Calcif Tissue Int (1992) 51:382-386
Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
Solubilization of Functional Receptors for Parathyroid Hormone and Parathyroid Hormone-Related Peptide from Clonal Rat Osteosarcoma Cells, ROS17/2.8 Susumu Uneno, 1'2 Takao Yamamuro, 2 Harald Jiippner, ~ Abdul-Badi Abou-Samra, 1 Henry T. Keutmann, 1 John T. Potts, Jr, 1 and Gino V. Segre I ~Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 021 t4, USA; and ~Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Kyoto University, Kyoto 606, Japan Received March 9, 1992, and in revised form May 22, 1992
Summary. 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~ 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 digitoninsolubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(136) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4azidobenzoate (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 1 0 - 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 [Nle 8"~8, Tyr 34] 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. Key words: Parathyroid hormone - PTH-related peptide Receptors - Solubilization - ROS 17/2.8 cells.
Specific receptors in the plasma membrane of target cells in bone and kidney play a key role in mediating the physiological effects of parathyroid hormone (PTH) [1-6]. Photoaffinity radiolabeling techniques have shown that PTH receptors in ROS 17/2.8 cells appear as a broad band of molecular size 80,000 daltons (Mr = 80kD) upon autoradiography following
Offprint requests to: G. V. Segre
SDS-PAGE [7, 8]. PTH receptors are glycoproteins with asparagine-linked oligosaccharides, and these receptors can be purified by wheat-germ agglutinin (WGA) affinitychromatography quite efficiently [8, 9]. Others have reported the apparent molecular weight of the major component of the PTH ligand-receptor complex to range from 60,000 daltons to 85,000 daltons [9-14]. Although photoaffinity radiolabeling techniques have been quite useful for labeling PTH receptors in intact cells [7, 8] and in membrane [9, 10], PTH binding to detergent-solubilized receptors has proved difficult to demonstrate; Malbon and Zull [15] in 1977 reported binding of tritiated bovine PTH to Triton X-100solubilized preparations from bovine renal cortex, and more recently, Nissenson et al. [16] solubilized a guanine nucleotide-sensitive hormone-receptor complex, after initially binding the radioiodinated ligand to particulate canine renal cortical membranes. Parathyroid hormone-related peptide (PTHrP) (synonym: parathyroid hormone-like protein (PTHLP, PLP)), has recently been isolated from several different human tumors and cloned [17, 18]. The sequence of its first 13 aminoterminal residues has close homology with that of PTH, though the remainder of its sequence differs extensively from that of PTH. The amino-terminal fragment of PTHrP, PTHrP(1-36), binds to the same receptors as does [Nle8'1s, Tyr34] bovine PTH(1-34) amide (NlePTH) in ROS17/2.8 cells [19, 20], in canine renal membrane [21], and in COS-7 cells transiently expressing cDNA encoding the opossum renal PTH receptor [22]. Photoreactive derivatives of PTHrP(136) label the same M~ = 80 kD receptor band in ROS17/2.8 cells [19, 20]. Therefore, the endocrine effects of PTHrP mimic those of PTH, presumably by activating the same receptors, although PTHrP is thought to function primarily as a locally secreted paracrine or autocrine [23]. In the present study, we used relatively low concentrations of the detergent, digitonin, to solubilize PTH receptors because we reasoned that these mild conditions were more likely to yield receptors that retained their capacity to specifically bind ligands. Using these conditions, we observed specific binding of radioiodinated PTHrP(I-36) to digitoninsolubilized receptors from ROS17/2.8 cells. The apparent molecular weight of the ligand-receptor complex, crosslinked with N-hydroxysuccinimyl-4-azidobenzoate (HSAB), is about Mr = 90 kD or slightly higher than of the ligandreceptor complex in intact cells that we previously reported (Mr = 80 kD) [7, 8, 19, 20].
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP Materials and Methods
Cells and Reagents ROS17/2.8 cells were a generous gift from Dr. Gideon A. Rodan (Merck Sharp and Dohme Laboratories, West Point, PA). NRK49F cells were from the American Tissue Culture Collection. NlePTH was from Bachem (Torrance, CA). PTHrP(1-36) was synthesized in our laboratory or it was a generous gift from Dr. Andrew F. Stewart (Yale University School of Medicine, New Haven, CT). Sodium nsI-iodide (2200 Ci/mmol) was from DuPont-New England Nuclear (Boston, MA). Digitonin was from Gallard-Schlesinger (Carle Place, NY). Dimethyl-sulfoxide (DMSO), dithiothreitol (DTT), 4-fluoro-3nitrophenyl azide (FNPA), and HSAB were from Pierce (Rockwood, IL). Electrophoresis reagents were from Bio-Rad (Richmond, CA). WGA-agarose and molecular weight standards were from Pharmacia (Uppsala, Sweden). Human ACTH(1-39), bovine insulin, N-acetyl-D-glucosamine, and protease inhibitors were from Sigma (St. Louis, MO). Tissue culture media and sera were from Gibco (Grand Island, NY).
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hour at 4~ with continuous shaking, and then centrifuged (250,000 • g, 1 hour at 4~ The supernatant, which contained solubilized PTH receptors, was directly used for photoaffinity radiolabeling assays, or was used after it had been partially purified by WGA affinity-chromatography.
Purification of Solubilized PTH Receptors by WGA Affnity-Chromatography The supernatant from the previous step was diluted 2.5-fold with 5 mM Tris-HCl, (pH 7.3) 5 mM HEPES, 150 mM NaC1, (WGA buffer), and incubated with 2 ml of WGA-agarose in a column equilibrated with WGA buffer containing 0.1% digitonin (WGA-digitonin buffer) for 1 hour at 4~ with gentle shaking. The column was washed extensively with WGA-digitonin buffer, and then eluted with 0.5 M N-acetyl-D-glucosamine in WGA-digitonin buffer. The eluate (6 ml) was concentrated and the sugars removed using Centricon-30 microconcentrators (Amicon, Beverly, MA). The eluate was then diluted with 25 mM HEPES (pH 7.5), 125 mM NaC1, 5 mM KC1, and 2 mM CaC12 (binding buffer) containing 0.1% digitonin.
