THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, No. 30, Issue of October 25, pp. 14795-14800,1987 Printed in U. S. A .

Characterization of Soluble and Particulate Parathyroid Hormone Receptors Using a Biotinylated Bioactive Hormone Analog*

(Received for publication, March 20, 1987)

Denise P. Brennan and Michael A. Levine From the Division of Endocrinology and Metabolism, Department of Medicine, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [NleS,Nle'8,Tyr34]bovine parathyroid hormone-( 1-34) (bPTH-(1-34)) amide was prepared by reacting the peptide with N-biotinyl-e-aminocaproic acid N-hy- droxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with de- tergent ((3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate) extracts of renal cortical mem- branes, and two membrane components were identi- fied. Labeling of these components was competitively inhibited by underivatized bPTH-( 1-34) or bPTH-(3- 34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-( 1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by in- cubating the membrane with biotinyl-bPTH-( 1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68- kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, por- cine, and canine renal membranes analyzed by two- dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin- agarose chromatography of solubilized membrane ex- tracts that had first been incubated with biotinyl- BPTH-( 1-34). Biotinyl-bPTH-( 1-34) should be useful as a tool for further characterization and purification of the PTH receptor.

The biological actions of PTH' on bone and kidney are mediated by binding of the hormone to specific receptors in the plasma membranes of its target cells (1). There is evidence that the hormone-receptor interaction leads to activation of

* This work was supported in part by Grant DK 34281 from the National Institutes of Health and by a grant from the Willen Drug Co. Portions of this work were presented at the 8th Annual Meeting of the American Society for Bone and Mineral Research, Anaheim, CA, June 21-24, 1986 (J. Bone Min. Res. (1986) 1, Suppl. 1, Abstr. 320). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' The abbreviations used are: PTH, parathyroid hormone; bio- tinyl-bPTH-(l-34), biotinyl-c-aminocaproyl-[Nle8,Nle'8,Tyr34]bovine parathyroid hormone-(l-34) amide; CHAPS, 3-[(3-~holamidopro- pyl)dimethylammonio]-1-propanesulfonate; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid DSS, disuccinimidyl suberate; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis; rPTH-(1-34), rat parathyroid hormone-(1- -34); bPTH-(3-34), [Nles,Nle'8,Tyr34]bovine parathyroid hormone- (3-34) amide. GTPrS, guanosine 5'-0-(thiotriphosphate); ACTH, adrenocorticotropin; PBS, phosphate-buffered saline.

adenylate cyclase and generation of CAMP, which ultimately produces many, although perhaps not all, of the biologic effects of PTH. PTH-binding components have been identi- fied in particulate membrane preparations by several groups using biologically active, photolabile PTH radioligands (2-4). These studies have demonstrated specific labeling of a highly conserved protein of apparent molecular mass 60-70 kDa.

Full investigation of the interactions of PTH with its re- ceptor and subsequent signal transmission will not be possible until the active receptor can be isolated from cell membranes and purified to homogeneity. Attempts to purify the PTH receptor have been hampered by difficulties in solubilizing the receptor in active form. Also, there has been no rapid and sensitive technique to identify the soluble receptor during various stages of purification.

We have undertaken the development of an affinity puri- fication technique for functional soluble PTH receptors. As a first step, we have developed a biotinylated derivative of bPTH-(1-34) to detect soluble recepcor. Because biotin has a high affinity (Kd = M) for egg white avidin and streptav- idin (5), biotinylated ligands provide a means for sensitive detection of receptor proteins. Biotinylated peptides have been used as specific probes for labeling and localizing several membrane receptors, including those for adrenocorticotropin (6), insulin (7), corticotropin-releasing hormone (8), gonado- tropin-releasing hormone (9), and calmodulin (10). Moreover, since functional insulin receptors can be purified with a biotinylated insulin derivative and avidin-Sepharose (11,12), interaction of avidin with biotin-labeled proteins should be an effective approach to isolating specific cellular compo- nents. We have synthesized a bioactive biotinylated bPTH- (1-34) analog and used it to characterize further the physi- cochemical properties of particulate and soluble PTH recep- tors.

