Biochemistry 1987, 26, 7825-7833 Simpkin, K. G., Chapman, C. R., & Coles, G. C. (1980) Exp. Parasitol. 49, 281-287. Smyth, J. D. (1954) Q. J. Microsc. Sci. 95, 139-154. Smyth, J. D., & Clegg, J. A. (1959) Exp. Parasitol. 8 , 286-323. Smyth, J. D., & Halton, D. W. (1982) The Physiology of Trematodes, 2nd ed., pp 90-99, Cambridge University Press, Cambridge, U.K. Stephenson, W. (1947) Parasitology 38, 128-139. Thangaraj, T., Nellaiappan, K., & Ramalingam, K. (1 982) Parasitology 85, 577-58 1. Threadgold, L. T., & Irwin, S. W. B. (1970) Z. Parasitenk. 35, 16-30. Waite, J. H. (1983) J. Biol. Chem. 258, 2911-2915.
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Waite, J. H. (1984) Anal. Chem. 56, 1935-1939. Waite, J. H. (1986) J. Comp. Physiol., B 156, 491-496. Waite, J. H., & Benedict, C. V. (1984) Methods Enzymol. 107, 391-413. Waite, J. H., Housley, T. J., & Tanzer, M. L. (1985) Biochemistry 24, 5010-5014. Wang, F. L., Su, Y. F., Yang, G. M., Wang, X. Z., Qiu, Z. Y., Zhou, X. K., & Hu, Z. Q. (1986) Mol. Biochem. Parasitol. 18, 69-72. Wharton, D. A. (1983) Parasitology 86, 85-97. Wong, Y.-C., Pustell, J., Spoerel, N., & Kafatos, F. C. (1985) Chromosoma 92, 124-135. Zurita, M., Bieber, D., Ringold, G., & Mansour, T. E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2340-2344.
The Canine Renal Parathyroid Hormone Receptor Is a Glycoprotein: Characterization and Partial Purification+ David B. Karpf,*it Claude D. Arnaud, Kathy King, Thomas Bambino, Jane Winer, Katalin Nyiredy, and Robert A. Nissensone Endocrine Unit, Veterans Administration Medical Center, and Departments of Medicine and Physiology, University of California, San Francisco, California 941 21 Received December 4, 1986; Revised Manuscript Received July 14, 1987
ABSTRACT: Covalent labeling of the canine renal parathyroid hormone receptor with [ 1251] bPTH( 1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a p1,4 linkage to N-acetyl-D-glucosamine. N o high mannose chains or 0-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62 000 [or 58 000 if the mass of bPTH(1-34) is excluded]. The binding of [1251]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.
I t is generally agreed that most of the effects of parathyroid hormone (PTH)’ on its major targets (kidney and bone) are mediated, at least in part, by an intrinsic membrane-bound receptor which is catalytically linked to adenylate cyclase through a stimulatory guanine nucleotide regulatory protein (G,) (Goltzman et al., 1978; Nissenson, 1982; Teitelbaum et al., 1982; Habener et al., 1984). Whereas the biological properties of this receptor have been characterized extensively, little information on its structure has been forthcoming since its initial identification by photoaffinity radiolabeling techniques (Coltrera et al., 1981; Draper et al., 1982). It has been necessary to covalently label the receptor in order to detect its presence after detergent solubilization, because disruption This work is supported by the Research Service of the Veterns Administration and by N I H Grants A M 35323 (R.A.N.) and A M 21614 (C.D.A.). * Address correspondence to this author at the VA Medical Center ( I I l N ) , San Francisco, CA 94121. t D.B.K. is an Associate Investigator of the Veterans Administration. sR.A.N. is an Associate Research Career Scientist of the Veterans Administration.
