


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  		Fig. 5. Analysis of Cross-Linking Efficiency for Bpa19-PTHrP(1–36) and 125I-Bpa19-[M3]PTH(1–21) on P1R Mutants Altered in the TM2 Region

A, 125I-Bpa19-PTHrP(1–36) was bound to COS-7 cells transiently transfected with the WT P1R, the mutants indicated, or with pcDNA1 vector control, and photoaffinity cross-linking was induced. Cells were lysed and, with the exception of P1R-R233A, equal amounts of specifically bound radioactivity (65,187 ± 67 cpm; 36,841 cpm for P1R-R233A) were analyzed by SDS-PAGE/autoradiography. For P1R-R233A, radioligand binding was inadequate for equal analysis. Relative cross-linking efficiency was reduced for P1R-A234V, P1R-V235A, and P1R-K240A, as judged by the lower relative band intensities for these mutant P1Rs (the relative cross-linking efficiency of P1R-R233A could not be accurately assessed by this analysis). B, The cross-linking efficiency of 125I-Bpa19-[M3]PTH(1–21) was analyzed as in A. Equal amounts of specifically bound radioactivity (79, 776 ± 133 cpm) were analyzed for each receptor, with the exceptions of P1R-R233A (<100 cpm), P1R-A234V (<100 cpm), P1R-I237A (684 cpm), and P1R-F238A (68,000 cpm), each of which exhibited radioligand binding levels that were inadequate for equal analysis. Relative cross-linking efficiency was reduced for the P1R-V235A and P1R-K240A mutants. The gels in panels A and B are each representative of three separate experiments. The nonspecifically bound radioactivity was determined from vector-transfected cells.

 
We then examined the cross-linking properties of Bpa19-[M3]PTH(1–21) (Table 1Go). This PTH(1–21) analog is a derivative of the modified PTH(1–20) analogs used in the previous functional (25) and NMR (26) studies that explored the role of residue 19 on ligand activity and peptide conformation. The Bpa19-[M3]PTH(1–21) analog cross-linked efficiently and specifically to the WT P1R, and digestive mapping analysis indicated that photo-insertion occurred within the same CNBr/LysC-defined interval, Leu232-Lys240, as it did with Bpa19-PTHrP(1–36) (data not shown). We therefore analyzed the capacity of Bpa19-[M3]PTH(1–21) to cross-link to the P1Rs altered by point mutation in the Leu232-Lys240 interval. With four of the mutants, P1R-R233A, P1R-A234V, P1R-I237A, and P1R-F238A, binding of the analog was too low (<=22% of P1R-WT, Fig. 5BGo) to permit an accurate assessment of cross-linking efficiency. With the remaining mutants, equal amounts of receptor-bound radioactivity were loaded onto the gels, and relative effects on cross-linking efficiency were assessed, as described above. Of these mutants, reduced cross-linking efficiency was observed for P1R-V235A and P1R-K240A (Fig. 5BGo). Similar to the findings with Bpa19-PTHrP(1–36), therefore, Bpa19-[M3]PTH(1–21) cross-links to a site within the extracellular portion of TM2 and requires Val235 and Lys240 for efficient photo-insertion.