EXPERIMENTAL PROCEDURES

Materials Synthetic bPTH-(1-34)-, bPTH-(3-34)-, and rPTH-(l-34)-pep-

tides were purchased from Bachem Corp. (Torrance, CA.) Oxidized rPTH-(1-34) was prepared by the peroxide method of Tashjian et al. (13). Human adrenocorticotropin, human insulin, CHAPS detergent, and radioimmunoassay-grade bovine serum albumin were obtained from Sigma. Biotinyl-c-aminocaproic acid N-hydroxysuccinimide es- ter was purchased from Behring Diagnostics. Reagents and dyes for colorimetric detection of biotin and biotin-labeled peptides (Bethesda Research Laboratories DNA Detection System) and streptavidin- agarose were obtained from Bethesda Research Laboratories. Nitro- cellulose (BA85) and a 96-well manifold were obtained from Schleicher & Schuell. Cartridges of ODS-silica (Sep-Pak C18) were purchased from Waters Associates (Milford, MA). DSS was pur- chased from Pierce Chemical Co. ["PIATP was obtained from Du Pont-New England Nuclear. GTP+ was obtained from Boehringer Mannheim. All other chemicals were the best commercial grade available.

14795

14796 PTH Receptor Studies with Biotinyl-bPTH-(l-34)

Methods PTH Bioassay-The biological activity of biotinylated derivatives

of bPTH-(1-34) was determined by activation of renal membrane adenylate cyclase (14). Porcine renal membranes (50-70 pg of protein) were incubated in a 100-pl final volume containing 50 mM HEPES (pH 7.6), 10 mM MgCI,, 30 mM KC], 1 mM DTT, 20 p~ CAMP, 0.1 mM GTPyS, an ATP-regenerating system (1.8 mg/ml creatine phos- phate and 0.2 mg/ml creatine phosphokinase), and 0.5 mM [ O I - ~ ~ P ] ATP (0.1 Ci/mmol). Other modulators were added as noted in the figure legends. After incubation for 20 min at 30 "C, [32P]cAMP was isolated and measured by the method of Salomon et al. (15).

Preparation of Purified Plasma Membranes-Renal cortical and hepatic plasma membranes were isolated by modification of previ- ously described methods (14,16). Minced tissue was dispersed at 4 "C in 3 volumes of Buffer A (10 mM Tris-HC1 (pH 7.5), 0.25 M sucrose, 1 mM EDTA) and homogenized with 10 strokes of a motor-driven smooth Teflon pestle. The homogenate was centrifuged at 2,200 X g for 15 min. The upper layer of the resulting "double-layered" pellet was resuspended in 1 volume of Buffer A with two strokes of the motor-driven Teflon pestle and layered onto discontinuous gradients of sucrose (39 and 37% (w/v)) prepared in 10 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA. After centrifugation for 90 min at 75,000 X g, each band and the pellet were collected, diluted with 5 volumes of 10 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA, and recentrifuged at 7,800 X g for 15 min to remove the sucrose. Each pellet was washed again and finally resuspended in a minimal volume of 50 mM Tris- HCI (pH 7.5) and stored at -70 "C until used. Material collected from the 37% sucrose interface was the source of purified membranes.

Human dermal fibroblasts were grown, and plasma membranes were isolated by a modification of a previously described method (17). Confluent fibroblast monolayers were washed three times with phos- phate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM so- dium chloride (pH 7.4)) a t room temperature and then swollen by incubation in 5 mM HEPES (pH 7.4), 0.5 mM EDTA for 30 min. Cells were detached by manual scraping, and the cell suspension was centrifuged at 27,000 X g for 20 min at 4 "C. The cell pellet was resuspended in 9 volumes of cold 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA and homogenized with 20 strokes of a Dounce (Type B) homogenizer. Plasma membranes were isolated by ultracentifugation through discontinuous sucrose density gradients (17). Purified plasma membranes were stored in 20 mM Tris-HC1 (pH 7.5), 0.25 M sucrose, 1 mM MgCl,, and 1 mM DTT at -70 "C until used.

PTH Receptor Solubilization-Purified membrane preparations were centrifuged at 48,000 X g for 30 min at 4 "C. The pellet was resuspended in 0.5% (v/v) CHAPS and 10 mM Tris-HC1 (pH 7.4) to a protein concentration of 7 mg/ml. The suspension was vortexed every 5-7 min at 23 "C for 30 min and then centrifuged at 48,000 X g for 90 min at 4 "C. The supernatant was used as the source of soluble receptors. Detergent and protein concentrations were varied system- atically in pilot experiments to arrive at these solubilization condi- tions.