Cell Culture Photoaffinity Radiolabeling of Solubilized PTH Receptors All ceils were grown in a humidified 95% air, 5% C O 2 atmosphere. ROS17/2.8 cells, a clonal osteoblastic cell line derived from a rat osteosarcoma, were maintained in Ham's F-12 medium, supplemented with 5% fetal bovine serum. The medium was renewed every 2-3 days until the cells reached confluence. Because cell surface receptors for PTH continue to increase after the cells reach confluence, the cells were grown for an additional 5-7 days and fed daily before study. NRK49F cells, a clonal fibroblastic cell line derived from normal rat kidney, were maintained in Dulbecco's modified Eagle's medium, supplemented with nonessential amino acids and 5% calf serum. NRK49F cells do not show any specific binding of PTH.
PTH Radioreceptor Assays and Photoaffinity Labeling of PTH Receptors in Intact Cells 125I-NlePTH and 125I-PTHrP(1-36) were prepared by the Chloramine-T method as described [7]. Radioreceptor assays with intact cells were performed as described [24] with minor modifications. Briefly, cells grown in 24-well plates, were rinsed twice with cold phosphate-buffered saline (PBS), incubated With approximately 100,000 cpm of 125I-NIePTH or 125I-PTHrP(1-36) in 250 ~xlof a buffer consisting of 50 mM Tris-HC1 (pH 7.5), 100 mM NaCI, 5 mM KC1, 2 mM CaCI2, 0.5% fetal bovine serum and 5% horse serum at 15~ for 4 hours. The cells then were rinsed four times with the same buffer, lysed with 500 ~1 of 0.5 M NaOH, and counted for bound radioactivity. Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 10 6 M of NIePTH or PTHrP(I-36)) from total binding (binding in the absence of competing peptide). PTH receptors in intact ROS17/2.8 cells were radiolabeled with photoreactive FNPA-derivatives of ~zSI-NlePTH or ~25I-PTHrP(136), as described elsewhere [7, 20].
Solubilization of Particulate Membrane Fraction Confluent cells in 175 cm 2 flasks were rinsed twice with cold PBS, scraped into 4 ml of an ice-cold buffer consisting of 10 mM HEPES (pH 7.4), 20% glycerol, 5 mM EDTA, 2 mg/ml bacitracin, 80 ~g/ml chymostatin, 1 mM TPCK, 10 p.g/ml leupeptin, 1 ~g/ml pepstatin A, 0.5 TIU/ml aprotinin, 1 mM PMSF (homogenizat!on buffer), homogenized with a Potter-Elvehjem glass homogenizer on ice, and centrifuged (250,000 • g, 30 rain at 4~ The pellet, which contained the particulate membranes, was resuspended in 4 ml of the same buffer and centrifuged again. The pellet was then resuspended in 1 ml of ice-cold homogenization buffer containing 0.25% digitonin (solubilization buffer) using a Dounce homogenizer, incubated (1
Aliquots of solubilized receptors were incubated with lzsI-PTHrP(lr 36) (5 • 10 lo M) in 200 ~1 of binding buffer containing 0.1% digitonin, 2 mg/ml bacitracin, 80 ~g/ml chymostatin, 10 ~g/ml leupeptin, 1 p.g/ml pepstatin A, and 0.5 TIU/ml aprotinin (16 hours at 4~ in the presence or absence of competing peptide. Then, 4 ~1 of 25 mM HSAB, dissolved in DMSO, was added (final concentration of HSAB was 0.5 mM), vortexed, andincubated on ice for 10 minutes in the dark. The reaction was quenched by the addition of 8 p.l of 1 M Tris-HCl (pH 7.5). The samples were then transferred to wells of a 24-well tissue culture plate and photolyzed for 20 minutes on ice, using a Blak Ray long-wave lamp (366 nm, 7 mW/cm z, UV Products, San Gabriel, CA) at a source-sample distance of 20 cm.
SDS-PAGE and Autoradiography After photolysis, samples were concentrated using Centricon-30 microconcentrators to a final volume of approximately 30 txl. The samples then were mixed with 10 ixl of 250 mM Tris-HC1 (pH 6.8), 8% SDS, 40% glycerol, 200 mM DTT (fourfold higher concentration than routine sample buffer for SDS-PAGE), boiled for 10 minutes, and analyzed by SDS-PAGE (7,5%--15% acrylamide), according to the method of Laemmli [25], with subsequent autoradiography at -80~ using DuPont Cronex Lightning Plus intensifying screens and Kodak X-Omat AR films.