We then investigated whether or not any of the point mutations in the cross-link interval functionally contribute to binding interactions involving residues at or near position 19 in the ligand. Each mutant P1R was assessed for its capacity to bind the radioligands 125I-[M3]PTH(1–21) and 125I-[M3]PTH(1–15). The mutations Leu232->Ala, Val235->Ala, and Val239->Ala resulted in little or no change in the binding of either radioligand (Table 2Go); the native residues at these sties, therefore, are not required for efficient binding of either the PTH(1–15) or PTH(1–21) analog. The mutations Arg233->Ala, Ala234->Val, Ile237->Ala, and Phe238->Ala resulted in reduced binding of both radioligands; these results suggest that the residues at these sites function as binding determinants for residues in the 1–15 portion of the ligand (Table 2Go). The Lys240->Ala mutation resulted in a 50% decrease in the binding of 125I-[M3]PTH(1–21) and a 45% increase in the binding of 125I-[M3]PTH(1–15) (Table 2Go); lysine-240, therefore, functions as a binding determinant for residues in the 16–21 region of the ligand. In competition assays in which 125I-[M3]PTH(1–15) was used as a tracer radioligand, the apparent binding affinities of [M]PTH(1–20) and [M]PTH(1–14) were more comparable to each other on P1R-K240A (IC50 = 220 ± 50 and 4,500 ± 400 nM, respectively) than they were on P1R-WT (IC50 = 50 ± 6 nM and 20,000 nM, respectively, Fig. 6Go, A and B). Similarly, the binding affinities of the constrained analogs [M3]PTH(1–19) and [M3]PTH(1–15) (23) on P1R-K240 were approximately equivalent on P1R-K240A (IC50 = 270 ± 30 and 360 ± 30 nM, respectively, P = 0.03) but differed by approximately 4-fold on P1R-WT (IC50 = 16 ± 3 and 61 ± 12 nM, respectively, P = 0.006; Fig. 6Go, C and D). The Lys240->Ala mutation, therefore, reduced the capacity of the P1R to distinguish, on the basis of binding affinity, between the PTH(1–19) and PTH(1–15) analogs. The results suggest, therefore, that Lys240 functions as a binding determinant for residues in the 16–19 region of the ligand.



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  		Fig. 6. Effect of the Lys240->Ala Substitution in the P1R on Apparent Binding Affinity of PTH Analogs

COS-7 cells transiently transfected with P1R-WT (A and C) or P1R-K240A (B and D) were incubated with 125I-[M3]PTH(1–15) tracer radioligand and varying concentrations of the unlabeled peptides [M]PTH(1–20) and [M]PTH(1–14) (A and B), or [M3]PTH(1–19) and [M3]PTH(1–15) (C and D). Cells were incubated for 4 h at 15 C. Specific binding was measured and expressed as a percentage of the specific binding observed in the absence of unlabeled ligand. Data are from three separate experiments, each performed in duplicate.

 
We used molecular modeling to address whether the ligand/receptor contacts suggested so far by cross-linking data for the N-terminal portion of the ligand are simultaneously compatible with a ligand/receptor interaction mode in which the N-terminal portion, residues 1–21, of native PTH binds to the receptor as a single {alpha}-helix (20, 26). We used for this analysis the ligand/receptor proximities of Val2/Met425 (8, 29), Lys13/Arg186 (9), and Glu19/Lys240 (this study). Because each of the three receptor residues is in or near a TM domain, TM6, TM1, and TM2, respectively, only the heptahelical portion of the receptor, extending N-terminally to Asp185, was used for the analysis. The x-ray crystallographic coordinates of rhodopsin (30, 31) were used as a template for the starting structure of the P1R heptahelical domain, and the x-ray crystallographic coordinates of PTH(1–34) (20), limited to residues 1–21, were used for the starting structure of the ligand. The ligand was initially docked to the receptor by positioning the three ligand contact residues within 10 Å of the cognate receptor residue, and the complex was then subjected to an iterative energy minimization process without applying constraints. In the resulting structure, shown in Fig. 7Go, the ligand remained fully helical and the side-chain of each ligand-contact residue remained close to its cognate receptor residue (Val2/Met425, 7.33 Å; Lys13/Arg186, 6.02 Å; Glu19/Lys240, 2.79 Å). Within the limits imposed by the assumptions of this model, therefore, three contacts suggested by cross-linking data for the 1–21 portion of PTH can be accommodated by a fully {alpha}-helical ligand structure.