Synthesis of Biotinyl-bPTH-(1-34)-The biotinylation reagent bio- tinyl-eaminocaproic acid N-hydroxysuccinimide ester was dissolved in dimethyl sulfoxide to 40 mM. The reaction was initiated by slow addition with vortexing of the biotinylation reagent to a suspension of 0.25 mM bPTH-(1-34) in 0.01 M sodium borate (pH 9.0), calculated to yield the desired molar ratio of biotin to peptide. The reaction mixture was incubated for 2 h at 23 "C with frequent vortexing. The reactants and products were separated using an ODs-silica chroma- tography cartridge. The cartridge was prewetted with water, followed by 50% (v/v) acetonitri1e:water and equilibrated with 10 mM sodium borate (pH 9.0) containing 0.1% (v/v) trifluoroacetic acid. After application of the reaction mixture, the column was washed with 8 volumes of equilibrating buffer. Unreacted biotinylation reagent was eluted with the same buffer containing 30% (v/v) acetonitrile, and the bPTH-(l-S4)-peptide was eluted with the same buffer containing 50% (v/v) acetonitrile. The eluate was lyophilized in the presence of 0.5% (w/v) bovine serum albumin (or mannitol in experiments where protein was measured), and the residue was stored in a desiccator a t -20 "C.

Visualization of Receptor Binding-Protein electroblots were in- cubated at room temperature in blocking buffer (100 mM Tris-HC1 (pH 7.5), 100 mM NaCl, 2 mM MgCl,, 5% (w/v) non-fat dry milk, and 0.05 (v/v) Tween 20) for 2-5 h. To detect biotinyl-bPTH-(l-34) binding to immobilized membrane components, blots were incubated for 10 min with streptavidin (2 pg/ml), washed three times, and incubated for 10 min with biotinylated calf intestinal alkaline phos-

phatase (1 pg/ml). Blots were washed three times to remove the unbound alkaline phosphatase, and chromogenic development pro- ceeded using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (18). To determine the concentration of biotinyl-bPTH- (1-34) to be used in binding studies, serial dilutions of biotinyl- bPTH-(1-34) were applied directly to a nitrocellulose filter under light vacuum using a dot-blot apparatus. A final concentration of 4- 17 nM biotinyl-bPTH-(l-34) was chosen as the ligand concentration for receptor studies.

We used the reaction buffer described for the adenylate cyclase assay, with the omission of the ATP-regenerating system and GTPyS, to bind the ligand to particulate receptors. Membrane protein (100 pl) was incubated in a final volume of 100 pl for 20 min at 23 "C in the presence of 17 nM biotinyl-bPTH-(l-34). To determine binding specificity, 20 p M underivatized bPTH-(1-34) was included in the incubation mixture. At the end of the incubation period, the reaction mixtures were centrifuged at 12,000 X g for 4 min at 4 "C, and the particulate membrane pellets were resuspended in 200 p1 of ice-cold PBS.

Biotinyl-bPTH-(l-34) was bound to soluble receptors at 23 "C for 40 min in a mixture containing 50-75 pg of soluble membrane protein, 17 nM biotinyl-bPTH-(1-34), and 0.1% (w/v) CHAPS in a final volume of 110 pl. To determine specific binding, underivatized bPTH (1-34), bPTH-(3-34), oxidized rPTH-(1-34), ACTH, or insulin (all at 20 p ~ ) was included in the binding reaction. The reaction was terminated by addition of 2 volumes of ice-cold PBS.

Cross-linking Protocols-The cross-linking agent DSS was dis- solved in dimethyl sulfoxide at a concentration of 0.1 M and used to attach biotinyl-bPTH-(1-34) covalently to membrane receptor pro- teins (19). Particulate membranes or soluble membrane extracts were incubated in the presence of 2 mM DSS for 15 min in an ice bath. Reactions containing particulate membranes were terminated by adding 5 volumes of ice-cold 10 mM Tris-HC1, 1 mM EDTA (pH 7.4) and then centrifuging at 4 "C for 4 min at 12,000 X g. This step was repeated an additional four times. Cross-linking reactions with solu- ble membrane extracts were terminated by adding 2 volumes of ice- cold PBS.

Electrophoresis-Soluble extracts or particulate membrane sam- ples were incubated at room temperature for 30 min in electrophoresis sample preparation buffer (1% SDS (w/v), 10% sucrose (w/v), 62.5 mM Tris-HC1 (pH 8.6), and 0.005% bromphenol blue) containing either 100 mM DTT or 5 mM iodoacetamide and subjected to electro- phoresis in the discontinuous buffer system described by Laemmli (20). Separating gels contained 7.5% acrylamide and 0.1% SDS.

Two-dimensional polyacrylamide gel electrophoresis was per- formed as described by O'Farrell (21).

Immediately after electrophoresis, proteins were electrophoreti- cally transferred from the slab gel to nitrocellulose in 50 mM Tris- HCI, 200 mM glycine, and 20% (v/v) methanol a t 200 mA for 10 h at room temperature. The lanes with transferred protein standards were cut off and stained with Amido Black 10B, whereas the lanes con- taining the transferred membrane proteins were analyzed for PTH- binding components as described above.

Ligand Affinity Blot Technique-A ligand affinity blotting proce- dure was developed to identify PTH-binding proteins in plasma membrane preparations analyzed by one- or two-dimensional electro- phoresis. Resolved proteins were electroblotted onto nitrocellulose, and the nitrocellulose blot was subsequently incubated in blocking buffer for 2-5 h at room temperature. Blots were then incubated in 50 mM HEPES (pH 7.5), 10 mM MgCl,, 30 mM KC1,l mM DTT, 3% (w/v) bovine serum albumin, and 4 nM biotinyl-bPTH for 1 h at 30 "C with shaking. To determine the specificity of receptor labeling, blots were coincubated with underivatized bPTH-(1-34) (13.5 p ~ ) and biotinyl-bPTH-(1-34) for 1 h. Alternatively, blots were incubated with 13.5 p~ underivatized peptide for 1 h followed by a 1-h incuba- tion with biotinyl-bPTH-(l-34).

All blots were rinsed in 100 mM Tris-HC1 (pH 7.5), 100 mM NaCl, 2 mM MgC12, and 0.05% (v/v) Triton X-100 prior to application of the biotin detection system.

Streptauidin-Agarose Precipitation of Soluble PTH-binding Com- ponents-Renal cortical membrane proteins that had been extracted with 0.5% (w/v) CHAPS were dialyzed overnight at 4 "C against 50 mM HEPES, 1 mM EDTA, 0.1% (w/v) CHAPS (pH 7.5). A precau- tionary "preclearance" step was performed to ensure removal of all endogenous biotin-containing proteins. Adsorption of biotin-contain- ing proteins to streptavidin-agarose was done batchwise. Membrane extract and an equal volume of a 50% slurry of streptavidin-agarose were incubated at 4 "C overnight with constant mixing on a low-

PTH Receptor Studies with Biotinyl-bPTH-(l-34) 14797

speed tube rotator. After incubation, the streptavidin-agarose suspen- sion was centrifuged at 3,000 X g for 5 min, and the supernatant was aspirated and placed on ice for subsequent binding experiments. To recycle the streptavidin-agarose, the packed matrix was washed with 1 M NaCl and 0.02 N HCI to remove nonspecifically adsorbed and specifically adsorbed (i.e. biotin-containing) proteins, respectively (12). The precleared extract was incubated with 17 nM biotinyl-PTH- (1-34) for 40 min at 23 "C. Biotinyl-bPTH-(l-34) was covalently linked to its receptor by the addition of DSS as described above. Biotinyl-bPTH-(l-34)-receptor complexes were specifically isolated by incubating the membrane extract with an equal volume of a 50% slurry of streptavidin-agarose (or Sepharose CL-4B as a control) overnight with rotation at 4 "C. To remove nonspecifically bound proteins, the resin was washed with 12 volumes of ice-cold 50 mM HEPES, 1 mM EDTA, 0.1% (w/v) CHAPS (pH 7.5). Biotinyl-bPTH- (1--34)-receptor complexes were desorbed by resuspension of the resin in SDS-containing electrophoresis sample preparation buffer and heating at 100 "C for 3 min. After heating, the suspension was centrifuged at 10,000 X g for 3 min, and aliquots of the supernatant were subjected to SDS-PAGE. Resolved proteins were transferred electrophoretically to nitrocellulose, and biotin-labeled proteins were detected as described above.

Biotin and Protein Determination-Protein concentration in the membrane preparations was measured by the method of Lowry et al. (22). Lyophilized biotinyl-bPTH-(l-34) residue was dissolved in 0.2 M sodium borate (pH 8.5), and the protein concentration was meas- ured with fluorescamine (23). Bovine serum albumin was used as the protein standard. The biotin content of biotinyl-bPTH-(l-34) was determined according to the method of Green (24).