Results
Bacitracin Inhibits Degradation of PTH/PTHrP W h e n either 125I-NlePTH or 125I-PTHrP(1-36) was incubated with the particulate m e m b r a n e fraction from R O S 17/2.8 cells (2 hours at 15~ approximately 75% of either tracer was degraded, as assessed by precipitation with 10% trichloroacetic acid (Table 1). Bacitracin inhibited this degradation m u c h more effectively than did any of the other p r o t e a s e inhibitors tested. Interestingly, E D T A (5 raM) p r o t e c t e d 125I-NlePTH better than it p r o t e c t e d 125I-PTHrP(1-36), presumably by inhibiting one or m o r e metallo-peptidases. The integrity of the radioligand was also tested by initially incubating it w i t h the p a r t i c u l a t e m e m b r a n e f r a c t i o n f r o m ROS17/2.8 cells, either in the p r e s e n c e or absence of bacitracin (2 mg/ml), and then rebinding of the radioligand to intact ROS17/2.8 cells (Table 2). Binding of " f r e s h " radioligand and that of the tracer, after it had b e e n p r e i n c u b a t e d
384
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP
Tahle 1. Effects of protease inhibitors on the degradation of ~zsINtePTH and ~25I-PTHrP(1-36) Protease Inhibitors
1zSI-NIePTH (%)
125I-PTHrP(1-36) (%)
None (without None Bacitracin Chymostatin TPCK Leupeptin Pepstatin A Aprotinin PMSF EDTA
13.6 + 0.1 79.1 • 0.2 25.4 +-_0.2 41.2 + 0.6 70.0 • 0.1 72.3 • 0.4 71.9 • 0.5 72.5 • 1.6 70.6 • 1.0 32.6 • 0.0
6.3 • 0.1 75.3 +-- 0.4 17.1 • 0.4 57.1 • 2.1 74.7 • 0.2 77.3 • 0.2 73.0 • 0.6 74.7 • 0.2 71.1 • 0.1 65.4 • 0.1
membrane) 2 mg/ml 80 ixg/ml 1 mM 10 ixg/ml 1 p~g/ml 0.5 TIU/ml 1 mM 5 mM
The percentage of the degraded radioligand was estimated after precipitation of the intact radioligand by addition of trichloroacetic acid (TCA). Approximately 100,000 cpm of 125I-NIePTH or ~25IPTHrP(I-36) was incubated with aliquots of particulate membrane fractions prepared from ROS 17/2.8 cells in 250 p~lof 50 mM Tris-HC1 pH 7.5, 100 mM NaCI, 5 mM KC1, 2 mM CaCla (Tris-containing binding buffer) in the presence or absence of protease inhibitors (2 hours, at 15~ The membranes then were pelleted by centrifugation at 4,000 x g for 30 minutes, and the supernatant was separated. Ice-cold TCA was added to the supernatant (final TCA concentration was 10% and final volume 1 ml), then centrifuged at 4,000 x g for 15 minutes at 4~ The supernatant and the pellet were separated and the radioactivity in each fraction was determined. The table shows the percentage of TCA-soluble radioactivity, which was calculated by dividing the radioactivity in the supernatant by the summed radioactivity present in both the supernatant and the pellet. Data represent mean • SD of duplicate determinations. Representative data of five experiments are shown Table 2. Binding of ~25I-NlePTHafter preincubation of the radioligand with the particulate membrane fraction from ROS 17/2.8 cells in the presence or absence of bacitracin Binding Tracer
Total (%)
Nonspecific (%)
Specific (%)
Fresh tracer Preincubated, bacitracin ( - ) Preincubated, bacitracin (+)
13.1 • 0.6
2.6 • 0.2
10.5
1.3 ~ 0.4
1.1 +-- 0.6
0.2
12.7 ~ 1.5
1.4 --- 0.1
11.3
Radioreceptor assays were performed with ROS17/2.8 cells in a 24well plate and 100,000 cpm/well of 12SI-NlePTHthat had been preincubated with the particulate membrane fraction prepared from ROS17/2.8 cells in Tris-containing binding buffer (see legend for Table 1) (2 hours at, 15~ in the presence or absence of 2 mg/ml of bacitracin. The results are compared with binding of I25I-NlePTH that had not been preincubated with membranes ("fresh tracer"). Data represent mean --- SD of quadruplicate determinations. Representative data of three experiments are shown with the particulate membrane fraction in the presence of bacitracin, were indistinguishable, whereas there was virtually no binding when the tracer had been preincubated with the membranes in the absence of bacitracin. The effectiveness of bacitracin for inhibiting degradation of IzSI-NtePTH was dose dependent (data not shown). Bacitracin was used at a concentration of 2 mg/ml in all subsequent studies unless otherwise noted, because higher concentrations interfered with ligand binding (data not shown).
PTHrP(1-36) Specifically Labels a Soluble Protein from ROS17/2.8 Membranes
M r =
90 kD
The particulate membrane fraction from ROS17/2.8 cells was
Fig. 1. Lanes A to E: The particulate membrane fraction was prepared from ROS17/2.8 cells and solubilized with 0,25% digitoniu. Binding of ~zsI-PTHrP(1-36) to the solubilized fraction was performed in the absence (Lane A), or in the presence of PTHrP(1-36) (Lanes B to E, 4 x 10-9M, 4 x 10-8M, 4 x 10-TM, and4 x 10 - 6 M, respectively). Samples were cross-linked with HSAB and subjected to SDS-PAGE and subsequent autoradiography as described in Materials and Methods. Lanes F and G: PTH/PTHrP receptors in intact ROS 17/2.8 cells were cross-|inked using photoderivatized, radiolabeled PTHrP(1-36) as described in Materials and Methods. Binding was performed in the absence (Lane F) or in the presence (Lane G) of 1 x 10-6 M of NlePTH,
solubilized with 0.25% digitonin, and it then was incubated with 125I-PTHrP(1-36) at 4~ After 16 hours, HSAB (final 0.5 mM) was added, and the cross-linking protocol (see methods) was followed. The samples then were subjected to SDS-PAGE and autoradiography. As shown in Figure 1, a broad band was observed that had a molecular size of approximately M r = 90 kD (Lane A). This band was not evident when the incubation was performed in the presence of excess unlabeled PTHrP(1-36) (4 • 10 -6 M, Lane E). This band was most intense, however, in the presence of 4 x 10 -9 M of unlabeled PTHrP(1-36) (Lane B), and progressively less intense in the presence of higher concentrations of the unlabeled hormone (lanes C-E). The apparent molecular weight of this band (Mr = 90 kD) was slightly higher than that of the PTH ligand-receptor complex in intact ROS 17/2.8 cells previously reported from our laboratory (M r = 80 kD, Lane F).
