Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses via the juxtamembrane region of the PTH-1 receptor.

The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21)NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.

Enhanced activity in parathyroid hormone-(1-14) and -(1-11): novel peptides for probing ligand-receptor interactions.

The amino-terminal portion of PTH is critical for PTH-1 receptor (P1Rc) activation. In exploring this component of the ligand receptor interaction, we recently showed that the agonist potency of the weakly active PTH-(1-14)NH(2) peptide can be enhanced by natural amino acid substitutions at several positions, including position 11 (normally leucine). Here we show that the potency of PTH-(1-14)NH(2) can be enhanced by using nonnatural amino acids that increase the length and polarizability of the position 11 side-chain. Thus, in LLC-PK(1) cells stably expressing high levels of the human P1Rc, [homoarginine([Har)(11)]PTH-(1-14)NH(2) was 30-fold more potent for cAMP production than was native PTH-(1-14)NH(2). Combining the homoarginine-11 substitution with other recently identified activity-enhancing substitutions yielded [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH-(1-14)NH(2), which was 1500-fold more potent than PTH-(1-14)NH(2) (EC(50) = 0.12 +/- 0.04 and 190 +/- 20 microM, respectively) and only 63-fold less potent than PTH-(1-34) (EC(50) = 1.9 +/- 0.5 nM). The even shorter analog [Ala(3),Gln(10),Har(11)]PTH-(1-11)NH(2) was also a full cAMP agonist (EC(50) = 3.1 +/- 1.5 microM). Receptor mutations at Phe(184) and Leu(187) located near the boundary of the amino-terminal domain and transmembrane domain-1 severely impaired responsiveness to the PTH-(1-11) analog. Overall, these studies demonstrate that PTH analogs of only 11 amino acids are sufficient for activation of the PTH-1 receptor through interaction with its juxtamembrane region.

Molecular properties of the PTH/PTHrP receptor.

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) is a G protein-coupled receptor (GPCR) that plays a key role in controlling blood Ca(2+) concentration and endochondral bone formation. This review focuses on the molecular mechanisms by which the receptor recognizes the PTH and PTHrP peptide ligands and transmits their signal across the cell membrane. The available data suggest that there are two principal components to the ligand-receptor interaction. First, a docking interaction between the C-terminal portion of PTH(1-34) and the N-terminal extracellular domain of the receptor; and second, a weaker interaction between the N-terminal portion of the ligand and the juxtamembrane region of the receptor, which induces signal transduction. A full understanding of these processes could lead to new PTH/PTHrP receptor ligands that are effective in controlling diseases of bone and mineral metabolism, such as osteoporosis.

Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1--36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking.

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1--34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1--36) and the P1R, we prepared analogs of [I(5),W(23),Y(36)]PTHrP-(1--36)-amide with individual p-benzoyl-l-phenylalanine (Bpa) substitutions at positions 22--35. When tested with LLC-PK(1) cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC(50) of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa(24)-substituted analog, similar to that of the parent compound. The radiolabeled Bpa(23)-, Bpa(27)-, Bpa(28)-, and Bpa(33)-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I(5),Bpa(23),Y(36)]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I(5),W(23),Bpa(27),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 96--102; [I(5),W(23),Bpa(28),Y(36)]PTHrP-(1--36)- amide cross-linked to residues 64--95; and [I(5),W(23), Bpa(33),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

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.

Selective and nonselective inverse agonists for constitutively active type-1 parathyroid hormone receptors: evidence for altered receptor conformations.

The spontaneous signaling activity of some G protein-coupled receptors and the capacity of certain ligands (inverse agonists) to inhibit such constitutive activity are poorly understood phenomena. We investigated these processes for several analogs of PTH-related peptide (PTHrP) and the constitutively active human PTH/PTHrP receptors (hP1Rcs) hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist PTHrP(5-36) functioned as a weak partial/neutral agonist with both mutant receptors but was converted to an inverse agonist for both receptors by the combined substitution of Leu(11) and D-Trp(12). The N-terminally intact analog [Bpa(2)]PTHrP(1-36)-a partial agonist with the wild-type hP1Rc-was a selective inverse agonist, in that it depressed basal cAMP signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The ability of [Bpa(2)]PTHrP(1-36) to discriminate between the two receptor mutants suggested that H223R and T410P confer constitutive receptor activity by inducing distinct conformational changes. This hypothesis was confirmed by the observations that: 1) the double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels that were 2-fold higher than those of either single mutant; and 2) hP1Rc-H223R and hP1Rc-T410P internalized (125)I-PTHrP(5-36) to markedly different extents. The overall results thus reveal that two different types of inverse agonists are possible for PTHrP ligands (nonselective and selective) and that constitutively active PTH-1 receptors can access different conformational states.

Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist.

The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.

Evaluating the signal transduction mechanism of the parathyroid hormone 1 receptor. Effect of receptor-G-protein interaction on the ligand binding mechanism and receptor conformation.

Ligand binding to the PTH1 receptor is described by a "two-site" model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3-34) and PTH(1-14): PTH(1-14), which binds predominantly to the J domain, only partially inhibited binding of 125I-PTH(3-34); and PTH(3-34), shown to bind predominantly to the N domain, only partially inhibited PTH(1-14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of 125I-PTH(3-34) with 30 microM guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1-36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and PTH(1-14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3-34) binding was GTPgammaS-insensitive. Finally, several observations suggest the receptor conformation is more "closed" at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1-14) and PTH(3-34). At RG PTH(1-14) better occluded binding of 125I-PTH(3-34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states.

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.

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 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.

Receptors for PTH and PTHrP: their biological importance and functional properties.

The type 1 receptor (PTH1R) for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) is a G protein-coupled receptor that is highly expressed in bone and kidney and mediates in these tissues the PTH-dependent regulation of mineral ion homeostasis. The PTH1R also mediates the paracrine actions of PTHrP, which play a particularly vital role in the process of endochondral bone formation. These important functions, the likely involvement of the PTH1R in certain genetic diseases affecting skeletal development and calcium homeostasis, and the potential utility of PTH in treating osteoporosis have been the driving force behind intense investigations of both the receptor and its peptide ligands. Recent lines of work have led to the identification of constitutively active PTH1Rs in patients with Jansen's metaphyseal chondrodysplasia, the demonstration of inverse agonism by certain ligand analogs, and the discovery of the PTH-2 receptor subtype that responds to PTH but not PTHrP. As reviewed herein, a detailed exploration of the receptor-ligand interaction process is currently being pursued through the use of site-directed mutagenesis and photoaffinity cross-linking methods; ultimately, such work could enable the development of novel PTH receptor ligands that have therapeutic value in treating diseases such as osteoporosis and certain forms of hypercalcemia.

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.

Studies of the N-terminal region of a parathyroid hormone-related peptide (1-36) analog: receptor subtype-selective agonists, antagonists, and photochemical cross-linking agents.

The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I5,W23,Y36]-PTHrP(1-36)NH2 analogs having stepwise deletions of residues 1-4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK1 cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I5,W23]-PTHrP(5-36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I5,W23,Y36]-PTHrP(1-36)NH2 that were singly modified with parabenzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa2]- and [Bpa4]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1-5 region of [I5,W23]-PTHrP(1-36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.

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.

Evidence for a ligand interaction site at the amino-terminus of the parathyroid hormone (PTH)/PTH-related protein receptor from cross-linking and mutational studies.

Low resolution mutational studies have indicated that the amino-terminal extracellular domain of the rat parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or PTHrP-(1-36). To further define ligand-receptor interactions, we prepared a fully functional photoreactive analog of PTHrP, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide ([Bpa23]PTHrP, where Bpa is p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad approximately 80-kDa complex yielded a radiolabeled approximately 9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23-63, Tyr23 being the presumed amino-terminus of the receptor. This assignment was confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of approximately 46 kDa, which was reduced to a sharp band of approximately 20 kDa upon deglycosylation. CNBr digestion of complexes formed with two additional rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated protein bands that were approximately 6 kDa in size, indicating that [Bpa23]PTHrP cross-links to amino acids 23-40 of the rP1R. This segment overlaps a receptor region previously identified by deletion mapping to be important for ligand binding. Alanine scanning of this region revealed two residues, Thr33 and Gln37, as being functionally involved in ligand binding. Thus, the convergence of photoaffinity cross-linking and mutational data demonstrates that the extreme amino-terminus of the rP1R participates in ligand binding.

Residues in the membrane-spanning and extracellular loop regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide.

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.