RESULTS

Biotin was incorporated into bPTH-(1-34) using the biotin derivative biotinyl-c-aminocaproic acid N-hydroxysuccinim- ide ester. The biotinylated peptide was specifically eluted from an ODs-silica column with 50% acetonitrile-containing 0.1% trifluoroacetic acid, whereas elution of the unreacted bioti- nylation reagent required 30% acetonitrile-containing 0.1% trifluoroacetic acid. The recovery of biotinyl-bPTH-( 1-34) under these conditions was typically 45-50%.

Biotinylation of bPTH-(1-34) a t a biotin:peptide molar ratio of 1O: l reduced bioactivity by 90%. In contrast, nearly full biological activity was preserved when biotinylation was performed at a molar ratio of 5:l (Fig. 1). Under these con- ditions, the biotin content of biotinyl-bPTH-(l-34), as deter- mined by the dye assay (24), was 1 mol of biotin/mol of peptide. As little as 350 fmol of biotinyl-bPTH-(l-34) im- mobilized on nitrocellulose paper could be detected easily with streptavidin-biotin-alkaline phosphatase chromogens. This degree of sensitivity is theoretically sufficient to permit visu- alization of approximately 20-50 pg of a receptor protein with

an apparent molecular mass of 68-70 kDa. Contaminating underivatized bPTH-( 1-34) might be the

cause of bioactivity in the ODs-silica cartridge eluate. There- fore, we incubated the eluate with streptavidin-agarose gel to remove biotinyl-bPTH-( 1-34) and found no detectable biotin or PTH bioactivity in the renal membrane adenylate cyclase assay (Fig. 1). In contrast, preincubation of the eluate with Sepharose CL-4B caused no loss of either biotin reactivity or adenylate cyclase stimulating activity. These experiments indicate that PTH bioactivity recovered following the bioti- nylation reaction results from the presence of the biotin- containing bPTH-(1-34) derivative.

Ligand Binding to PTH Receptors-Treatment of biotinyl- bPTH-( 1-34)-labeled porcine renal membranes with DSS resulted in the covalent attachment of biotinyl-bPTH-(1-34) to a diffuse protein band of 68-70 kDa (Fig. 2, lane A) . The presence of 20 PM underivatized bPTH-(1-34) during the incubation abolished nearly completely the appearance of labeled bands (Fig. 2, lane B ) . These findings indicate that binding of biotinyl-bPTH-( 1-34) to the 68-70-kDa protein(s) was specific.

The effect of detergent solubilization of membranes on binding of biotinyl-bPTH to PTH receptor protein is shown in Fig. 3. Receptor solubilization was achieved by extraction of purified renal membranes with 0.5% CHAPS, and binding assays were performed in the presence of 0.1% CHAPS. Under these conditions, biotinyl-bPTH-(l-34) specifically labeled two bands of apparent molecular mass 68 and 70 kDa (lane A ) , which were markedly reduced in intensity when solubilized receptors were coincubated with biotinyl-bPTH-( 1-34) and either 20 PM bPTH-(1-34) (lane B ) or 20 PM bPTH-(3-34). In contrast, labeling of these two bands was not affected when the binding incubation was performed in the presence of 20 PM oxidized rPTH-(1-34), 20 pM ACTH, or 20 PM insulin. Soluble receptors that were covalently attached to biotinyl- bPTH by DSS could also be specifically precipitated with streptavidin-agarose gel (lane D), but not with Sepharose CL- 4B (lane C).

A B Mr

(x~o-~)

100

40

20

I I I I I I I I I I I

10-9 10-8 10-7 10-6 10-5 10-4

PEPTIDE CONCENTRATION la) FIG. 1. Effect of treatment with streptavidin-agaroseon the

bioactivity of biotinyl-bPTH-(1-34). Biotinyl-bPTH-(1-34) was incubated with either streptavidin-agarose resin (0) or Sepharose CL-4B resin (0). The material not adsorbed to the resin was compared to the native bPTH-(l-34)-peptide (0) for bioactivity in the porcine renal adenylate cyclase system. Each point represents the mean of triplicate determinations.

43,

30,

20.1,

FIG. 2. Identification of a PTH-binding component in por- cine renal membranes. Renal cortical membranes were incubated with biotinyl-bPTH-(l-34) in the presence ( l a n e A ) or absence ( l a n e B ) of 20 p~ underivatized peptide. After treatment with DSS to attach the peptide to its receptor, samples were resolved by SDS- PAGE and transferred to nitrocellulose, and the biotin detection system was applied to the blots. The migration positions of marker proteins of known molecular weights are indicated.

PTH Receptor Studies with Biotinyl-bPTH-(l-34) 14798

A

( z i 3 ,

94,

67 43.

30. rn

B C D

FIG. 3. Identification of solubilized PTH-binding compo- nents and precipitation of the hormone-receptor complexes with streptavidin-agarose. Extracts obtained by treatment of porcine renal membranes with 0.5% CHAPS were incubated with streptavidin-agarose resin to remove endogenous biotin-containing proteins. The unadsorbed extract was then incubated with biotinyl- bPTH-(1-34) in the presence ( l a n e A ) or absence ( l a n e B ) of 20 PM underivatized peptide and treated with DSS. The labeled extract was incubated with Sepharose CL-4B ( l a n e C ) or streptavidin-agarose ( l a n e D).

43,

20.1 -

FIG. 4. Porcine renal cortical membranes resolved by SDS- PAGE and electroblotted onto nitrocellulose. Proteins immobi- lized on the blots were incubated with biotinyl-bPTH-(l-34) only (lanes A and B, without and with CHAPS treatment, respectively), preincubated with 20 p~ underivatized bPTH-(1-34) prior to incu- bation with biotinyl-bPTH-(1-34) ( l a n e C), or coincubated with biotinyl-bPTH-(1-34) and 20 p~ underivatized bPTH-(1-34) ( l a n e D). Application of the colorimetric biotin detection system allowed visualization of proteins that bound biotinyl-bPTH-(l-34).

Receptors for PTH were also identified by means of a ligand affinity blot technique. Plasma membrane proteins, resolved by slab gel electrophoresis, were transferred to nitrocellulose; and the electroblot was incubated with biotinyl-bPTH-( 1-34). An intensely stained band of apparent molecular mass 68-70 kDa was present in the porcine renal membrane (Fig. 4, lane A) . A doublet band of similar molecular mass, but less intense staining, was present in the CHAPS-solubilized porcine renal membrane extract (Fig. 4, lane B ) .

In addition, the ligand affinity blot procedure revealed a third, less intensely reacting, PTH-binding component a t 150 kDa (Fig. 4, lunes A and B ) . We observed no quantitative or

~ B C D E F Mr

94- 67-

4 3 ,

30- 20.1-

FIG. 5. Binding of biotinyl-bPTH-(l-34) to receptor com- ponents in membranes from different tissues and species. Plasma membrane preparations were solubilized with CHAPS (lanes A-E) or SDS ( l a n e F ) , resolved by SDS-PAGE, and electroblotted onto nitrocellulose. All lanes were incubated with 4 nM biotinyl- bPTH-(1-34). Lanes A-E, CHAPS extracts of canine, porcine, and human renal cortical, rat hepatic, and human eythrocyte plasma membrane origin, respectively; lane F, SDS-solubilized plasma mem- branes from cultured human fibroblasts of dermal origin.

pH 4.0 5.0 6.0 7.0

Mr

94. 67.

. -

p1r3)

43,

30-

20.1,

FIG. 6. Binding of biotinyl-bPTH-(1-34) to human renal membrane components after two-dimensional electrophore- sis. Triton X-100-extracted plasma membranes were subjected to two-dimensional electrophoresis, followed by transfer to nitrocellu- lose. The blot was probed with biotinyl-bPTH-(l-34).

qualitative differences in the labeling patterns of the three PTH-binding components when we analyzed porcine renal membrane extracts under reducing or nonreducing conditions of electrophoresis.

Labeling of these bands was specific, as inclusion of excess competing underivatized bPTH-(l-34)-peptide with biotinyl- bPTH-(1-34) in the electroblot incubation resulted in com- plete depletion of labeling of the 68- and 70-kDa bands and near complete depletion of the 150-kDa band (Fig. 4, lane D). Moreover, preincubation of electroblots with excess underiv- atized bPTH-(1-34) (13.5 PM), but not with 20 PM insulin or 20 PM ACTH, prior to incubation with biotinyl-bPTH-( 1-34) also resulted in near complete inhibition of labeling of the three bands (Fig. 4, lane C). Finally, these three bands were visible only when electroblots were incubated with biotinyl- bPTH-(1-34) prior to application of the biotin detection system. In contrast, several bands of lower molecular weight could occasionally be identified with the biotin detection

PTH Receptor Studies with Biotinyl-bPTH-(1-34) 14799

system alone. These bands probably represent biotin-contain- ing proteins present in the membrane preparation (12, 25). The quantity and staining intensity of these bands increased when less pure membrane preparations were analyzed (data not shown).