Binding of the Radioligand to Receptors after Partial Purification by WGA Affinity-Chromatography Partial purification of the solubilized receptors by WGA affinity-chromatography greatly reduced nonspecific binding. When these partially purified receptors were incubated with JzsI-PTHrP(1-36) and then cross-linked with HSAB, a broad band was observed with an apparent molecular size of M r 90 kD, in the absence of competing peptide (Fig. 2, Lane A). Again, it was most intense in the presence of a low concentration of unlabeled PTHrP(1-36) Lanes B-C, 10- lo M_10-9 M) or NlePTH (Lane G, I • 10 -9 M). Increasing concentrations of PTHrP(1-36) progressively reduced the intensity of this band (Lanes D-E, 10 -8 M-10 - 7 M), and 1 • 10 -6 M of the peptide caused its complete disappearance (Lane F). NlePTH (1 • 10 -6 M) also markedly reduced the density of this radiolabeled band (Lane H), although it remained detectable. Neither human insulin nor ACTH(1-39) (both at 1 • 10-5 M) reduced the intensity of labeling of the M r = 90 kD band (Lanes I and J); in fact, the intensity of this band was
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP
Fig. 2. The particulate membrane fraction was prepared from ROS17/2.8 cells solubilized with 0.25% digitonin, and then partially purified by WGA affinity-chromatography, as described in Materials and Methods. Aliquots of the eluate were photoaffinity radiolabeled using 12sI-PTHrP(1-36) and HSAB. Binding of t~sI-PTHrP(I-36) was performed in the absence of any competing peptide (Lane A), or in the presence of PTHrP(1-36) (Lanes B to F, 1 x 10-1o M, 1 x 10 -9 M, 1 x 10 -8 M, 1 x 10 _7 M , and 1 x 10 6 M, respectively) or NlePTH (Lanes G and H, 1 x 10 -9 M and 1 x 10 -6 M) or human ACTH(1-39) (Lane I, 1 x 10 -6 M) or bovine insulin (Lane J, 1 x 10 -6 M). Samples were subjected to SDS-PAGE and subsequent autoradiography.
increased, compared with that observed in t h e absence of any competing ligand (Lane A). The observation that the M r = 90 kD band is most intense when the binding is conducted in the presence of insulin, ACTH(1-39), or low concentrations of either PTHrP(1-36) or NIePTH suggests that these peptides protect either the radioligand or the solubilized receptors from degradation. This protection, in turn, allows more of the radioligand to bind to the receptors. As a control, solubilized membranes were prepared from NRK49F, that did not bind PTH, and they were subjected to purification on WGA-agarose columns. Subsequent binding studies, which were performed with 125I-PTHrP(I-36) and both the "flow-through" fractions and the fractions eluted with N-acetyl-D-glucosamine, failed to show any specific binding of the radioligand (data not shown).
Discussion
We have demonstated specific binding of 12sI-PTHrP(1-36) to PTH/PTHrP receptors that have been solubilized from ROS17/2.8 cells with 0.25% digitonin, in the presence of protease inhibitors. The apparent molecular size of the ligandreceptor complex is approximately Mr = 90 kD, and is slightly higher than the apparent receptor size (M r = 80 kD) our laboratory has previously reported using photoaffinitylabeling techniques in intact ROS17/2.8 cells [7, 8, 19, 20]. It seems likely that this discrepancy relates to differences in methodologies used in these studies. Previously, we used radioiodinated ligands of amino-terminal fragments of either PTH or PTHrP which contained photoreactive groups only on one of the lysine residues within the sequence of the radioligands. The receptors on intact ROS17/2.8 cells were then cross-linked, prior to solubilization with SDS. The conditions used to solubilize the receptors with digitonin are far gentler than those previously used to solubilize the ligandreceptor complex from intact cells. Solubilization with digitonin may have resulted in the solubilization of the receptors with a portion of the adjacent membrane. Additionally, cross-linking by photolysis of the photoderivatized, radioiodinated ligand to receptors on intact cells probably occurs at a single receptor site in close proximity to the single F N P A derivatized lysine residue of the radioligand. Cross-linking
385
with HSAB, however, is far less specific. The reagent initially couples to many available lysine residues; thus, the larger apparent size of the receptor described in this paper is likely to have resulted from cross-linking of the ligand to multiple sites within the receptors. We were unable to reproducibly bind FNPA-derivatized, radioiodinated ligands to solubilized receptors. There are several possible explanations. As we previously reported, these photoderivatized, radioiodinated ligands have a lower affinity for the receptor in intact cells than do their underivatized parent molecule [20, 26, 27]. Additionally, highaffinity binding requires association of the ligand-occupied receptor with stimulatory guanine-nucleotide binding proteins (G-proteins) [10, 16]. Solubitization of the receptors may disturb these interactions at one or more steps. F o r example, the integrity of the receptor itself, once removed from the membrane, may be sufficiently altered to lower its capacity to bind the ligand with high affinity. Solubilization is also likely to disrupt the association of the receptors with G-proteins. Our data do not allow us to distinguish between these alternatives. However, the failure o f N l e P T H and PTHrP(1-36) to bind equivalently to the solubilized receptors (Figs. 1,2), as they do with intact cells [19, 20], suggests that subtle modifications of the r e c e p t o r ' s binding domain(s) have taken place during solubilization. Our initial efforts to solubilize functionally active PTH/ PTHrP receptors has been successful. In these initial experiments, we intentionally used conditions calculated to solubilize only a portion of the available receptors with the expectation that the receptors would be more likely to retain their capacity to bind the ligand. Isolation and purification of functional soluble receptors in high yield will enable detailed characterization of their biochemical properties.
References
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ated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS17/2.8 cells. J Biol Chem 263:8557-8560 Jtippner H, Abou-Samra AB, Uneno S, Keutmann HT, Potts JT Jr, Segre GV (1990) Preparation and characterization of [N'~-(4azido-2-nitrophenyl)Ala~, Tyr36]-parathyroid hormone-related peptide (1-36) amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS17/2.8 cells. Biochemistry 29:6941-6946 Orloff JJ, Wu TL, Heath HW, Brady TG, Brines ML, Stewart AF (1989) Characterization of canine renal receptors for parathyroid hormone-like protein associated with humoral hypercalemia of malignancy. J Biol Chem 264:6097-6103 Jtippner H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF Jr, Hock J, Potts JT Jr, Kronenberg HM, Segre GV (1991) A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024-1026 Martin TJ, Ebeling PR (1990) A novel parathyroid hormonerelated protein: role in pathology and physiology. In: Peterlik M, Bronner F (eds) Molecular and cellular regulation of calcium and phosphate metabolism. John Wiley & Sons, New York, pp 1-37 Yamamoto I, Shigeno C, Potts JT Jr, Segre GV (1988) Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. Endocrinology 122:1208-1217 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Shigeno C, Hiraki Y, Keutmann HT, Stern AM, Potts JT Jr, Segre GV (1989) Preparation of a photoreactive analog of parathyroid hormone [Nle 8, Lys(N-~-4-azido-2-nitrophenyl)~3, Nle TM, Tyr 34] bovine parathyroid hormone (1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors. Anal Biochem 179:268-273 Jtippner H, Abou-Samra AB, Uneno S, Schipani E, Keutmann HT, Potts JT Jr, Segre GV (1990) Properties of amino-terminal parathyroid hormone-related pepties modified at positions 1113. Peptides 11:1139-1142
Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
Solubilization of Functional Receptors for Parathyroid Hormone and Parathyroid Hormone-Related Peptide from Clonal Rat Osteosarcoma Cells, ROS17/2.8 Susumu Uneno, 1'2 Takao Yamamuro, 2 Harald Jiippner, ~ Abdul-Badi Abou-Samra, 1 Henry T. Keutmann, 1 John T. Potts, Jr, 1 and Gino V. Segre I ~Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 021 t4, USA; and ~Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Kyoto University, Kyoto 606, Japan Received March 9, 1992, and in revised form May 22, 1992
Summary. 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~ 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 digitoninsolubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(136) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4azidobenzoate (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 1 0 - 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 [Nle 8"~8, Tyr 34] 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. Key words: Parathyroid hormone - PTH-related peptide Receptors - Solubilization - ROS 17/2.8 cells.
Specific receptors in the plasma membrane of target cells in bone and kidney play a key role in mediating the physiological effects of parathyroid hormone (PTH) [1-6]. Photoaffinity radiolabeling techniques have shown that PTH receptors in ROS 17/2.8 cells appear as a broad band of molecular size 80,000 daltons (Mr = 80kD) upon autoradiography following
Offprint requests to: G. V. Segre
SDS-PAGE [7, 8]. PTH receptors are glycoproteins with asparagine-linked oligosaccharides, and these receptors can be purified by wheat-germ agglutinin (WGA) affinitychromatography quite efficiently [8, 9]. Others have reported the apparent molecular weight of the major component of the PTH ligand-receptor complex to range from 60,000 daltons to 85,000 daltons [9-14]. Although photoaffinity radiolabeling techniques have been quite useful for labeling PTH receptors in intact cells [7, 8] and in membrane [9, 10], PTH binding to detergent-solubilized receptors has proved difficult to demonstrate; Malbon and Zull [15] in 1977 reported binding of tritiated bovine PTH to Triton X-100solubilized preparations from bovine renal cortex, and more recently, Nissenson et al. [16] solubilized a guanine nucleotide-sensitive hormone-receptor complex, after initially binding the radioiodinated ligand to particulate canine renal cortical membranes. Parathyroid hormone-related peptide (PTHrP) (synonym: parathyroid hormone-like protein (PTHLP, PLP)), has recently been isolated from several different human tumors and cloned [17, 18]. The sequence of its first 13 aminoterminal residues has close homology with that of PTH, though the remainder of its sequence differs extensively from that of PTH. The amino-terminal fragment of PTHrP, PTHrP(1-36), binds to the same receptors as does [Nle8'1s, Tyr34] bovine PTH(1-34) amide (NlePTH) in ROS17/2.8 cells [19, 20], in canine renal membrane [21], and in COS-7 cells transiently expressing cDNA encoding the opossum renal PTH receptor [22]. Photoreactive derivatives of PTHrP(136) label the same M~ = 80 kD receptor band in ROS17/2.8 cells [19, 20]. Therefore, the endocrine effects of PTHrP mimic those of PTH, presumably by activating the same receptors, although PTHrP is thought to function primarily as a locally secreted paracrine or autocrine [23]. In the present study, we used relatively low concentrations of the detergent, digitonin, to solubilize PTH receptors because we reasoned that these mild conditions were more likely to yield receptors that retained their capacity to specifically bind ligands. Using these conditions, we observed specific binding of radioiodinated PTHrP(I-36) to digitoninsolubilized receptors from ROS17/2.8 cells. The apparent molecular weight of the ligand-receptor complex, crosslinked with N-hydroxysuccinimyl-4-azidobenzoate (HSAB), is about Mr = 90 kD or slightly higher than of the ligandreceptor complex in intact cells that we previously reported (Mr = 80 kD) [7, 8, 19, 20].
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP Materials and Methods
Cells and Reagents ROS17/2.8 cells were a generous gift from Dr. Gideon A. Rodan (Merck Sharp and Dohme Laboratories, West Point, PA). NRK49F cells were from the American Tissue Culture Collection. NlePTH was from Bachem (Torrance, CA). PTHrP(1-36) was synthesized in our laboratory or it was a generous gift from Dr. Andrew F. Stewart (Yale University School of Medicine, New Haven, CT). Sodium nsI-iodide (2200 Ci/mmol) was from DuPont-New England Nuclear (Boston, MA). Digitonin was from Gallard-Schlesinger (Carle Place, NY). Dimethyl-sulfoxide (DMSO), dithiothreitol (DTT), 4-fluoro-3nitrophenyl azide (FNPA), and HSAB were from Pierce (Rockwood, IL). Electrophoresis reagents were from Bio-Rad (Richmond, CA). WGA-agarose and molecular weight standards were from Pharmacia (Uppsala, Sweden). Human ACTH(1-39), bovine insulin, N-acetyl-D-glucosamine, and protease inhibitors were from Sigma (St. Louis, MO). Tissue culture media and sera were from Gibco (Grand Island, NY).
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hour at 4~ with continuous shaking, and then centrifuged (250,000 • g, 1 hour at 4~ The supernatant, which contained solubilized PTH receptors, was directly used for photoaffinity radiolabeling assays, or was used after it had been partially purified by WGA affinity-chromatography.
Purification of Solubilized PTH Receptors by WGA Affnity-Chromatography The supernatant from the previous step was diluted 2.5-fold with 5 mM Tris-HCl, (pH 7.3) 5 mM HEPES, 150 mM NaC1, (WGA buffer), and incubated with 2 ml of WGA-agarose in a column equilibrated with WGA buffer containing 0.1% digitonin (WGA-digitonin buffer) for 1 hour at 4~ with gentle shaking. The column was washed extensively with WGA-digitonin buffer, and then eluted with 0.5 M N-acetyl-D-glucosamine in WGA-digitonin buffer. The eluate (6 ml) was concentrated and the sugars removed using Centricon-30 microconcentrators (Amicon, Beverly, MA). The eluate was then diluted with 25 mM HEPES (pH 7.5), 125 mM NaC1, 5 mM KC1, and 2 mM CaC12 (binding buffer) containing 0.1% digitonin.
Cell Culture Photoaffinity Radiolabeling of Solubilized PTH Receptors All ceils were grown in a humidified 95% air, 5% C O 2 atmosphere. ROS17/2.8 cells, a clonal osteoblastic cell line derived from a rat osteosarcoma, were maintained in Ham's F-12 medium, supplemented with 5% fetal bovine serum. The medium was renewed every 2-3 days until the cells reached confluence. Because cell surface receptors for PTH continue to increase after the cells reach confluence, the cells were grown for an additional 5-7 days and fed daily before study. NRK49F cells, a clonal fibroblastic cell line derived from normal rat kidney, were maintained in Dulbecco's modified Eagle's medium, supplemented with nonessential amino acids and 5% calf serum. NRK49F cells do not show any specific binding of PTH.
PTH Radioreceptor Assays and Photoaffinity Labeling of PTH Receptors in Intact Cells 125I-NlePTH and 125I-PTHrP(1-36) were prepared by the Chloramine-T method as described [7]. Radioreceptor assays with intact cells were performed as described [24] with minor modifications. Briefly, cells grown in 24-well plates, were rinsed twice with cold phosphate-buffered saline (PBS), incubated With approximately 100,000 cpm of 125I-NIePTH or 125I-PTHrP(1-36) in 250 ~xlof a buffer consisting of 50 mM Tris-HC1 (pH 7.5), 100 mM NaCI, 5 mM KC1, 2 mM CaCI2, 0.5% fetal bovine serum and 5% horse serum at 15~ for 4 hours. The cells then were rinsed four times with the same buffer, lysed with 500 ~1 of 0.5 M NaOH, and counted for bound radioactivity. Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 10 6 M of NIePTH or PTHrP(I-36)) from total binding (binding in the absence of competing peptide). PTH receptors in intact ROS17/2.8 cells were radiolabeled with photoreactive FNPA-derivatives of ~zSI-NlePTH or ~25I-PTHrP(136), as described elsewhere [7, 20].
Solubilization of Particulate Membrane Fraction Confluent cells in 175 cm 2 flasks were rinsed twice with cold PBS, scraped into 4 ml of an ice-cold buffer consisting of 10 mM HEPES (pH 7.4), 20% glycerol, 5 mM EDTA, 2 mg/ml bacitracin, 80 ~g/ml chymostatin, 1 mM TPCK, 10 p.g/ml leupeptin, 1 ~g/ml pepstatin A, 0.5 TIU/ml aprotinin, 1 mM PMSF (homogenizat!on buffer), homogenized with a Potter-Elvehjem glass homogenizer on ice, and centrifuged (250,000 • g, 30 rain at 4~ The pellet, which contained the particulate membranes, was resuspended in 4 ml of the same buffer and centrifuged again. The pellet was then resuspended in 1 ml of ice-cold homogenization buffer containing 0.25% digitonin (solubilization buffer) using a Dounce homogenizer, incubated (1
Aliquots of solubilized receptors were incubated with lzsI-PTHrP(lr 36) (5 • 10 lo M) in 200 ~1 of binding buffer containing 0.1% digitonin, 2 mg/ml bacitracin, 80 ~g/ml chymostatin, 10 ~g/ml leupeptin, 1 p.g/ml pepstatin A, and 0.5 TIU/ml aprotinin (16 hours at 4~ in the presence or absence of competing peptide. Then, 4 ~1 of 25 mM HSAB, dissolved in DMSO, was added (final concentration of HSAB was 0.5 mM), vortexed, andincubated on ice for 10 minutes in the dark. The reaction was quenched by the addition of 8 p.l of 1 M Tris-HCl (pH 7.5). The samples were then transferred to wells of a 24-well tissue culture plate and photolyzed for 20 minutes on ice, using a Blak Ray long-wave lamp (366 nm, 7 mW/cm z, UV Products, San Gabriel, CA) at a source-sample distance of 20 cm.
SDS-PAGE and Autoradiography After photolysis, samples were concentrated using Centricon-30 microconcentrators to a final volume of approximately 30 txl. The samples then were mixed with 10 ixl of 250 mM Tris-HC1 (pH 6.8), 8% SDS, 40% glycerol, 200 mM DTT (fourfold higher concentration than routine sample buffer for SDS-PAGE), boiled for 10 minutes, and analyzed by SDS-PAGE (7,5%--15% acrylamide), according to the method of Laemmli [25], with subsequent autoradiography at -80~ using DuPont Cronex Lightning Plus intensifying screens and Kodak X-Omat AR films.
Results
Bacitracin Inhibits Degradation of PTH/PTHrP W h e n either 125I-NlePTH or 125I-PTHrP(1-36) was incubated with the particulate m e m b r a n e fraction from R O S 17/2.8 cells (2 hours at 15~ approximately 75% of either tracer was degraded, as assessed by precipitation with 10% trichloroacetic acid (Table 1). Bacitracin inhibited this degradation m u c h more effectively than did any of the other p r o t e a s e inhibitors tested. Interestingly, E D T A (5 raM) p r o t e c t e d 125I-NlePTH better than it p r o t e c t e d 125I-PTHrP(1-36), presumably by inhibiting one or m o r e metallo-peptidases. The integrity of the radioligand was also tested by initially incubating it w i t h the p a r t i c u l a t e m e m b r a n e f r a c t i o n f r o m ROS17/2.8 cells, either in the p r e s e n c e or absence of bacitracin (2 mg/ml), and then rebinding of the radioligand to intact ROS17/2.8 cells (Table 2). Binding of " f r e s h " radioligand and that of the tracer, after it had b e e n p r e i n c u b a t e d
384
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP
Tahle 1. Effects of protease inhibitors on the degradation of ~zsINtePTH and ~25I-PTHrP(1-36) Protease Inhibitors
1zSI-NIePTH (%)
125I-PTHrP(1-36) (%)
None (without None Bacitracin Chymostatin TPCK Leupeptin Pepstatin A Aprotinin PMSF EDTA
13.6 + 0.1 79.1 • 0.2 25.4 +-_0.2 41.2 + 0.6 70.0 • 0.1 72.3 • 0.4 71.9 • 0.5 72.5 • 1.6 70.6 • 1.0 32.6 • 0.0
6.3 • 0.1 75.3 +-- 0.4 17.1 • 0.4 57.1 • 2.1 74.7 • 0.2 77.3 • 0.2 73.0 • 0.6 74.7 • 0.2 71.1 • 0.1 65.4 • 0.1
membrane) 2 mg/ml 80 ixg/ml 1 mM 10 ixg/ml 1 p~g/ml 0.5 TIU/ml 1 mM 5 mM
The percentage of the degraded radioligand was estimated after precipitation of the intact radioligand by addition of trichloroacetic acid (TCA). Approximately 100,000 cpm of 125I-NIePTH or ~25IPTHrP(I-36) was incubated with aliquots of particulate membrane fractions prepared from ROS 17/2.8 cells in 250 p~lof 50 mM Tris-HC1 pH 7.5, 100 mM NaCI, 5 mM KC1, 2 mM CaCla (Tris-containing binding buffer) in the presence or absence of protease inhibitors (2 hours, at 15~ The membranes then were pelleted by centrifugation at 4,000 x g for 30 minutes, and the supernatant was separated. Ice-cold TCA was added to the supernatant (final TCA concentration was 10% and final volume 1 ml), then centrifuged at 4,000 x g for 15 minutes at 4~ The supernatant and the pellet were separated and the radioactivity in each fraction was determined. The table shows the percentage of TCA-soluble radioactivity, which was calculated by dividing the radioactivity in the supernatant by the summed radioactivity present in both the supernatant and the pellet. Data represent mean • SD of duplicate determinations. Representative data of five experiments are shown Table 2. Binding of ~25I-NlePTHafter preincubation of the radioligand with the particulate membrane fraction from ROS 17/2.8 cells in the presence or absence of bacitracin Binding Tracer
Total (%)
Nonspecific (%)
Specific (%)
Fresh tracer Preincubated, bacitracin ( - ) Preincubated, bacitracin (+)
13.1 • 0.6
2.6 • 0.2
10.5
1.3 ~ 0.4
1.1 +-- 0.6
0.2
12.7 ~ 1.5
1.4 --- 0.1
11.3
Radioreceptor assays were performed with ROS17/2.8 cells in a 24well plate and 100,000 cpm/well of 12SI-NlePTHthat had been preincubated with the particulate membrane fraction prepared from ROS17/2.8 cells in Tris-containing binding buffer (see legend for Table 1) (2 hours at, 15~ in the presence or absence of 2 mg/ml of bacitracin. The results are compared with binding of I25I-NlePTH that had not been preincubated with membranes ("fresh tracer"). Data represent mean --- SD of quadruplicate determinations. Representative data of three experiments are shown with the particulate membrane fraction in the presence of bacitracin, were indistinguishable, whereas there was virtually no binding when the tracer had been preincubated with the membranes in the absence of bacitracin. The effectiveness of bacitracin for inhibiting degradation of IzSI-NtePTH was dose dependent (data not shown). Bacitracin was used at a concentration of 2 mg/ml in all subsequent studies unless otherwise noted, because higher concentrations interfered with ligand binding (data not shown).
PTHrP(1-36) Specifically Labels a Soluble Protein from ROS17/2.8 Membranes
M r =
90 kD
The particulate membrane fraction from ROS17/2.8 cells was
Fig. 1. Lanes A to E: The particulate membrane fraction was prepared from ROS17/2.8 cells and solubilized with 0,25% digitoniu. Binding of ~zsI-PTHrP(1-36) to the solubilized fraction was performed in the absence (Lane A), or in the presence of PTHrP(1-36) (Lanes B to E, 4 x 10-9M, 4 x 10-8M, 4 x 10-TM, and4 x 10 - 6 M, respectively). Samples were cross-linked with HSAB and subjected to SDS-PAGE and subsequent autoradiography as described in Materials and Methods. Lanes F and G: PTH/PTHrP receptors in intact ROS 17/2.8 cells were cross-|inked using photoderivatized, radiolabeled PTHrP(1-36) as described in Materials and Methods. Binding was performed in the absence (Lane F) or in the presence (Lane G) of 1 x 10-6 M of NlePTH,
solubilized with 0.25% digitonin, and it then was incubated with 125I-PTHrP(1-36) at 4~ After 16 hours, HSAB (final 0.5 mM) was added, and the cross-linking protocol (see methods) was followed. The samples then were subjected to SDS-PAGE and autoradiography. As shown in Figure 1, a broad band was observed that had a molecular size of approximately M r = 90 kD (Lane A). This band was not evident when the incubation was performed in the presence of excess unlabeled PTHrP(1-36) (4 • 10 -6 M, Lane E). This band was most intense, however, in the presence of 4 x 10 -9 M of unlabeled PTHrP(1-36) (Lane B), and progressively less intense in the presence of higher concentrations of the unlabeled hormone (lanes C-E). The apparent molecular weight of this band (Mr = 90 kD) was slightly higher than that of the PTH ligand-receptor complex in intact ROS 17/2.8 cells previously reported from our laboratory (M r = 80 kD, Lane F).
Binding of the Radioligand to Receptors after Partial Purification by WGA Affinity-Chromatography Partial purification of the solubilized receptors by WGA affinity-chromatography greatly reduced nonspecific binding. When these partially purified receptors were incubated with JzsI-PTHrP(1-36) and then cross-linked with HSAB, a broad band was observed with an apparent molecular size of M r 90 kD, in the absence of competing peptide (Fig. 2, Lane A). Again, it was most intense in the presence of a low concentration of unlabeled PTHrP(1-36) Lanes B-C, 10- lo M_10-9 M) or NlePTH (Lane G, I • 10 -9 M). Increasing concentrations of PTHrP(1-36) progressively reduced the intensity of this band (Lanes D-E, 10 -8 M-10 - 7 M), and 1 • 10 -6 M of the peptide caused its complete disappearance (Lane F). NlePTH (1 • 10 -6 M) also markedly reduced the density of this radiolabeled band (Lane H), although it remained detectable. Neither human insulin nor ACTH(1-39) (both at 1 • 10-5 M) reduced the intensity of labeling of the M r = 90 kD band (Lanes I and J); in fact, the intensity of this band was
S. Uneno et al.: Solubilization of Functional Receptors for PTH and PTHrP
Fig. 2. The particulate membrane fraction was prepared from ROS17/2.8 cells solubilized with 0.25% digitonin, and then partially purified by WGA affinity-chromatography, as described in Materials and Methods. Aliquots of the eluate were photoaffinity radiolabeled using 12sI-PTHrP(1-36) and HSAB. Binding of t~sI-PTHrP(I-36) was performed in the absence of any competing peptide (Lane A), or in the presence of PTHrP(1-36) (Lanes B to F, 1 x 10-1o M, 1 x 10 -9 M, 1 x 10 -8 M, 1 x 10 _7 M , and 1 x 10 6 M, respectively) or NlePTH (Lanes G and H, 1 x 10 -9 M and 1 x 10 -6 M) or human ACTH(1-39) (Lane I, 1 x 10 -6 M) or bovine insulin (Lane J, 1 x 10 -6 M). Samples were subjected to SDS-PAGE and subsequent autoradiography.
increased, compared with that observed in t h e absence of any competing ligand (Lane A). The observation that the M r = 90 kD band is most intense when the binding is conducted in the presence of insulin, ACTH(1-39), or low concentrations of either PTHrP(1-36) or NIePTH suggests that these peptides protect either the radioligand or the solubilized receptors from degradation. This protection, in turn, allows more of the radioligand to bind to the receptors. As a control, solubilized membranes were prepared from NRK49F, that did not bind PTH, and they were subjected to purification on WGA-agarose columns. Subsequent binding studies, which were performed with 125I-PTHrP(I-36) and both the "flow-through" fractions and the fractions eluted with N-acetyl-D-glucosamine, failed to show any specific binding of the radioligand (data not shown).
Discussion
We have demonstated specific binding of 12sI-PTHrP(1-36) to PTH/PTHrP receptors that have been solubilized from ROS17/2.8 cells with 0.25% digitonin, in the presence of protease inhibitors. The apparent molecular size of the ligandreceptor complex is approximately Mr = 90 kD, and is slightly higher than the apparent receptor size (M r = 80 kD) our laboratory has previously reported using photoaffinitylabeling techniques in intact ROS17/2.8 cells [7, 8, 19, 20]. It seems likely that this discrepancy relates to differences in methodologies used in these studies. Previously, we used radioiodinated ligands of amino-terminal fragments of either PTH or PTHrP which contained photoreactive groups only on one of the lysine residues within the sequence of the radioligands. The receptors on intact ROS17/2.8 cells were then cross-linked, prior to solubilization with SDS. The conditions used to solubilize the receptors with digitonin are far gentler than those previously used to solubilize the ligandreceptor complex from intact cells. Solubilization with digitonin may have resulted in the solubilization of the receptors with a portion of the adjacent membrane. Additionally, cross-linking by photolysis of the photoderivatized, radioiodinated ligand to receptors on intact cells probably occurs at a single receptor site in close proximity to the single F N P A derivatized lysine residue of the radioligand. Cross-linking
385
with HSAB, however, is far less specific. The reagent initially couples to many available lysine residues; thus, the larger apparent size of the receptor described in this paper is likely to have resulted from cross-linking of the ligand to multiple sites within the receptors. We were unable to reproducibly bind FNPA-derivatized, radioiodinated ligands to solubilized receptors. There are several possible explanations. As we previously reported, these photoderivatized, radioiodinated ligands have a lower affinity for the receptor in intact cells than do their underivatized parent molecule [20, 26, 27]. Additionally, highaffinity binding requires association of the ligand-occupied receptor with stimulatory guanine-nucleotide binding proteins (G-proteins) [10, 16]. Solubitization of the receptors may disturb these interactions at one or more steps. F o r example, the integrity of the receptor itself, once removed from the membrane, may be sufficiently altered to lower its capacity to bind the ligand with high affinity. Solubilization is also likely to disrupt the association of the receptors with G-proteins. Our data do not allow us to distinguish between these alternatives. However, the failure o f N l e P T H and PTHrP(1-36) to bind equivalently to the solubilized receptors (Figs. 1,2), as they do with intact cells [19, 20], suggests that subtle modifications of the r e c e p t o r ' s binding domain(s) have taken place during solubilization. Our initial efforts to solubilize functionally active PTH/ PTHrP receptors has been successful. In these initial experiments, we intentionally used conditions calculated to solubilize only a portion of the available receptors with the expectation that the receptors would be more likely to retain their capacity to bind the ligand. Isolation and purification of functional soluble receptors in high yield will enable detailed characterization of their biochemical properties.
References
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