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  		Fig. 7. Model of PTH (1–21) Bound to the P1R

Residues 1–21 of human PTH are shown in {alpha}-helical conformation (20 ) and bound to the extracellular portion of the heptahelical bundle of the P1R. The residues comprising the seven TM helical domains of the receptor were determined by HHMTOP. The seven helical domains were spatially arranged using SWISS-MODEL version 3.5 and the x-ray crystallographic structure of rhodopsin (30 31 ) as a template. The ligand was docked to the receptor manually, using proximity between Val2, Lys13, and Glu19 in the ligand and Met425, Arg186, and Arg240, respectively, in the P1R as initial positioning points. The complex was then subjected to iterations of energy minimization without constraints until a steady-state structure was reached. The atoms of PTH(1–21), in space-filled format, are colored orange, except for the side-chains of Val2, Lys13, and Glu19, which are colored by atom type (white, carbon; red, oxygen; blue, nitrogen). The seven TM helices of the receptor, in wire-frame format, are colored as follows: TM1, yellow; TM2, dark blue; TM3, green; TM4, red; TM5, white; TM6, magenta; and TM7, light blue. The side-chain atoms of Met425, Arg186, and Arg240 in the P1R are shown in space-filled format and in the color of their respective TM domain (Met425, magenta; Arg186, yellow; and Arg240, dark blue).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study we explored the role of residue 19 in PTHrP and PTH in the P1R-interaction process and we specifically tested the hypothesis that this residue interacts with the P1R J domain (25). We used for our analysis the photochemical cross-linking approach, in which we employed a PTHrP(1–36) analog and a conformationally constrained PTH(1–21) analog, each of which was modified with the photolabile amino acid analog, Bpa, at position 19. This work extends prior studies in which Bpa-modified PTH(1–34) and PTHrP(1–36) analogs have been used to map sites of physical proximity between other sites in the ligand (e.g. positions 1, 2, 23, 27, 27, 28, and 33) and specific sites or regions in the P1R (4, 5, 7, 8, 9, 29, 32). Both of our current Bpa19-modified PTHrP(1–36) and PTH(1–21) analogs cross-linked efficiently and specifically to the P1R, and for each ligand, the photo-insertion site was mapped to the nine-amino acid segment (Leu232-Lys240), which comprises the extracellular portion of TM2. The results, therefore, were fully consistent with our starting hypothesis that residue 19 interacts with the receptor’s J domain.

Within the (Leu232-Lys240) interval, we observed diminished cross-linking efficiency for both analogs with the mutant P1Rs altered by point mutation at Val235 and Lys240, and for the PTHrP(1–36) analog, with the mutant altered at Ala234 (poor binding of the PTH(1–21) analog to P1R-A234V precluded cross-linking analysis). The actual photo-insertion site for the Bpa-19 moiety cannot be definitively determined from our current mapping methods, and it may be that any or all of the three residues, Ala234, Val235, and Lys240, contribute to the photo-reaction mechanism. In any event, the overall results clearly define a rather narrow region of the P1R that is in proximity to residue 19 of the bound ligand. They also suggest that a modified conformationally constrained N-terminal PTH fragment analog, such as [M3]PTH(1–21), interacts with the receptor in a fashion similar to that used by a relatively unmodified ligand, such as PTHrP(1–36).

Using radioligand binding assays we explored the functional contribution that the individual receptor residues in the Bpa19-cross-link interval make to the ligand-interaction process. Some of the point mutations in this interval, including Val235->Ala, had little or no effect on the binding of either 125I-[M3]PTH(1–21) or 125I-[M3]PTH(1–15), and several others: Arg233->Ala, Ala234->Val, Ile237->Ala, and Phe238->Ala, reduced binding of both 125I-[M3]PTH(1–21) and 125I-[M3]PTH(1–15). The native residues at the former sites do not appear to contribute importantly to ligand-binding, at least concerning the 1–14 portion of the ligand, whereas those at the latter sites contribute to binding via potentially indirect mechanisms that affect the binding site for the 1–15 portion of the ligand. The Lys240->Ala mutation was unique in that it reduced by 40% the total binding of 125I-[M3]PTH(1–21) yet increased, by 45%, the total binding of 125I-[M3]PTH(1–15). In competition assays, the Lys240->Ala mutation reduced the apparent binding affinity of [M3]PTH(1–19) more than it did the affinity of [M3]PTH(1–15) (17-fold vs. 6-fold). Lysine-240, therefore, appears to contribute to ligand-binding affinity via a mechanism that involves residues in the 16–19 region of the ligand.

Our initial hypothesis that residue 19 in the ligand interacts with the P1R J domain was based primarily on the observation that the Glu19->Arg substitution in various PTH analogs enhances signaling potency (~10- to 100-fold) on P1R-delNt (25). We therefore investigated whether the affinity-enhancing effect of the Arg19 substitution was mediated by Lys240. This possibility was ruled out, as we found that [M]PTH(1–20)-Glu19 and [M]PTH(1–20) (Arg19) exhibited the same (~20-fold) difference in binding affinities on P1R-K240A (IC50 = 5500 ± 1, 100, and 220 ± 40 nM, respectively, P = 0.009, n = 4) as they did on P1R-WT (IC50 = 850 ± 30 and 50 ± 6 nM, respectively, P = 0.0001, n = 4). We also found that Ser236, one helical turn below Lys240, did not mediate the enhancing effect of Arg19, as again, an approximately 20-fold difference in affinity was observed for [M]PTH(1–20)-Glu19 and [M]PTH(1–20) on P1R-S236A (IC50 = 920 ± 120 and 47 ± 3 nM, respectively, P = 0.002, n = 4). Other residue(s) in the P1R J domain may, therefore, mediate the enhancing effect that Arg19 has on receptor-binding affinity. Alternatively, the effect could involve intramolecular changes in ligand conformation, as suggested by the increased helicity observed for [M]PTH(1–20), vs. that of [M]PTH(1–20)-Glu19, in the recent NMR study of Piserchio et al. (26). It may be that residue 19 plays multiple, and potentially overlapping, roles in the ligand-receptor interaction process, including contact to the receptor J domain and modulation of ligand conformation.

The mapping of the contact site for the Bpa19-modified analogs to the extracellular portion of TM2 is particularly noteworthy in light of prior mutational studies on this helix of the P1R and of other class-2 GPCRs. In the opossum P1R, Turner et al. (28) showed that Ser229, Arg233, and Ser236 (in the human P1R position numbering), by forming a polar face on TM2, contribute to PTH(1–34) binding affinity and signaling potency. Turner et al. also found that Ile237 in the opossum P1R serves as a "selectivity filter," because its mutation to Asn (the corresponding secretin receptor residue) unmasks responsiveness to secretin (33). Mutations at Arg233 in the P1R, which is two helical turns below and on the same helical face as Lys240, impairs binding affinity and signaling potency for PTH(1–34) agonist ligands, possibly via a mechanism involving interhelical interactions with the conserved Gln451 in TM7 PTH(3–34) (27). The residues corresponding to lysine-240 and arginine-233 in the P1R are highly conserved in the class-2 GPCRs (34), and studies on the receptors for secretin (35), vasoactive intestinal peptide (15), and glucagon (13, 36) have shown that these sites contribute to agonist-binding affinity and potency in these class-2 receptors. Closer to the intracellular terminus of TM2, mutation of the conserved His223 to Arg in the P1R (37), and the equivalent mutation in several other class-2 receptors (38, 39, 40, 41) results in constitutive receptor signaling activity. The combined data suggest, therefore, that TM2 of the class-2 GPCRs plays a key role in ligand-recognition and receptor activation; in the case of the P1R, this role appears to involve residue 19 of the ligand.