In order to compare physical properties of PTH receptors in several different tissues and species, we analyzed CHAPS- solubilized membrane proteins from human, canine, and por- cine kidney, rat liver, human erythrocyte, and SDS-solubilized membrane proteins from cultured human fibroblasts. The solutions were analyzed, simultaneously, by the ligand affinity blot technique (Fig. 5). All three binding components were present in each of the membrane preparations, except human erythrocyte.

Two-dimensional Gel Electrophoresis-Ligand affinity blot analysis of PTH-binding proteins resolved by two-dimen- sional gel electrophoresis revealed a pattern consistent with that of solubilized membranes from human, canine, and por- cine renal cortex. The ligand affinity blot in Fig. 6 shows labeling of the 68- and 70-kDa PTH-binding components in a sample of human renal cortical membrane. Each component yielded multiple spots of PI 6.1-6.3 (68 kDa) and 6.2-6.7 (70 kDa). Similar analyses of canine and porcine renal cortical membranes showed a consistent shift toward a more acidic charge for the 68-kDa protein spots (data not shown).

DISCUSSION

Previous studies of the PTH receptor have used lZ5I-labeled photolabile derivatives of bPTH-(1-34) to identify PTH- binding components in membranes and cells from several species and target tissues (2-4). These studies demonstrated that there is a similar, if not identical, single membrane component of 60-70 kDa in all tissues examined and that this component can be specifically and covalently labeled by the photolabile bPTH radioligands. In order to analyze further the PTH receptor and to develop an approach to the affinity purification of the receptor protein, we prepared a biotinylated derivative of bPTH-(1-34). Although Niendorf et al. (26) had used biotinylated bPTH-( 1-84) to visualize PTH-binding sites on cultured kidney cells, we considered specific features of this ligand undesirable for our study. First, sequence 1-84 of bPTH recognizes not only receptor components that spe- cifically bind bPTH-(1-34), but also a second class of low affinity receptors that exhibit specific binding for bovine or human PTH-(53-84)-peptides (27). We therefore elected to use sequence 1-34 of bPTH as a receptor probe so that ligand binding would be limited to the high affinity PTH receptors that are linked to adenylate cyclase (1). Second, biotinyl- bPTH-( 1-84) has only 10% of the bioactivity of the underiv- atized peptide (26) and may possess receptor-binding char- acteristics that differ from those of the native hormone. In contrast, biotinylation of the oxidation-resistant bPTH ana- log bPTH-(1-34) (28) at a biotin:peptide molar ratio of 5:l produced a derivative with 85100% receptor binding and adenylate cyclase stimulating activity. Third, a major goal of this work was to prepare a biotinylated peptide that would detect the PTH receptor as well as provide the basis for purification of the receptor by affinity chromatography. Di- rect attachment of biotin to proteins such as insulin (7, 11) can interfere with the subsequent interaction of biotin-protein with avidin. We therefore interposed a 7-atom e-aminocaproic acid linkage between biotin and bPTH-(1-34). Similar spacer arms have been used to eliminate steric hindrances to avidin binding (29) and to increase avidin-biotinyl-protein stability (30).

We used biotinyl-bPTH-(1-34) to visualize PTH receptor

immobilized on nitrocellulose blots. This “ligand affinity blot” technique demonstrated specific binding of biotinyl-bPTH- (1-34) to several proteins in particulate and solubilized mem- branes, including a doublet band at 68-70 kDa. Similar, if not identical, binding proteins of 68 and 70 kDa were observed in solubilized membranes from classical PTH target tissues, including porcine, canine, and human kidney, as well as nonconventional PTH target tissues, including rat liver (31) and human dermal fibroblast (32) (Fig. 4). Significantly, no binding was observed to membrane components of human erythrocytes, which lack PTH receptors. These findings, as well as the results of two-dimensional electrophoresis, confirm and extend the results of previous studies (4) which indicate that the physicochemical properties of the PTH receptor are highly conserved.

Immobilized receptors for several hormones, including PTH (33), have been identified by incubation of the nitrocellulose blot first with ligand and subsequently with an antibody to the ligand. Because inhibition of ligand binding by an excess of “unlabeled” competitor cannot be demonstrated by this “immunoblot” method, binding achieved under these condi- tions cannot be rigorously proved to be specific. Hence, it is not possible to characterize binding proteins identified by ligand immunoblot as specific receptors. In contrast, we used a biotin-labeled hormone analog in a direct approach to identify PTH receptors that had been immobilized on nitro- cellulose filters. The success of this approach implies that the immobilized PTH receptor is renaturable after SDS-PAGE and/or blotting (34) and that the receptor is capable of binding PTH in an oligomeric or membrane-bound form (35).

