RESUMO
ROS17/2.8 cells, a cell line derived from a rat osteosarcoma, have abundant receptors for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). A particulate membrane fraction was prepared from these cells and it was solubilized using relatively mild conditions with digitonin (0.25%), a nonionic detergent. When radioligands of both PTH and PTHrP were incubated with this membrane fraction in the absence of any protease inhibitor at 15 degrees C, approximately 75% of these radioligands were degraded within 2 hours. This degradative activity was inhibited more effectively by bacitracin than by any of several other protease inhibitors tested. The digitonin-solubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(1-36) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), as cross-linker. When an aliquot of the reaction solution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, a broad band was observed that had an apparent molecular size of 90,000 daltons (M(r) = 90 kD). This band was no longer seen when the binding was conducted in the presence of 10(-6) M of unlabeled PTHrP(1-36), and it was decreased in density when binding was conducted in the presence of 10(-6) M of unlabeled [Nle8,18, Tyr34] bovine PTH(1-34) amide (NlePTH). The solubilized receptors retained their capacity to bind the radioligand after partial purification by wheat-germ agglutinin affinity-chromatography. The use of relatively mild detergent conditions thus offers a means to solubilize receptors that retain their capacity to bind PTH and PTHrP.
Assuntos
Proteínas de Neoplasias/metabolismo , Osteossarcoma/química , Hormônio Paratireóideo/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/química , Animais , Autorradiografia , Bacitracina/farmacologia , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Proteína Relacionada ao Hormônio Paratireóideo , Inibidores de Proteases/farmacologia , Ensaio Radioligante , Ratos , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Receptores de Hormônios Paratireóideos , Solubilidade , Células Tumorais CultivadasRESUMO
Biological properties of amino-terminal PTHrP analogues modified in the region 11-13 were examined using ROS 17/2.8 cells. [Leu11,D-Trp12,Arg13,Tyr36]PTHrP(1-36)amide had a 17-fold lower binding affinity for the receptor (apparent Kd: 5 x 10(-8) M) than [Tyr36]PTHrP(1-36)amide or [Arg11,13,Tyr36]PTHrP(1-36)amide (apparent Kd for both: 2 x 10(-9) M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu11,D-Trp12,Arg13,Tyr36,Cys38]PTHrP(7-3 8) and PTHrP(7-34)amide had similar receptor affinities (apparent Kds: 5 x 10(-8) M and 8 x 10(-8) M), while that of [Nle8,18,Tyr34]bPTH(7-34)amide was more than 10-fold lower (apparent Kd: 2 x 10(-6) M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1-36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly12.
Assuntos
Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , AMP Cíclico/biossíntese , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Ensaio Radioligante , Receptores de Superfície Celular/metabolismo , Receptores de Hormônios Paratireóideos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo , Hormônio Paratireóideo/síntese química , Fragmentos de Peptídeos/síntese química , Proteínas/síntese química , Marcadores de Afinidade/síntese química , Linhagem Celular , Reagentes de Ligações Cruzadas/síntese química , Cinética , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas/metabolismo , Proteínas/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores de Hormônios ParatireóideosRESUMO
[Nle8,18,Tyr34]bPTH-(1-34)amide (NlePTH) was biotinylated using sulfosuccinimidyl 6-(biotinamido)hexanoate, in dimethyl sulfoxide, and the multiple resulting peptides peaks were separated by reverse-phase high performance liquid chromatography. Their biological activities were compared with those of NlePTH, the parent compound, in radioreceptor and cAMP accumulation bioassays using rat osteosarcoma 17/2.8 cells; the earliest two eluting products, bioPTH 1 and 2, were equipotent, a third, bioPTH 3, was only 10% as potent, and the remaining, later eluting derivatives all were less than 0.1% as active. Competitive avidin binding assays using [3H]biotin suggested that bioPTH 1 and 2 had a single biotin congener per molecule, while bioPTH 3 contained two biotin residues. Upon Edman degradation, bioPTH 1 contained biotin on the lysine at position 13 of NlePTH; bioPTH 2's biotin was on the lysine at position 26 (or 27) and bioPTH 3 had biotins on lysines at both positions 13 and 26 (or 27). Avidin tagged with 125I, peroxidase, or fluorescein isothiocyanate was detected on bone-derived cells which had been incubated initially with bioPTH 2 (1, 10, and 100 nM) for 4 h, but not when NlePTH (1 microM) was added with bioPTH 2. A fluorescence-activated cell sorter detected a symmetrical shift in fluorescence of bone-derived cells incubated with 10 nM of bioPTH 2 and 10 micrograms/ml fluorescein isothiocyanate-avidin. Addition of a 30-fold molar excess of NlePTH, or omission of bioPTH 2, completely reversed this fluorescence shift, and no shift in fluorescence was seen with cells lacking PTH receptors. This fully active, high affinity biotinylated PTH-derivative should prove useful in the study of PTH receptor-bearing cells.