0006-2960/87/0426-7825$01.50/0
of the receptor-(;, complex shifts the receptor to a very low affinity state for agonist binding (Goltzman et al., 1978; Teitelbaum et al., 1982), and no ligands (either PTH agonists or antagonists) of sufficient affinity to be useful for detection of the low-affinity state of the PTH receptor are currently I Abbreviations: PTH, parathyroid hormone; b, bovine; h, human; VIP, vasoactive intestinal peptide; HSAB, N-succinimidyl 4-azidobenzoate; Tris-HCI, tris(hydroxymethy1)aminomethane hydrochloride; HEPES, N-(2-hydroxyethyl)piperazine-N’2-ethanesulfonic acid; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid trisodium salt; GTP, guanosine 5’-triphosphate; NEM, N-ethylmaleimide; PMSF, phenylmethanesulfonyl fluoride; WGA, wheat-germ agglutinin; S-WGA, succinylated wheat-germ agglutinin; RCAI, Ricinus communis agglutinin I; Con A, concanavalin A; PNA, peanut agglutinin; UEA-F, UIex europaeus agglutinin I; GlcNAc, N-acetyl-D-glucosamine; Gal, o-(+)-galactose; Man, methyl a-D-mannopyranoside; GTP, guanosine 5’-triphosphate; Gpp(NH)p, guanylyl imidodiphosphate; GDPPS, guanosine 5’-0-(2-thicdiphosphate);App(NH)p, adenylyl imidcdiphosphate; G,, stimulatory guanine nucleotide regulatory component of adenylate cyclase; Iodo-Gen, 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril; Triton, Triton X-100; kDa, kilodaltons; DMSO, dimethyl sulfoxide; Ig, immunoglobulin.
0 1987 American Chemical Society
7826 B I O C H E M I S T R Y available. Furthermore, there have been no reports to date of even initial stages of purification of this low-abundance membrane protein. This is due in large part to the lack of suitable reagents (i.e., anti-receptor antibodies) as well as the inability to follow binding activity of the solubilized receptor throughout various purification steps. Recent work in our laboratory, using the heterobifunctional cross-linking reagent HSAB, has identified a predominant M? 85 000 PTH binding protein in canine renal plasma membranes isolated in the presence of protease inhibitors, which decreases in apparent mass to M , 70000 in the absence of protease inhibitors (Nissenson et al., 1987). Minor bands also are seen on SDS-PAGE with M,s of 170 000, 135 000, 5 5 000, and C14400. Labeling of all bands can be inhibited by the inclusion in the binding reaction of as little as 1 nM unlabeled PTH, which correlates well with the Kd of the receptor in binding competition assays and with the K , for adenylate cyclase activity (Teitelbaum et al., 1982). Labeling also is fully inhibited by coincubation with 100 pM Gpp(NH)p or GDPpS but not with 100 pM GMP or App(NH)p, as would be expected of a receptor that is coupled to G,. The predominant M , 85 000 band appears to represent the binding component of the physiologically relevant PTH receptor. Many transmembrane hormone receptors recently have been shown to be glycosylated, including the adenylate cyclase linked receptors for glucagon (Iyengar & Herberg, 1984; Herberg et al., 1984; Iyengar & Herberg, 1985; Iwanij & Hur, 1985), VIP (Nguyen et al., 1986), and P-adrenergic agents (Stiles et al., 1984; Cervantes-Olivier et al., 1985). The presence of carbohydrate on receptors has allowed the use of lectin affinity chromatography for their purification (Hedo, 1984) and in the case of the cyclase-linked glucagon receptor has provided a method for assaying the free, solubilized receptor unassociated with G, (Herberg et al., 1984). If the PTH receptor likewise is glycosylated, these or similar techniques could significantly enhance its further characterization and purification. In particular, lectin affinity chromatography might provide a means of studying the solubilized PTH receptor disassociated from G,. The results of the present study indicate that the canine renal PTH receptor is a glycoprotein containing several complex N-linked carbohydrate chains, which account for approximately 23 kDa of the apparent molecular mass of the receptor on SDS-PAGE. Terminal sialic acid residues are present on at least one of the carbohydrate chains and may be in close proximity to the site of hormone binding. Finally, we report the use of WGA affinity chromatography in the partial purification of the covalently labeled PTH receptor. MATERIALS AND METHODS Materials. All agarose-bound lectins, as well as free WGA, S-WGA, RCAI, Con A, PNA, and UEA-F, were from Vector Laboratories (Burlingame, CA). Neuraminidase from Clostridium perfringens (EC 3.2.1.18) (160 units/mg), PMSF, pepstatin, leupeptin, NEM, N-acetyl-D-glucosamine, D-(+>galactose, L-fucose, methyl a-D-mannopyranoside, and N,N'-diacetylchitobiose were from Sigma Chemical Co. (St. Louis, MO). HSAB, Iodo-Gen, and Triton X-100 were from Pierce Chemical Corp. (Rockford, IL), aprotinin was from Molecular weight estimates for receptor components correspond to the actual position of labeled bands on SDS-PAGE without correction for the contribution of ['251]bPTH(1-34) and HSAB, unless otherwise indicated. Assuming a 1:l:l stoichiometry for ['2sI]bPTH(1-34): HSAB:binding protein, the estimates of receptor M , would be -4500 less than those presented.