The present identification of two, rather than one, 60-70- kDa PTH receptor proteins represents an apparent discrep- ancy between our results and those of Coltrera et (11. (2), Draper et al. (3), and Goldring et al. (4). It is doubtful that the doublet receptor proteins are an artifact of the affinity ligand technique, as a 68-70-kDa doublet is detected when biotinyl-bPTH-( 1-34) is chemically attached to receptor pro- teins prior to analysis by SDS-PAGE. Moreover, a similar doublet is observed when 4-fluoro-3-nitrophenyl azide-’T- bPTH-(1-34) is used to label ROS 17/2.8 cells by the pho- toaffinity approach (36). Whether the two 68-70-kDa PTH receptor forms are the result of post-translational differences, such as glycosylation (lo), or differences in the primary protein structure and whether they exhibit unique kinetic properties is not known. Analysis of these two receptor forms by two-dimensional electrophoresis demonstrated that each protein resolves as a distinct array of multiple spots over the range of pH 6.1-6.8, with the 68-kDa components more acidic than the 70-kDa components (Fig. 6).

In addition to the 68-70-kDa PTH receptor, we observed a third binding component of approximately 150 kDa when membranes were analyzed by the ligand affinity blot proce- dure (Fig. 4). This protein is similar in molecular size to detergent-solubilized membrane components previously iden- tified by Malbon and Zull (37, 38) in bovine renal cortex, by Weinshank and Luben (39) in mouse bone cells, and by Nissenson et al. (40) in canine renal cortex. The physicochem- ical relationship of the 150-kDa receptor to the 68-70-kDa doublet is unknown. However, as the relative amount of each of the three receptor proteins remained constant under reduc- ing or nonreducing conditions of electrophoresis, it is unlikely that the smaller 68-70-kDa proteins are subunits released from the larger molecule by cleavage of disulfide bonds.

Finally, we observed specific binding of biotinyl-bPTH-(l- 34) to receptors solubilized from porcine renal membranes by treatment with CHAPS. Pilot experiments showed that 0.5%

14800 PTH Receptor Studies with Biotinyl-bPTH-(l-34)

(8 mM) CHAPS was nearly optimal for solubilization of 11. Finn, F. M., Titus, G., Horstman, D., and Hofmann, K. functional receptors. However, it was necessary to reduce the Proc. Natl. Acad. Sci. U. S. A. 8 1 , 7328-7332 CHAPS concentrations to 0.1% (1.6 mM) in the labeling assay 12. Kohanski, R. A., and Lane, M. D. (1985) J. Biol. Chem

in order to obtain specific binding of biotinyl-bPTH-(l-34) 13, Tashjian, A. H., J ~ . , ontjes, D. A., and M ~ ~ ~ ~ ~ , p. L. to soluble receDtors. These results are consistent with earlier Riochpmktrv 3-11 75-1 182

5014-5025

(1984)

. 260,

(1964)

findings described by Simmonds et al. (41) for the opiate receptor. Whereas 10 mM CHAPS was optimal for solubili- zation of the opiate receptor, maximal binding activity oc- curred at 1.0 mM CHAPS (41). The apparent inhibitory effect of high concentrations of CHAPS on receptor activity may explain the inability of Nissenson et al. (40) to demonstrate high affinity binding of lz51-bPTH-(1-34) to PTH receptors solubilized from canine renal cortical membranes by treat- ment with CHAPSO.

In summary, we have introduced biotin into a PTH analog with preservation of bioactivity of the derivative. In addition to its usefulness as a receptor probe, a further application of biotinyl-bPTH-( 1-34) may be in the affinity purification of a functional PTH receptor. Indeed, in this investigation, we have demonstrated specific binding of soluble biotinyl-bPTH- (1-34)-PTH receptor complexes to a streptavidin-agarose af- finity matrix. Using a strategy based on the high affinity of the heterobifunctional ligand biotinyl-bPTH-( 1-34) for both the soluble PTH receptor and immobilized streptavidin, affin- ity isolation of PTH receptors is an attainable goal. The purification of active PTH receptors will provide the first step in the study of their structure and mechanism of action. Such studies will ultimately shed light on the phenomenon of receptor densensitization and PTH resistance.

Acknowledgments-We gratefully acknowledge Jazz Thrower and Michele Pass for excellent secretarial assistance. We thank Dr. Si- meon Margolis for critical reading of the manuscript.

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