Our cross-linking data on residue 19 provided a new distance constraint for modeling the PTH·P1R complex, specifically in terms of the J domain component of the interaction, which, in previous models (4, 19, 20, 42), has been assumed to principally include residues in the 1–13 region of the ligand. Our current data now suggest that this component of the interaction extends C terminally in the ligand to include residue 19. One uncertainty in modeling the PTH·P1R complex concerns the conformational state of the bound ligand. In the unbound state, the structures determined for PTH(1–34) analogs vary considerably, particularly for residues at or about position 19, which may be flexible (43, 44), helical (20, 26, 45, 46, 47), or involved in a turn (48, 49). Our modeling analysis shows that the contacts suggested so far by cross-linking methods for residues in the 1–19 portion of PTH [e.g. residues 2 (Refs. 8 and 29), 13 (Ref. 9), and 19 (herein)] are compatible with an interaction mode in which this portion of the ligand binds to the J domain as a single {alpha}-helix. In the model, the ligand appears to fit into a groove along the extracellular face of the heptahelical bundle, with the N terminus of the ligand abutting the extracellular end of TM5 (white helix in Fig. 7Go) and the C terminus resting between the extracellular ends of TMs 1 and 2 (yellow and blue, respectively, in Fig. 7Go). Importantly, valine-2 of the ligand, a key activation residue, is positioned within the TM5/TM6/extracellular loop 3 region, which is known to contain determinants of ligand-dependent signaling (8, 50).

As models of the PTH·P1R complex are further refined with additional cross-linking constraints, they are likely to yield new insights into how the P1R, and potentially other class-2 GPCRs, function. In regards to cross-linking data available for other class-2 GPCRs, Assil et al. (18) identified a proximity between lysine 16 of sauvagine and the second extracellular loop of the CRF-1 receptor; a result that suggests similarity between the CRF·CRFR complex and the PTH·P1R complex. Data for the secretin receptor are particularly interesting, in that a benzophenone group at each of four different residue positions in secretin(1–27), 6 (Ref. 51), 18 (Ref. 52), 22 (Ref. 53), and 26 (Ref. 53), cross-linked to the first 40 amino acids of the receptor’s N-terminal domain. These results differ considerably from the PTH/P1R system, in which four N-terminal residues of the ligand, 1 (Ref. 7), 2 (Refs. 8 and 29), 13 (Ref. 9), and 19 (this study), cross-link to the P1R J domain and four C-terminal residues, 23 (Ref. 5), 27, 28, and 33 (Ref. 4) cross-link to the P1R N domain. Cross-linking studies by Behar et al. (54) on the PTH-2 receptor suggest that the ligand-interaction topology used by this receptor mirrors that used by the PTH-1 receptor. Further work is clearly needed to elucidate the similarities and differences in the ligand/receptor interaction mechanisms used by the different class-2 receptors. Models on these receptors could ultimately facilitate the design of high-affinity, nonpeptide mimetic ligands. For the P1R, such molecules have yet to be described, but a potent P1R-selective agonist mimetic would be of considerable interest, particularly because PTH(1–34) has recently been shown to be an effective treatment for osteoporosis (55, 56).

In conclusion, we have used the photochemical cross-linking approach to identify a physical contact between residue 19 of a PTHrP(1–36) analog, as well as of a conformationally constrained PTH(1–21) analog, and the extracellular end of TM2 of the PTH-1 receptor. Lysine-240, at the extracellular end of TM2, contributes to photo-insertion efficiency, as well as to binding interactions involving ligand residues (16, 17, 18, 19). The newly identified contact permits refinement of models of the PTH·P1R complex, particularly for the J portion of the receptor, which now appears to accommodate at least the 1–19 portion of the ligand, binding as a single {alpha}-helical structure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Peptides and Reagents
Peptide sequences are shown in Table 1Go. All peptides were synthesized by the Protein and Peptide Core Facility at Massachusetts General Hospital (Boston, MA) using the solid-phase method on PerkinElmer (Boston, MA) Model 430A or 431A synthesizers. Peptides were purified by reverse-phase chromatography and their compositions were confirmed by amino acid analysis and mass spectroscopy.

