Alessandro Bisello

Parathyroid Hormone-Receptor Interactions Identified Directly by Photocross-linking and Molecular Modeling Studies

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc) was carried out by photoaffinity scanning studies. Photoreactive analogs of PTH singularly substituted with a p-benzoylphenylalanine (Bpa) at each of the first six N-terminal positions have been prepared. Among these, the analog [Bpa1,Nle8,18,Arg13,26,27,l-2-Nal23,Tyr34]bPTH-(1–34)NH2(Bpa1-PTH-(1–34)) displayed in vitroactivity with potency similar to that of PTH-(1–34). The radioiodinated analog 125I-Bpa1-PTH-(1–34) cross-linked specifically to the hPTH1-Rc stably expressed in human embryonic kidney cells. A series of chemical and enzymatic digestions of the hPTH1-Rc–125I-Bpa1-PTH-(1–34) conjugate suggested that a methionine residue (either Met414 or Met425) within the contact domain hPTH1-Rc-(409–437), which includes the transmembrane helix 6 and part of the third extracellular loop, as the putative contact point. Site-directed mutagenesis (M414L or M425L) identified Met425 as the putative contact point. Molecular modeling of the hPTH1-Rc together with the NMR-derived high resolution structure of hPTH-(1–34), guided by the cross-linking data, strongly supports Met425, at the extracellular end of transmembrane helix 6, as the residue interacting with the N-terminal residue of the hPTH-(1–34). The photocross-linking and molecular modeling studies provide insight into the topologic arrangement of the receptor-ligand complex.

Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves beta-arrestin2. Real-time monitoring by fluorescence microscopy.

Endocytosis and intracellular trafficking of the human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1–34) agonist bound to the hPTH1-Rc internalized rapidly at 37u2009°C via clathrin-coated vesicles, whereas fluorescent PTH-(7–34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was immunolocalized to the cell membrane and, to a lesser extent, the cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitorN-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent protein-β-arrestin2 fusion protein (β-Arr2-GFP), PTH agonists stimulated β-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1–34)-hPTH1Rc complexes and β-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves β-arrestin mobilization and targeting to clathrin-coated vesicles. Our results also indicate that receptor occupancy, rather than receptor-mediated signaling, is necessary, although not sufficient, for endocytosis of the hPTH1-Rc. Activation of PKC, however, is absolutely required.

Photoaffinity Cross-linking Identifies Differences in the Interactions of an Agonist and an Antagonist with the Parathyroid Hormone/Parathyroid Hormone-related Protein Receptor

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactivel-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa1- and Bpa2-PTHrP, respectively) displayed high affinity binding. Bpa1-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa2-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1–6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH3 group of Met425 in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa2-PTHrP also cross-links to a proximal site within the receptor domain Pro415–Met425. Unlike the antagonist Bpa2-PTHrP, the potent agonist Bpa2-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH3 group of Met425 (similar to Bpa1-PTHrP and Bpa1-PTH). Taken together, these results suggest that the antagonist Bpa2-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast‐derived N‐cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N‐cadherin expression was localized prominently in well‐differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS‐2 and rat ROS osteoblast‐like cells showed a relationship between N‐cadherin messenger RNA expression and cell‐to‐cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His‐Ala‐Val (HAV) adhesion motif of N‐cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell‐to‐cell contact in rat osteoblastic TRAB‐11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS‐2 cells. Transient transfection of SaOS‐2 cells with a dominant‐negative N‐cadherin mutant (NCADΔC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast‐derived N‐cadherin was inhibited by either treatment with HAV or transfection with NCADΔC. Together, these results strongly support a role for N‐cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation. (J Bone Miner Res 2000;15:198–208)

Direct Identification of Two Contact Sites for Parathyroid Hormone (PTH) in the Novel PTH-2 Receptor using Photoaffinity Cross-Linking

Direct examination of the interacting sites between PTH and the human PTH2 receptor (PTH2R) was conducted by photoaffinity cross-linking followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. Photoreactive analogs of PTH, individually substituted with an L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in cells stably expressing recombinant PTH2R. One highly bioactive analog, [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]PTH-(1-34)NH 2 (Bpa1-PTH), was chosen for cross-linking studies. In addition, a PTH analog in which the photoreacive moiety is at the mid-region position 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2R. The minimal digestion-restricted domain containing the contact site (contact domain) for 125I-Bpa1-PTH is in the sixth transmembrane domain and part of the third extracellular loop, spanning residues Ser364-Met395 of the receptor. This domain was further confirmed and refined by cross-linking 125I-Bpa1-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M,M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen bromide identified a single amino acid (position 380) as the putative contact point. The contact domain for 125I-K13 is located in the N-terminal extracellular tail of the receptor (in the C-terminal portion) and spans Gln138-Met147. Further validation of this contact domain was accomplished by photocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met425 and Phe173-Met189 as the contact sites for Bpa1-PTH and K13, respectively. These studies demonstrate that both receptor subtypes, PTH1- and PTH2-receptors, use analogous sites for interaction with positions 1 and 13 in PTH.

Conformational studies of a bicyclic, lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–Asp17, Lys26–Asp30)]PTHrP(1–34)NH2, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly12 and Arg19. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright

Structure-Function Relationship Studies on Parathyroid Hormone (PTH) 1–34 Analogs Containing β-Amino Acid Residues in Positions 11, 12, and 13

Parathyroid hormone (PTH) is an 84 amino acid residue peptide, which plays a key physiological role in the regulation of calcium levels in serum. Virtually, all bone-relevant activities of PTH are encoded in the fully active N-terminal 1–34 sequence [1]. On the basis of our previous results on a series of active and inactive analogs of the PTH(1–34) [2], we suggested that the structural elements essential for biological activity are an N-terminal and a C-terminal helical segments connected by hinges or flexible points around positions 12 and 19. To probe this hypothesis, in the present work we synthesized by solid phase methods, and characterized the following bPTH(1–34) analogs containing β-amino acid residues at positions 11, 12 and 13.

Local Conformation Around Position 12 of the (1–34) Fragment of Parathyroid Hormone Probed by Substitution with Aib Residues

Parathyroid hormone (PTH) controls the homeostasis of calcium ions in the blood. Its N-terminal 1–34 fragment is sufficient to reproduce all the physiological functions elicited by the intact hormone. The biologically relevant conformation of PTH(l-34) is thought to include two critical points of flexibility around residue 12 and residue 19 [1]. These two hinges are believed to connect two α-helical regions at either end of the molecule. In order to assess the relationship between biological activity and local conformation around Gly12, we prepared and studied several PTH(1–34) analogs containing Aib residues: [Aib11], [Aib12], [Aib11,12] or [Aib12,13].