K A R P F ET A I . .
Mobay Chemical Corp. (New York, NY), heat-inactivated Staphylococcus aureus (IgGsorb) was from Enzyme Center, Inc. (Waltham, MA), and bovine PTH(1-34) (5000 units/mg) was from Bachem (Torrance, CA). Centricon 10 microconcentrators were from Amicon (Danvers, MA), and Spectropor 2 dialysis tubing was from Spectrum Medical Industries (Los Angeles, CA). NP-40 was from Particle Data Labs, Ltd. (Elmhurst, IL). Acrylamide gradient gels (5-15% and 520%) for SDS-PAGE were purchased from Isolab (Barberton, OH). Na'251was from Amersham, Inc. (Arlington Heights, IL), and Nal3lI was from Cintichem, Inc. (Tuxedo, NY). Purified endoglycosidase F (Endo F) was the very generous gift of Drs. John H. Elder and Stephen Alexander, Scripps Clinic and Research Foundation; its preparation has been previously described (Elder & Alexander, 1982). Extensive use of this enzyme preparation with hundreds of proteins has failed to reveal any proteolytic activity in the presence of EDTA (J. Elder, personal communication). All other chemicals were from Sigma. Isolation of Canine Renal Plasma Membranes. Canine renal cortical plasma membranes were isolated by a published modification (Nissenson & Arnaud, 1979) of the technique of Fitzpatrick et al. (1969). Such preparations are enriched 7-1 0-fold (over crude homogenates) in ouabain-sensitive Na',K+-ATPase. Kidneys were processed in buffers containing either leupeptin (45 pg/mL) and EDTA (1 .O mM) or a mixture of protease inhibitors consisting of PMSF (10 pg/mL), leupeptin (5 pg/mL), pepstatin ( 5 pg/mL), aprotinin (10 units/mL), NEM (1.0 mM), and EDTA (1.0 mM). Membranes prepared with both combinations manifested essentially identical autoradiographic patterns on SDS-PAGE following covalent labeling. Membranes were stored at -80 "C and were stable with respect to [1251]bPTH(1-34)binding activity for at least 6 months. Iodination of Canine Renal Plasma Membranes. Canine renal plasma membranes were iodinated with NaiZ5Iand Iodo-Gen as previously described (Markwell & Fox, 1978). Plasma membranes (500 pg) in 100 pL of 50 mM Tris-HC1, 50 mM HEPES (pH 7.5), 2 mM MgCl,, and 7.5 pL (5 mCi) of NaiZ5Iwere added to a plastic microfuge tube onto which 100 pg of Iodo-Gen had previously been evaporated. Iodination was allowed to proceed for 18 min at room temperature, at which point the reaction mixture was diluted to 1.5 mL with ice-cold 50 mM Tris-HCI, 50 mM HEPES (pH 7.5), 0.1% BSA, and 2 mM MgC1,. This solution was immediately transferred to a glass centrifuge tube containing 6 mL of ice-cold 50 mM Tris-HC1, 50 mM HEPES (pH 7.5), 0.25 M NaI, and 0.1% BSA, and the membranes were washed by centrifiguation and resuspension three times prior to storage at -80 "C. [ i 2 5 1 ] b P T H ( 1 - 3 4 )and [ 1 3 1 1 ] b P T H ( I - 3 4Receptor ) Cross-Linking. Biologically active, electrolytically labeled [1251]bPTH(1-34)and [1311]bPTH(1-34)were prepared and purified by high-performance liquid chromatography as previously described (Nissenson et al., 1986). Approximately 1.O pCi/mL of [1251]bPTH(1-34)was incubated for 2 h at 30 "C with canine renal membranes (250 pg/mL) in a solution consisting of 50 mM Tris-HC1, 50 mM HEPES (pH 7 . 3 , 2.0 mM MgC12,and 0.1% BSA. Incubates were microcentrifuged at 4 "C, and the pellets were resuspended in ice-cold 50 mM sodium phosphate (pH 7.6) containing 0.1% BSA. After centrifugation, this wash step was repeated. Membrane pellets were washed an additional time with 50 mM sodium phosphate (pH 7.6, no BSA) and were resuspended in this buffer. The heterobifunctional cross-linking reagent HSAB in DMSO was
