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Examination of parathyroid hormone antisera for the presence of receptor antibodies
Molecular and Cellular Endocrinolo~,, 67 (1989) 139-147 Elsevier Scientific Publishers Ireland, Ltd.
139
MOLCEL 02169
Examination of parathyroid hormone antisera for the presence of receptor antibodies James E. Zull, J a c i n t a C h u a n g a n d Susan K. Smith Department of Biology, Case Western Reserve University, ~'leveland, OH 44106, U.S.A.
(Received 31 May 1989; accepted 16 August 1989)
Key words: Parathyroid hormone antiserum; Receptor antibody; (Bovine kidney)
Summary Proteins from bovine kidney membranes were separated by denaturating polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper. The blots were immunostained with parathyroid hormone (PTH) antisera, and the effect of the presence of PTH on immunostaining was determined. Immunostaining of membrane proteins by two specific antisera was altered by PTH. With one antiserum, the immunostaining of two specific proteins (apparent mass 90 and 105 kDa) was prevented by PTH. With the second antiserum the immunostaining of a 150 kDa protein was prevented by the hormone. These effects ,sere strongest with the 90 and 150 kDa proteins and these were investigated further. Antibody binding was prevented either by co-incubation or by preincubation of the blots with P'rH, followed by washing and subsequent exposure to the anti,era. Concentrations of PTH as low as 1 nM prevented antibody binding to the 90 kD, species, but somewha~ higher PTH conceatrations were required with the 150 kDa protein. Oxidation of the PTH methionir, e residues in the amino terminal segment of PTH, and deletion of the first nine residues in the hormone greatly reduced the competition with the 90 kDa protein, but had no effect on immunostaining of the 150 kDa species. The 35-84 fragment of PTH was not a competitor for the 90 kDa species, while the 1-34 fragment was ineffective with the 150 kDa protein. The antiserum labeling the 90 kDa species was also found to possess antagonist activity in activation of renal membrane adenylyl cyclase by PTH and by the biologically active 1-34 fragment of the hormone. The antiserum against the 150 kDa species did not have antagonist activity in this assay. It is concluded that the 90 kDa protein identified in this study may be a receptor related to the functions of the amino terminal domain of PTH, while the 150 kDa protein may be either a specific binding protein for the carboxy terminal portions of the hormone, or a protein with homology with PTH which cross-reacts with the PTH antisen~m. While the study shows that P'I'H antisera may contain anti-idiotypic antibodies against putative PTH receptors, more work is required to define reproducible conditions for generation of such antibodies.
Introduction Address for correspondence: James E. Zull, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. Supported by NIH Grant No. AM-28247.
Putative parathyroid hormone (PTH) receptors have been identified in plasma membrane frac-
0303-7207/89./$03.50 © 1989 Elsevier Scientific Publishers Ireland, Ltd.
14o tions and cells from a variety of tissues (Coltrera et al., 1981; Draper et al., 1982; Goldring et al., 1984; Weinshank et al., 1985a,b; Nissenson et al., 1987; Wright et ,xl., 1987; Shigeno et al., 1988). Cross-linking studies have led to identification of an 85 kDa species, which appears to be linked to a guanosine triphosphate (GTP) binding protein (Nissenson et al., '1987). This protein is thought to be associated with the activation of target cell adenylyl cyclase. Other studies which utilized cross-linking approaches have also identified possible PTH receptor species with a mass of 80-90 kDa (Brennan and Levine, 1987; Wright et al., 1987). A putative receptor with a mass of 150 kDa has also been identified in bone cell membranes (Weinshank and Luben, 1985). These potential PTH receptors are all specific for the amino terminal domain of PTH, which has known biological activities. Membrane and cellular binding of carboxyl terminal fragments of PTH has also been detected, implying the existence of receptors for proteins which may specifically bind this portion of the PTH molecule (McKee and Murray, 1985; Rao and Murray, 1985). However, a biological function for these receptors has not been identified. In a previous study we utilized PTH antisera to detect PTH binding proteins on nitrocellulose blots of denaturing polyacrylamide gels (Chuang et al., 1987). This 'ligand blotting' approach (Gershoni et al., 1983; Schwabe et al., 1988) led to the identification of the/~.subunit of the mitochondrial ATPase as a specific PTH binding protein (Laethem and ZuU, 1988). In that work we also observed a 54 kDa protein which was stained with the PTH antisera, but whose staining was reduced by the hormone, i.e. it appeared that the hormone and the antibodies were competing for binding to this protein on the blots. These findings suggested the possibility that PTH antisera might contain antibodies against PTH binding proteins. Indeed, with other hormones and cell surface ligands, receptor antibodies have been shown to be present in ligand antisera (Flier et al., 1975; Sege and Peterson, 1978; Schreiber et al., 1980; Schechter et al., 1982; Wasserman et al., 1982; Cleveland et al., 1985). These antibodies arise through an anti-idiotypic pathway (Jerne et al., 1982; Schechter et al., 1982,
1984; Elias et al., 1984) and in several of these cases, direct efforts to obtain antibodies against ligand receptors by immunization with ligands proved successful. Here we describe additional studies of the interactions of antibodies in PTH antisera with isolated kidney membrane proteins on nitrocellulose blots. This work has identified three proteins which either bind PTH on such blots, or bind antibodies against PTH. In one case, an antiserum was found to contain antagonist activity in the renal membrane adenylyl cyclase assay for PTH. While these results indicate that anti-receptor antibodies may be obtained by immunization with PTH, most PTH antisera were found not to be antagonistic to the action of the hormone and not to contain antibodies against PTH binding proteins. Thus, more study of conditions necessary for generation of these antibodies is required. Methods and materials
Parathyroid hormone and related peptides Bovine PTH free of oxidized forms was pu~fled from defatted glandular tissue as described (Frelinger and Zull, 1984). Oxidized forms of the hormone were prepared by hydrogen peroxide oxidation and separation on high performance liquid chromatography (HPLC). The 9-84 and 19-84 fragments were obtained by CNBr cleavage of PTH and its oxidized forms (Frelingor and Zull, 1986). The 35-84 fragment of PTH was obtained by cathepsin D digestion (Zull and Chuang, 1985). The 1-34 PTH fragment was purchased from Bachem (Torrence, CA, U.S.A.). Antisera The antisera used for the work were raised in our laboratory by immunization of rabbits and guinea pigs with highly purified bovine PTH, or were kindly provided by Dr. L. Mallette (Houston Medical Center, HoL)ston, TX, U.S.A.) Membrane preparations Isolated bovine kidney membrane fractions were prepared as described in earlier work (Zull et al., 1977). Two types of preparation are used: a partially purified preparation which has not been separated on sucrose gradients (PPM), and a highly
141
purified preparation which is separated on a linear sucrose gradient (HPM).
Electrophoresis, electrobiotting, and immunostaining These methods were described earlier (Chuang et al., 1987). The specificity and sensitivity of the antisera used were also determined on dot-blots as described.
Experiments and results In earlier work we used several PTH antisera to demonstrate direct PTH binding to blotted membrane proteins, i.e. PTH was required for immunostaining of this protein on the blots (Chuang et al., 1987). In those studies, however, we also noted the presence of a protein which was stained with the antiserum, and whose staining was reduced by PTH, Therefore, in further studies with immunostaining of blotted membranes, we looked for proteins whose immunostaining might either be enhanced or prevented by PTH. This involved examination of 26 antisera in all. Each antiserum was used to stain blotted kidney membrane proteins in the presence and absence of PTH.
Detection and properties of a 90 kDa PTH binding protein With several of the antisera we were able to detect staining of the/3-subunit of the mitochondrial ATPase which contaminates most membrane preparations (Laethem and Zull, 1988). This staining occurred only in the presence of PTH. However, in one serum (RSL) we observed strong staining of two additional proteins which was inhibited by PTH. This result and its specificity are shown in Fig. 1. As indicated, the mass of these two proteins is 90 and 105 kDa, and the staining of these proteins was inhibited by the hormone and some of its fragments. At the concentrations of peptides used in this experiment, native hormone, oxidized hormone, the 1-34 fragment, and the 9-84 fragment all reduced the staining of both proteins, although the 1-34 fragment was not as effective as native PTH with the 105 kDa species. On the other hand, the 19-84 and 35-84 fragments were much less effective in preventing staining of the 90 kDa species, but still prevented most of the staining of the 105 kDa protein.
