Minimization of parathyroid hormone. Novel amino-terminal parathyroid hormone fragments with enhanced potency in activating the type-1 parathyroid hormone receptor

The amino-terminal and carboxyl-terminal portions of the 1-34 fragment of parathyroid hormone (PTH) contain the major determinants of receptor activation and receptor binding, respectively. We investigated how the amino-terminal signaling portion of PTH interacts with the receptor by utilizing analogs of the weakly active fragment, rat (r) PTH(1-14)NH(2), and cells transfected with the wild-type human PTH-1 receptor (hP1R-WT) or a truncated PTH-1 receptor which lacked most of the amino-terminal extracellular domain (hP1R-delNt). Of 132 mono-substituted PTH(1-14) analogs, most having substitutions in the (1-9) region were inactive in assays of cAMP formation in LLC-PK1 cells stably expressing hP1R-WT, whereas most having substitutions in the (10-14) region were active. Several substitutions (e.g. Ser(3) --> Ala, Asn(10) --> Ala or Gln, Leu(11) --> Arg, Gly(12) --> Ala, His(14) --> Trp) enhanced activity 2-10-fold. These effects were additive, as [Ala(3),(10,12),Arg(11), Trp(14)] rPTH(1-14)NH(2) was 220-fold more potent than rPTH(1-14)NH(2) (EC(50) = 0.6 +/- 0.1 and 133 +/- 16 micrometer, respectively). Native rPTH(1-11) was inactive, but [Ala(3,10), Arg(11)]rPTH(1-11)NH(2) achieved maximal cAMP stimulation (EC(50) = 17 micrometer). The modified PTH fragments induced cAMP formation with hP1R-delNt in COS-7 cells as potently as they did with hP1R-WT; PTH(1-34) was 6,000-fold weaker with hP1R-delNt than with hP1R-WT. The most potent analog, [Ala(3,10,12),Arg(11), Trp(14)]rPTH(1-14)NH(2), stimulated inositol phosphate production with hP1R-WT. The results show that short NH(2)-terminal peptides of PTH can be optimized for considerable gains in signaling potency through modification of interactions involving the regions of the receptor containing the transmembrane domains and extracellular loops.     

Novel parathyroid hormone (PTH) antagonists that bind to the juxtamembrane portion of the PTH/PTH-related protein receptor

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.     

Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: selectivity of a modified PTH(1-15) radioligand for GalphaS-coupled receptor conformations

Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, (125)I-[Aib(1,3),Nle8,Gln10,homoarginine11,Ala12,Trp14,Tyr15]rPTH(1-15)NH2, (125)I-[Aib(1,3),M]PTH(1-15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed (125)I-PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of (125)I-[Aib(1,3),M]PTH(1-15) to the wild-type PTHR was strongly (approximately 90%) inhibited by guanosine 5'-O-(3-thio)triphosphate (GTPgammaS), whereas the binding of (125)I-PTH(1-34) was only mildly (approximately 25%) inhibited by GTPgammaS. Of these two radioligands, only (125)I-[Aib(1,3),M]PTH(1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPgammaS. Binding of (125)I-[Aib(1,3),M]PTH(1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (approximately 20%) inhibited by GTPgammaS, as was the binding of (125)I-PTH(1-34). In membranes prepared from cells lacking Galpha(S) via knockout mutation of Gnas, no binding of (125)I-[Aib(1,3),M]PTH(1-15) was observed, but binding of (125)I-[Aib(1,3),M]PTH(1-15) was recovered by virally transducing the cells to heterologously express Galpha(S). (125)I-PTH(1-34) bound to the membranes with or without Galpha(S). The overall findings confirm the hypothesis that (125)I-[Aib(1,3),M]PTH(1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to Galpha(S)-containing heterotrimeric G proteins, whereas the binding of (125)I-PTH(1-34) can occur in the absence of such coupling. Thus, (125)I-[Aib(1,3),M]PTH(1-15) appears to function as a selective probe of Galpha(S)-coupled, active-state PTHR conformations.     

Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues

The N-terminal fragment of PTH(1-34) is critical for PTH1 receptor activation. Various modifications of PTH(1-14) have been shown to result in a considerable increase in signaling potency [Shimizu et al. (2000) J. Biol. Chem. 275, 21836-21843]. Our structural investigations revealed an unusually stable helical structure of the signaling domain (1-14), where residues 6 (Gln) and 10 (Gln or Asn) were located on the same face of the alpha-helix. To test whether a stable N-terminal alpha-helix is required for productive interaction with PTH1 receptor, we designed two conformationally restricted PTH(1-14) analogues, each containing a lactam bridge at positions 6 and 10. Specifically, substitutions Gln(6)-->Glu(6) and Asn(10)-->Lys(10) were introduced into the most potent [Ala(1,3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH2 agonist. Both the Glu(6)-Lys(10) and Lys(6)-Glu(10) lactam-bridged analogues were characterized to examine the importance of orientation of the lactam. According to biological studies [Shimizu et al. (2003) Biochemistry 42, 2282-2290], none of the 6/10 substituted analogues (linear or cyclic) remained as active as the parent peptide. However, relative to their corresponding linear peptides, lactam-bridged analogues either maintained potency or showed 6-fold improvement. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the structural differences between the linear and cyclic PTH(1-14) fragments, supporting the hypothesis that an alpha-helix is the preferred bioactive conformation of the N-terminal fragment of PTH. In addition, our results demonstrate that the structural order of the very first residues (1-4) of the signaling domain plays a significant role in PTH action.     

Zinc(II)-mediated enhancement of the agonist activity of histidine-substituted parathyroid hormone(1-14) analogues

Previous studies on parathyroid hormone (PTH)(1-14) revealed that residues (1-9) played a dominant role in stimulating PTH-1 receptor-mediated increases in cAMP formation. In the present study, we examined the effects of installing a metal-binding motif in the (10-14) region of rat PTH(1-14) on the peptide's agonist activity. We found that substitution of histidine for the native asparagine at position 10 of PTH(1-14) provided a peptide that was approx. 8-fold more potent as an agonist in the presence of divalent zinc salts than it was in the absence of the metal. This enhancement in potency was dependent on the native histidine at position 14, the concentration of Zn(II) utilized, and did not occur with other divalent metal ions. The zinc-activated [His(10)]-PTH(1-14) peptide was blocked by a classical PTH-1 receptor antagonist, PTHrP(7-36), and did not activate the PTH-2 receptor. The zinc-mediated enhancing effect did not require the large N-terminal extracellular domain of the PTH-1 receptor. Although we were able to demonstrate that [His(10)]-PTH(1-14) binds Zn(II) using (1)H-NMR, our spectroscopic studies (circular dichroism and nuclear magnetic resonance) were not consistent with the notion that zinc enhanced the activity of [His(10)]-PTH(1-14) simply by inducing a helical structure in the 10-14 region. Rather, the data suggest that the enhancement in cAMP potency arises from the formation of a ternary complex between [His(10)]-PTH(1-14), a zinc atom, and the extracellular loop/transmembrane domain region of the PTH-1 receptor.     

The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1-34).

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182-190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1-34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.     

Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand

Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 --> Arg yielded hP1Rc-[Arg(11)]Tether(1-11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg(11)]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation.     

The (1-14) fragment of parathyroid hormone (PTH) activates intact and amino-terminally truncated PTH-1 receptors.

Recent mutagenesis and cross-linking studies suggest that residues in the carboxyl-terminal portion of PTH(1-34) interact with the amino-terminal extracellular domain of the receptor and thereby contribute strongly to binding energy; and that residues in the amino-terminal portion of the ligand interact with the receptor region containing the transmembrane helices and extracellular loops and thereby induce second messenger signaling. We investigated the latter component of this hypothesis using the short amino-terminal fragment PTH(1-14) and a truncated rat PTH-1 receptor (r delta Nt) that lacks most of the amino-terminal extracellular domain. The binding of PTH(1-14) to LLC-PK1 or COS-7 cells transfected with the intact PTH-1 receptor was too weak to detect; however, PTH(1-14) dose-dependently stimulated cAMP formation in these cells over the dose range of 1-100 microM. PTH(1-14) also stimulated cAMP formation in COS-7 cells transiently transfected with r delta Nt, and its potency with this receptor was nearly equal to that seen with the intact receptor. In contrast, PTH(1-34) was approximately 100-fold weaker in potency with r delta Nt than it was with the intact receptor. Alanine scanning of PTH(1-14) revealed that for both the intact and truncated receptors, the 1-9 segment of PTH forms a critical receptor activation domain. Taken together, these results demonstrate that the amino-terminal portion of PTH(1-34) interacts with the juxtamembrane regions of the PTH-1 receptor and that these interactions are sufficient for initiating signal transduction.     

Modifications of position 12 in parathyroid hormone and parathyroid hormone related protein: toward the design of highly potent antagonists

Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.     

Mutational analysis of the receptor-activating region of human parathyroid hormone.

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.     

Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially activated by mammalian and fugufish parathyroid hormone-related peptide.

To further explore the evolution of receptors for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), we searched for zebrafish (z) homologs of the PTH/PTHrP receptor (PTH1R). In mammalian genes encoding this receptor, exons M6/7 and M7 are highly conserved and separated by 81-84 intronic nucleotides. Genomic polymerase chain reaction using degenerate primers based on these exons led to two distinct DNA fragments comprising portions of genes encoding the zPTH1R and the novel zPTH3R. Sequence comparison of both full-length teleost receptors revealed 69% similarity (61% identity), but less homology with zPTH2R. When compared with hPTH1R, zPTH1R showed 76% and zPTH3R 67% amino acid sequence similarity; similarity with hPTH2R was only 59% for both teleost receptors. When expressed in COS-7 cells, zPTH1R bound [Tyr(34)]hPTH-(1-34)-amide (hPTH), [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), and [Ala(29),Glu(30), Ala(34),Glu(35), Tyr(36)]fugufish PTHrP-(1-36)-amide (fuguPTHrP) with a high apparent affinity (IC(50): 1.2-3.5 nM), and was efficiently activated by all three peptides (EC(50): 1.1-1.7 nM). In contrast, zPTH3R showed higher affinity for fuguPTHrP and hPTHrP (IC(50): 2.1-11.1 nM) than for hPTH (IC(50): 118.2-127.0 nM); cAMP accumulation was more efficiently stimulated by fugufish and human PTHrP (EC(50): 0.47 +/- 0.27 and 0.45 +/- 0.16, respectively) than by hPTH (EC(50): 9.95 +/- 1.5 nM). Agonist-stimulated total inositol phosphate accumulation was observed with zPTH1R, but not zPTH3R.     

Molecular determinants of tuberoinfundibular peptide of 39 residues (TIP39) selectivity for the parathyroid hormone-2 (PTH2) receptor. N-terminal truncation of TIP39 reverses PTH2 receptor/PTH1 receptor binding selectivity

Tuberoinfundibular peptide of 39 residues (TIP39) and the parathyroid hormone-2 (PTH2) receptor form part of an extended family of related signaling molecules that includes the PTH1 receptor, which responds to PTH and PTH-related protein. TIP39 does not appreciably activate the PTH1 receptor, but in this study it is shown to bind the receptor with moderate affinity (59 nm). In this study, we investigated the molecular determinants of both ligand and receptor for the PTH2 receptor selectivity of TIP39 and quantitatively evaluated the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. A chimeric receptor composed of the N-terminal extracellular domain of the PTH1 receptor and the remainder (juxtamembrane domain) of the PTH2 receptor (P2-NP1) was fully activated by TIP39 (E(max) = 98% of the rPTH-(1-34), E(max), EC(50) = 2.0 nm). This receptor chimera bound TIP39 with an equivalent affinity to the wild-type PTH2 receptor (2. 3 and 2.0 nm, respectively). The reciprocal chimeric receptor (P1-NP2) was not activated by TIP39 and bound the ligand with an affinity equivalent to that of the PTH1 receptor. Thus, the juxtamembrane receptor domain specifies the signaling and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. Removing six N-terminal residues of TIP39 eliminated activation of the PTH2 receptor and reduced binding affinity 70-fold. In contrast, this truncation increased affinity for the PTH1 receptor 10-fold, reversing the PTH2/PTH1 receptor binding selectivity and resulting in a high affinity interaction of TIP-(7-39) with the PTH1 receptor (6 nm). These findings can be explained by a strong interaction between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH2 receptor, with the corresponding domain of the PTH1 receptor acting as a selectivity barrier against high affinity binding of TIP39. As a result, TIP-(7-39) is a highly potent, selective antagonist for the PTH1 receptor.     

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.     

The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation

Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.