GLYCOPROTEIN STRUCTURE OF THE RENAL PTH RECEPTOR
added to a final concentration of 0.5 mM, and incubation was carried out for 10 min on ice in the dark. The reaction was terminated by the addition of 2 M Tris-HC1 (pH 7.5). Samples were transferred to 24-well cluster plates and subjected to photolysis for 20 min using a Blak-Ray ultraviolet lamp (emission maximum 365 nm) at a distance of 8 cm. The membranes then were washed once with 50 mM sodium phosphate (pH 7.6). Cross-linking with [1311]bPTH(1-34)was carried out in an identical fashion, except that approximately 50 pCi of 12SI-labeledmembranes were added either following photolysis or prior to binding with ['311]bPTH(1-34). Identical results were obtained with the two procedures. Membrane protein concentrations were determined by the method of Lowry et al. (1951). Solubilization. After cross-linking,membrane pellets were resuspended at a detergent:protein ratio of 1O:l w/w in solubilization buffer consisting of 20 mM Tris-HC1 (pH 7.4), 150 mM NaC1, 1.0 mM EDTA, and 1.0% Triton X-100. When the sample was to be chromatographed on Con A, PNA, or UEA-F, EDTA was withheld from the solubilization buffer and the appropriate divalent cations were added at a final concentration of 1.O mM, as these lectins require, respectively, Ca2+ and Mn2+, Ca2+and MgZ+,or Ca2+alone in order to maintain binding activity. The resuspended cross-linked pellets were agitated on an orbital shaker for 1.0 h at room temperature, after which the nonsoluble material was separated by centrifugation at 200000g for 30 min at 4 "C. When this technique was used, greater than 85% of the cross-linked radioactivity was routinely solubilized. Lectin Affinity Chromatography. The agarose-bound lectins used in this study and their carbohydrate specificities are as follows: WGA (terminal sialic acid, terminal and internal N-acetybglucosamine); S-WGA (terminal and internal N-acetyl-D-glucosamine); RCAI (terminal and penultimate D-galactose linked P1,4 to N-acetyl-D-glucosamine); Con A (terminal and internal a-D-mannose); PNA (terminal Dgalactose linked Pl,3 to N-acetylgalactosamine; and UEA-F (terminal a-L-fucose. Lectin columns with bed volumes of 1.O mL were regenerated by washing with 50 mL of buffer A containing 0.01% SDS. For WGA, S-WGA, and RCAI buffer A consisted of 20 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 1.O mM EDTA, and 0.1% Triton X-100. For the remaining lectins the EDTA was withheld, and the following divalent cations (1 mM) were added: Ca2+ and Mn2+ (Con A), Ca2+ and Mg2+(PNA), or Ca2+alone (UEA-F). After the SDS wash, the columns were washed with 25 mL of buffer A containing 300 mM of the appropriate haptenic sugar (buffer B), followed by at least 200 mL of buffer A alone. All steps were carried out at 4 OC. Solubilized preparations of covalently labeled membranes were applied to the columns and recycled twice. The material that failed to adsorb to the column constituted the "run-through". The columns were then washed with 25-50 mL of the appropriate buffer A in order to remove the remainder of the nonspecifically bound material. The final 1.0 mL of wash never contained greater than background radioactivity. Specifically bound radioactivity was eluted by 1.O-mL washes of buffer B at 0.1 mL/min (this fraction constitutes the "sugar eluate"), with approximately 90% of the bound counts eluting in the first 1.0-mL wash. The specificity of lectin binding was assessed by the addition of the specific haptenic sugar to the solubilized covalently labeled receptor prior to chromatography. When lectin column fractions were assessed by SDS-PAGE and autoradiography, equivalent aliquots of the run-through and sugar-eluate fractions were