Assuntos
Biotina , Citometria de Fluxo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Superfície Celular/metabolismo , Animais , Avidina/metabolismo , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Lisina/análise , Osteossarcoma/metabolismo , Hormônio Paratireóideo/isolamento & purificação , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ensaio Radioligante , Ratos , Receptores de Hormônios Paratireóideos , Teriparatida/análogos & derivados , Células Tumorais CultivadasRESUMO
We and others have recently shown that amino terminal sequences of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), which share a 62% homology within the first 13 residues, bind to the same receptor on ROS 17/2.8 cells. The remaining PTHrP sequence is markedly different from PTH, suggesting that receptor binding may be dependent on the first 13 amino acids of either peptide. However, since the amino acid residues 14-34 have previously been recognized as an important binding domain for PTH, conformational similarity within this portion's secondary structure of both peptides could contribute to their capacity to bind to the same receptor. To test this hypothesis, we synthesized [Tyr36,Cys38]PTHrP-(14-38) and [Tyr34]bPTH(14-34)NH2, and studied binding of both peptides to the common PTH/PTHrP receptor on ROS 17/2.8 cells. Radioiodinated, HPLC-purified [Nle8,18, Tyr34]bPTH(1-34)NH2 (NlePTH) and [Tyr36]PTHrP-(1-36)NH2 were used to functionally define receptor binding requirements. [Tyr36,Cys38]PTHrP(14-38) and [Tyr34]bPTH(14-34)NH2 competed with 125I-NlePTH for binding sites on ROS 17/2.8 cells with apparent Kds of 10 microM and 50 microM respectively. Both peptides also competed with 125I-[Tyr36]PTHrP(1-36)NH2 with apparent Kds of 30 microM and 10 microM respectively. In the same assay system, NlePTH and [Tyr36,Cys38]PTHrP(1-38)inhibited binding of either radioiodinated ligand with apparent Kds of 0.3 and 1.0 nM. These studies indicate that although [Tyr34]bPTH(14-34)NH2 and [Tyr36,Cys38]PTHrP(14-38) share virtually no sequence homology, their secondary structures must be sufficiently similar to permit binding to a common PTH/PTHrP receptor.
Assuntos
Sequência de Bases , Proteínas de Neoplasias/genética , Hormônio Paratireóideo/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Homologia de Sequência do Ácido Nucleico , Ligação Competitiva , Linhagem Celular , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/genética , Ensaio Radioligante , Receptores de Hormônios ParatireóideosRESUMO
The effects of PTH on the binding and mitogenic activity of epidermal growth factor (EGF) were studied in clonal, PTH- and EGF-responsive mouse osteoblastic cell line, MC3T3-E1. Treatment of cells with synthetic rat PTH-(1-34) at 10(-10)-10(-8) M resulted in a dose-related decrease (maximally 30% relative to the control value) in the specific binding of a biologically active, 125I-iodolabeled derivative of recombinant human EGF ([125I]iodo-EGF). The PTH-induced decrease in EGF binding was time dependent, requiring at least 4 h of PTH treatment at 37 C for a maximal effect, completely reversible after cessation of PTH exposure and specific only to biologically active PTH. Scatchard analysis of saturation binding data revealed that the PTH-induced reduction of EGF binding was accounted for by a proportional decrease in the available EGF-binding sites without an alteration in binding affinity (Kd = 0.7-0.8 nM). PTH treatment resulted ina concomitant decrease in mitotic responsiveness to EGF (maximally 40-50% of control). Both of these down-regulatory effects of PTH were closely mimicked by forskolin, cholera toxin, or (Bu)2cAMP. Addition of either colchicine or cytochalasin-B during PTH treatment completely abolished the PTH-induced reduction of EGF binding. These data indicate that PTH down-regulates EGF receptors and reduces the mitotic responsiveness to EGF, probably via a cAMP-dependent cytoskeleton-mediated mechanism(s) in osteoblastic MC3T3-E1 cells.
Assuntos
AMP Cíclico/fisiologia , Receptores ErbB/metabolismo , Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Crânio/citologia , Animais , Células Clonais , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Camundongos , Mitose/efeitos dos fármacosRESUMO
The effect of transforming growth factor beta (TGF-beta) on cellular proliferation of osteoblastic MC3T3-E1 cells was studied with particular emphasis on its effect on modulation of epidermal growth factor (EGF) receptors. In other cells, TGF-beta has been reported to augment EGF receptors. Exposure of MC3T3-E1 cells to TGF-beta initially increased cell surface EGF receptor levels and decreased the rate of DNA synthesis. The initial elevation of EGF receptor levels was due to increased receptor number per cell, not to changes in binding affinity. On the contrary, prolonged exposure (longer than 40 h) resulted in a decrease in EGF receptor and an increase in the rate of DNA synthesis. Thus, the effects of TGF-beta on these cells appears to be biphasic, reflecting complex mechanisms of action; the early effects of TGF-beta may be consistent with cellular differentiation to the osteoblastic phenotype with decreased cellular proliferation, whereas chronic exposure of these cells to TGF-beta stimulated cellular proliferation and inhibited osteoblastic phenotype expression. It is not likely that stimulation of cellular proliferation was through elevation of EGF receptor levels, because TGF-beta did not enhance the stimulatory effect of EGF on cellular proliferation. Thus, we conclude that TGF-beta possesses a stimulatory effect on the cellular proliferation of osteoblastic MC3T3-E1 cells independent of its modulative effect on EGF receptor level.
Assuntos
Receptores ErbB/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Fatores de Crescimento Transformadores/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais/efeitos dos fármacos , DNA/biossíntese , Camundongos , Osteoblastos/citologiaRESUMO
[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides.