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.     

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.     

Role of amino acid side chains in region 17-31 of parathyroid hormone (PTH) in binding to the PTH receptor

The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.     

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.     

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.     

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.     

Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.     

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

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.     

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.     

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.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.     

Autoregulation by the juxtamembrane region of the human ephrin receptor tyrosine kinase A3 (EphA3)

Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamembrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and leu(261) (of the first extracellular loop) of the human PTH1 receptor

In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel "contact site" between hPTH1-Rc and Lys(27) of PTH(1-34). Lys(27) is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys(27). The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, (125)I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between (125)I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L(261) or R(262) of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the (125)I-K27-[L261M]hPTH1-Rc conjugate and free (125)I-K27 and (ii) the failure to cross-link (125)I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L(261) is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys(27) (in PTH) to L(261) (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH-PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.     

Tritiated photoactivatable analogs of the native human thrombin receptor (PAR-1) agonist peptide, SFLLRN-NH2.

Six photoactivatable analogs of the human thrombin receptor activating peptide (TRAP), SFLLRN-NH2, were synthesized by substituting the photoactive amino acid, p-benzoylphenylalanine (Bpa), into each position of the peptide sequence. Platelet aggregation assays indicated that the peptides with Bpa substitutions at positions 3 to 6 retained agonist activity. These peptides were prepared in tritiated form as potential thrombin receptor photoaffinity labels. The [3H]Bpa-containing analogs were constructed by resynthesizing the peptides with the amino acid, 4-benzoyl-2',5'-dibromophenylalanine (Br2Bpa), and subjecting the purified peptides to Pd-catalyzed tritiodebromination. The radiochemical yields for the reductive tritiation were < 2% for peptides with [3H]Bpa in the third and fourth positions, and between 7 and 16% for the peptides with substitutions at the fifth and sixth positions. The low yields were due to over-reduction of the Bpa carbonyl group and nonspecific degradation during reductive tritiation. This report describes the first use of Br2Bpa for the preparation of tritiated photoactivatable peptides.     

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.