VOL. 2 6 , NO. 24, 1987
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utilized so that direct comparisons could be made. Neuraminidase Treatment. Cross-linked membranes were washed once with 100 mM sodium acetate (pH 5.1) at 25 OC, followed by centrifugation at 18OOOg for 10 min. Membranes were then resuspended in the same buffer containing 0.1 mM PMSF, 3 pg/mL pepstatin, and 5 mM EDTA. Neuraminidase was added at a concentration of 1.0 unit/mL to all except the control sample, and incubation was performed for 14 h at 37 "C. An aliquot of control and neuraminidasetreated membranes was removed, centrifuged at 1SOOOg, washed once with 20 mM Tris-HC1 (pH 7.4), and solubilized in electrophoresis buffer for subsequent SDS-PAGE or in solubilization buffer for lectin chromatography. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Samples to undergo SDS-PAGE were dialyzed against 20 mM Tris-HC1 (pH 7.4) and 0.1% SDS at 4 OC overnight, utilizing Spectropor 2 dialysis tubing with a molecular weight cutoff of 12000-14000. Samples were concentrated by Centricon 10 centrifugation, adjusted to a final concentration of 76.3 mM Tris-HC1 (pH 6.8), 9.8% glycerol, 1.06%SDS, and 0.46% dithiothreitol, and incubated at 37 "C for 10 min. Boiling of samples was avoided since this led to apparent aggregation, with retention of the samples in the 3% stacking gel during electrophoresis. Samples then were subjected to SDS-PAGE according to the method of Laemmli (1970), using gradient slab gels containing either 5-15% or 5-20% acrylamide in the separating gel. Following electrophoresis the gels were fixed and stained with Coomassie blue G-250, destained, and dried with a Bio-Rad gel dryer prior to autoradiography at -80 "C. The dried gels were exposed to Kodak X-Omat film in cassettes equipped with a single intensifying screen (Cronex Lightening-Plus). Molecular weights were determined from plots of log molecular weight versus log RF (relative migration, measured at the center of the band). The molecular weight markers used (Bio-Rad) were myosin ( M , 200 000), P-galactosidase (MI 116 000), phosphorylase b (MI 92 500), bovine serum albumin ( M , 66 000), ovalbumin (MI 45 000), carbonic anhydrase (MI 31 000), soybean trypsin inhibitor (MI 21 500), and lysozyme (MI 14400). Endoglycosidase F Treatment. Covalent labeled receptor that had been purified by WGA chromatography was dialyzed overnight and subjected to SDS-PAGE on a 5-20% gradient gel as described above. Upon completion of the electrophoresis the sample lane was sliced on a Bio-Rad Model 195 electric gel slicer, and the slices were counted on a Packard Multi-Prias 4 y counter. The slices containing the 85-kDa receptor component were pooled and transferred to the sample cup of an 1 x 0 Model 1750 sample concentrator, overlaid with 10 mM NH4HC03(pH 8.6) and 0.02% SDS, and electroeluted at 1 W for 12 h at 4 O C . An identical procedure was carried out for the 55-kDa component. Recovery of components from the gel slices by this technique averaged >70%. The electroeluted material was dialyzed overnight against 100 mM sodium phosphate (pH 6.1), 50 mM EDTA, and 0.1% SDS at 4 'C and then concentrated by Centricon 10 centrifugation at room temperature for 60 min. Endo F treatment was then performed according to the protocol of Elder and Alexander (1982). The isolated 85- and 55-kDa receptor species were each divided into two equal aliquots, and the volume was adjusted to 90 pL with 100 mM sodium phosphate (pH 6.1), 50 mM EDTA, 1.0% P-mercaptoethanol, and 0.1% SDS and incubated at 30 OC for 3 min. The samples were then made 1.O% with respect to NP-40, and 30 units/mL of Endo F or buffer alone was added. The samples were incubated at 37