The following reagents were purchased from the indicated vendors: Na125I (specific activity, 2000 Ci/mmol), NEN Life Science Products (Boston, MA); DMEM, trypsin/EDTA, penicillin G/streptomycin, and horse serum, Life Technologies, Inc. (Rockville, MD); fetal bovine serum, HyClone Laboratories (Logan, UT); Tricine, Sigma (St. Louis, MO); trifluoroacetic acid, Pierce (Rockford, IL); CNBr and endopeptidase LysC, Serva Fine Chemicals/Boehringer Ingelheim (Heidelberg, Germany); PNGase F, New England Biolabs Inc. (Beverly, MA); 14C-methylated protein molecular mass markers, Amersham Pharmacia Biotech (Piscataway, NJ); diethylaminoethyl-dextran, Pharmacia (Uppsala, Sweden); Biomax MS film, Eastman Kodak Co. (Rochester, NY).

Mutagenesis of the P1R
Mutations were introduced into the P1R by site-directed mutagenesis using uracil-containing single-stranded plasmid DNA encoding the WT human P1R cDNA as a template and oligonucleotides encoding the desired mutations as mutagenic primers (11, 57). Mutations were verified by automated nucleotide sequence analysis of cesium chloride-purified plasmid DNA.

Cell Culture/DNA Transfection
Cells were cultured at 37 C in a humidified atmosphere (95% air/5% carbon dioxide) in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. For photoaffinity cross-linking studies, COS-7 cells were cultured in six-well plates (6 cm diameter wells), and transfected using diethylaminoethyl-dextran (50) and 800 ng cesium chloride-purified plasmid DNA per well when the cell monolayer reached approximately 90% of confluence. For radioreceptor-binding assays, COS-7 cells were cultured in 24-well plates and transfected using Fugene 6 (Roche Biosciences, Palo Alto, CA) and 200 ng plasmid DNA per well when the cell monolayer reached approximately 50% of confluence. COS-7 cells were used for experiments 4 d after transfection. HKRK-B7 cells (58), which express the human P1R via stable DNA transfection, were cultured in 24-well plates and used for functional assays 2–3 d after the cell monolayer became confluent.

Radiolabeling of Peptides
Radiolabeled peptides were prepared by chloramine-T iodination and purified via HPLC using a 30–50% acetonitrile/H2O gradient in 0.1% trifluoroacetic acid over 30 min.

Radioligand Binding and cAMP Accumulation Assays
Radioligand binding assays were preformed as previously described (23). In brief, a 125I-labeled radioligand (~100,000 cpm per well of a 24-well plate) was incubated with whole cells expressing either the WT or a mutant P1R in the absence or presence of varying concentrations (3 x 10-9 to 1 x 10-5 M) of unlabeled peptide. After a 4-h incubation at 15 C, the binding mixture was removed by aspiration, the cells were rinsed three times with binding buffer, lysed in NaOH, and the entire lysate was counted for {gamma}-irradiation. Intracellular cAMP accumulation assays were performed as previously described (23). In brief, cells in 24-well plates were treated with ligand in 3-isobutyl-1-methylxanthine-containing buffer for 30 min at room temperature, the media was removed, the cells were lysed by freezing the plates on dry ice and adding 0.5 ml of 50 mM HCl to the wells. The cAMP in the lysates, diluted to 2.5 ml with H2O, was measured by RIA.

Photoaffinity Labeling of the P1Rs
COS-7 cells transiently expressing either the WT or a mutant P1R were incubated with a 125I-labeled, Bpa19-modified ligand analog (3 x 106 cpm per well of a six-well plate) for 6 h at 4 C. The cells were rinsed twice with ice-cold binding buffer, 1 ml of binding buffer was added, and the plate was placed on ice under an UV light source (Blak Ray long-wave lamp, 366 nm, 7000 microwatts/cm2; UV Products, San Gabriel, CA) at a source-to-target distance of 5 cm for 15 min. The cells were then lysed using binding buffer containing 1% Triton X-100 and the lysate was clarified by a brief centrifugation at 1500 x g. The supernatant was then mixed 1:1 (vol:vol) with 2x SDS-PAGE sample buffer to attain final concentrations of 4% SDS, 80 mM Tris-HCl (pH 6.8), 20% glycerol, 0.2% bromphenol blue, and 100 mM dithiothreitol.

SDS-PAGE Analysis/Purification
For visualization of the intact cross-linked ligand·receptor complexes, the samples in SDS-PAGE sample buffer were incubated at room temperature for 2 h and then subjected to SDS-PAGE (10% acrylamide:3% Bis-Acryl) performed according to the method of Laemmli (59). The gels were dried and developed by autoradiography at -80 C. For purification of the complexes, samples were subjected to SDS-PAGE performed as described above but without drying; the lanes of the wet gels were cut into sections (~1 cm x 0.5 cm), which were counted in a {gamma} counter, and sections with peak radioactivity were subjected to electroelution in a dialysis bag (molecular mass cutoff, 12,000 Da) at 100 V for 2 h. The eluates were concentrated using Centricon-10 tubes (Millipore Co., Bedford, MA) and then subjected to digestion and size analysis, as described below.

Chemical/Enzymatic Cleavage
For cleavage of the ligand·receptor complexes at methionine residues, the gel-purified samples were incubated in a solution of CNBr (100 mM) and formic acid (70%) at 20 C for 24 h. After digestion, the CNBr and formic acid were removed by repetitive lyophylizations. For cleavage of the complexes at lysine residues, the samples were incubated with endopeptidase LysC at 37 C for 24 h. For removal of Asn-linked glycosyl groups, the samples were treated with PNGase F (2500 U) for 3 h at 37 C.

Fragment Size Analysis by Tricine-SDS-PAGE
Digested or mock-digested samples were suspended in SDS-PAGE sample buffer and incubated at room temperature for 2 h. The samples were then analyzed by Tricine-SDS-PAGE (12% acrylamide:3.4% Bis-acrylamide) performed according to the method of Schagger and von Jagow (60). Autoradiographic analysis of the dried gels was performed at -80 C.

Data Calculation
Calculations were performed using Microsoft Excel. Nonlinear regression analysis of binding data was performed using the four-parameter equation: yp = minimum + [(maximum - minimum)/(1 + (IC50/x)slope)], and the Excel Solver function to optimize parameters (61, 62). The statistical significance between two data sets was assessed using a two-tailed Student’s t test, assuming unequal variances for the two sets.

Molecular Modeling
The seven TM domains of the P1R were defined using HHMTOP version 2.0 (G. E. Tusnady, Institute of Enzymology, Budapest, Hungary). The seven TM domains were fit to the rhodopsin x-ray crystallographic structure (30, 31) using SWISS-MODEL version 3.5, and the resulting atomic coordinate file was subsequently manipulated using SwissPdbViewer version 3.7 (63). To incorporate the cross-link between residue 13 of PTH and Arg186 of the P1R (9), the P1R segment (Asp185-Gly188) was manually ligated to Met189 at the extracellular terminus of TM1. For the PTH ligand structure, the atomic coordinate file of the crystal structure of native human PTH(1–34) (20) was used, adopting only the coordinates for residues 1–21. The ligand was manually docked to the receptor using three ligand/receptor proximities that have been suggested by cross-linking analyses, Val2/Met425 (8, 29), Lys13/Arg186 (9), and Glu19/Lys240 (this work), as positioning points. Energy minimization was performed using the GROMOS96 program of SwissPdbViewer.


   FOOTNOTES
 
This work was supported by NIH Grant DK-11794.

Abbreviations: Bpa, para-Benzoyl-L-phenylalanine; CNBr, cyanogen bromide; CRF, corticotropin-releasing factor; G protein-coupled receptor; J, juxtamembrane; NMR, nuclear magnetic resonance; P1R, type-1 PTH receptor; PNGase, peptide N-glycosidase; TM, transmembrane helix; WT, wild-type.

Received for publication July 15, 2003. Accepted for publication August 19, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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