Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are calciotropic hormones interacting with a shared seven-transmembrane domain G protein-coupled receptor, which is located predominantly in bone and kidney. To map the interface of the bimolecular interaction between hormone and receptor, we designed and radioiodinated a bioactive, photoreactive PTH agonist, (125)I-[Nle(8,18),Lys13(epsilon-p-(3-I-Bz)Bz),L-2-Nal(23),Arg(26,2 7),Tyr34] bPTH-(1-34)NH2 ((125)I-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys13. All other lysyl residues are substituted by argynyls. The analog photocrosslinks specifically to the recombinant hPTH/PTHrP receptor stably transfected into human embryonic kidney cells (HEK-293/C-21 cells, approximately 400,000 receptors per cell), generating a diffuse approximately 87-kDa band on SDS/PAGE autoradiography. To identify the "contact domain" within the hPTH/PTHrP receptor involved in binding of (125)I-all-R-K13, the radiolabeled band containing the ligand-receptor conjugate was subjected to chemical and enzymatic cleavage. Two independent pathways of sequential digestion were used: Route A, lysyl endopeptidase C, then endo-N-glycosidase F, followed by cyanogen bromide; Route B, cyanogen bromide followed by endo-N-glycosydase F. The identified domain is in contact with position 13 in (125)I-all-R-K13 and corresponds to amino acids 173-189 of the hPTH/PTHrP receptor, located at the C-terminal region of the N-terminal extracellular domain.

The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.

We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.

Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.

Activity of two synthetic amphiphilic peptides and magainin-2 against herpes simplex virus types 1 and 2.

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin-1 (mod-1) and modelin-5 (mod-5), and of the natural antibacterial peptide magainin-2 (mag-2) against herpes simplex viruses type 1 (HSV-1) and 2 (HSV-2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod-1 displayed a strong antiviral effect against HSV-1 and HSV-2, with 50% effective dose (ED50) values of 4.6 and 4.1 micrograms/mL, respectively. Mag-2, mod-5 and a mixture of both had no significant inhibitory effect. Addition of mod-1 up to a concentration of 100 micrograms/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue-exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod-1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod-1 may be an effective topical antiviral agent against herpes viruses.

Refolding of the isolated extracellular domain of the erythropoietin receptor.

Refolding of the soluble recombinant binding-active extracellular domain of the murine erythropoietin receptor (sEPO-R) was achieved with greater than 90% recovery either from urea/NaCI (1.5/0.5 M) or guanidine HCI (1 M). An expression plasmid encoding the extracellular plasma region of the human EPO-R (sEPO-R) was transfected into COS cells, and the sEPO-R so produced was labelled with [35S]methionine and purified by EPO-affinity chromatography on a EAH-Sepharose 4B-EPO column. Rapid rates of refolding were required for recovery of the native proteins. Refolding was evaluated by rebinding of the sEPO-R to the EPO affinity column.

Structure-function studies of amphiphilic antibacterial peptides.

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Augmentation of the antibacterial activity of magainin by positive-charge chain extension.

Novel analogs of the broad-spectrum antimicrobial peptide magainin-2 were obtained by extension of its chain through addition of segments of positively charged amino acids to either its N or its C terminus and by increasing its helicity. The activity of magainin-2 toward American Type Culture Collection strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was most considerably enhanced by these modifications, whereas, in general, its low hemolytic capacity was not or was only slightly affected. The antibacterial potencies of magainin-2 and its derivatives were more evident following decreases of pH from 7.2 to 6 and 5.

All-D-magainin: chirality, antimicrobial activity and proteolytic resistance.

All-D-magainin-2 was synthesized to corroborate experimentally the notion that the biological function of a surface-active peptide stems primarily from its unique amphiphilic alpha-helical structure. Indeed, the peptide exhibited antibacterial potency nearly identical to that of the all-L-enantiomer. Being highly resistant to proteolysis and non-hemolytic all-D-magainin might have considerable therapeutic importance.