7828 B I O C H E M I S T R Y OC for 18 h, after which they were prepared for SDS-PAGE as described above. Receptor Binding Assays. PTH binding in canine renal plasma membranes was determined in triplicate as described previously (Nissenson et al., 1985), except that membranes were incubated for 60 min at 30 OC in the absence or presence of increasing concentrations of free lectins prior to the 2-h incubation with [i251]bPTH(1-34). At the highest lectin concentration, controls were included that contained a 100-fold excess of each lectin’s specific saccharide hapten; in the case of WGA and S-WGA this was N,N’-diacetylchitobiose ( N acetyl-D-glucosaminyl-~l,4-N-acetyl-~-glucosamine). Additionally, bound and free hormone was separated by microcentrifugation rather than by filtration. Blank binding was negligible, and nonspecific binding, which was assessed in the presence of 1 pM bPTH(1-34), was routinely 75%) of the adsorbed Iz5Iradioactivity was eluted with the specific sugar GlcNAc. SDS-PAGE of the GlcNAc eluate
G L Y C O P R O T E I N S T R U C T U R E O F T H E R E N A L PTH R E C E P T O R 0
VOL. 26, N O . 24, 1 9 8 7
7829
A
85*
-81
-115
a 0
e STARTING MATERIAL
E,
R
E,
,R
E,
,R
RCA
S-WGA
WGA
B 13585-
55-
1
-- a
8
4
135+
65+
---
MATER STARTlffiR IA b,-
E CON
R
E
PNA
,R E, UEA
FIGURE 2: Lectin affinity chromatography of CRPMs covalently labeled with [ 1251J bPTH( 1-34). Following solubilizing of the covalently labeled receptor, chromatography was performed on a panel of agarose-bound lectins as described in the legend for Figure 1. Equal aliquots of the nonretained material (run-through) and the specifically eluted material (sugar elutate) were run on SDS-PAGE, and the gel was subjected to autoradiography. R = run-through; E = sugar eluate. (A) Results with WGA, S-WGA, and RCA (RCA ). The dots are molecular weight standards that were overlaid with JC1. (B) Results with CON (Con A), PNA, and UEA (UEA-F). Molecular weights shown are X 1000.
Table I: Affinity of the PTH Receptor for Various Solid-Phase Lectins" % of applied radioactivity lectin n specifically eluted WGA 13 11.7 f 1.0 S-WGA 4 9.7 i 2.5 RCA, 4 8.0 f 2.2 Con A 3 0.9 f 0.1 PNA 3 0.4 i 0.4 UEA 3 0.4 i 0.2 "Canine renal membranes were covalently labeled with ['2sI]bPTH(1-34). solubilized, and chromatographed over various lectin affinity columns as outlined under Materials and Methods. The radioactivity that was eluted only with each lectin's specific sugar is given as a percentage of the applied radioactivity (lo' to 5 X IO5 cpm). The results are the mean f SEM of n exmriments.
revealed the presence of the lzI-labeled 85-,135-,and 55-kDa membrane components that were present in the starting materia13 (Figure 2A). Our previous studies have provided ev~~
~
~
~
_
_
_
_
The pattern routinely seen following Triton X-100 solubilization is somewhat different from that found when cross-linked membranes are immediately analyzed by SDS-PAGE. In the latter condition there is (in membranes prepared with protease inhibitors) a clearly dominant 85-kDa band, a less prominent band of