Fig. 1. The staining of the 90 and 105 kDa proteins by the RSL antiserum and the inhibition of staining by PTH and its fragments. Individual nitrocellulose strips from blots of highly purified membranes were incubated with the RSL antiserum in the presence of 100 nM concentrations of the following peptides: lane C, no peptide; lane D, native PTH; lane E, oxidized PTH; lane F, 1-34 PTH peptide, lane G, 9-84 PTH peptide; lane H, 19-84 PTH peptide; and lane I, 35-84 PTH peptide. The blots were then developed as described in the Methods section (first antibody dilution •l/100). Lane A shows the molecular weight markers and lane B shows the proteins stained with Amido Black.
As with most of our PTH antisera, this serum contained antibodies directed primarily against regions of the PTH molecule which are carboxyterminal to residue 34. This is shown in Fig. 2. Under identical conditions, no detectable reaction with the 1-34 fragment was observed at amounts as high as 1/zg on the blots, while as little as 1 ng of the 35-84 fragment could be detected. Other studies (Frelinger, 1986) established that the hormone oxidized at methionines 8 and 18, and the 19-84 and 9-84 fragments of the' hormone all react as well as the 35-84 fragment with RSL. Dilution of the RSL antiserum showed that the staining of the 90 kDa protein was considerably stronger than that of the 105 kDa species. Since only limited amounts of this serum were available, it was used at dilutions which led only to staining of the 90 kDa protein for additional studies. Fig. 3 shows that the inhibition of antibody binding could be observed by preincubation of the blots with PTH, followed by washing and then exposure to the antiserum. If the, blots were first exposed to the antiserum, then washed and exposed to PTH the effect of the hormone was significantly reduced. However, in this protocol the staining was
142
1°°t 80
60,
\
40,
1
20. (~ 0.5
I 1 .o (Peptide)
1 o.o nM
1oo.o
Fig. 4. The effect of different conceW.-ations of competing peptides on staining the 90 kDa protein. The strips were co-incubated with the antiserum and the indicated concentration of competing peptides as described in Fig. 2. The intensity of staining of the 90 kDa band was then determined by densitometry. Fig. 2. Specificity of the RSL antiserum for the carboxy terminal segment of PTH. The 1-34 and 35-84 fragments of the hormone were applied to the blots in the amounts indicated. The blots were then developed with first and second antibody as described (Chuang et al., 1987).
generally quite light because of the additional washes involved following exposure to the antiserum.
-, 97K
9OK"
- , 68K
" 43K
ABODEF
G
H
Fig. 3. Effect of preincubation with PTH or with RSL on the immunostaining of the 90 kDa protein. Strips A and B were incubated with the antiserum for 2 h, then either washed with buffer for 2 h (A), or incubated with 100 nM PTH for 2 h (B). Strips C and D were incubated with buffer for 2 h (C) or with 100 nM PTH for 2 h (D), then washed with buffer for 2 h, then exposed to the antiserum for 2 h. Strips E and F were treated as in Fig. 2, i.e. co-incubation of antiserum and buffer (E) or antiserum and PTH (F) for 2 h. Lanes G and H show the total protein staining and the molecular weight markers, respectively (first antibody dilutions were 1/1000).
The effects of different concentrations of PTH and its fragments on staining of the 90 kDa species are presented in Fig. 4. This study clearly shows that the competitive effect of PTH is greatest with the native hormone. However, unexpectedly, the 1-34 fragments was not as effective as the oxidized form or its 9-84 fragment. On the other hand, the 35-84 fragment had no effect over the concentration range investigated. In additional studies we attempted to demonstrate binding of iodinated PTH directly to the blotted 90 kDa protein by autoradiography. However, using this approach, the radioactive hormone was found to bind strongly and non-specifically to the nitrocellulose blots and the background levels of radioactivity were too high to show selective binding to any particular protein. In our earlier work (Chuang et al., 1987), the RSL antiserum was also used but we did not observe the 90 and 105 kDa proteins. However, those studies were conducted with partially purified membranes (PPM), while the experiments described above were conducted with highly purified membranes (HPM). Further examination of the localization of the 90 kDa protein was conducted and the amounts of this protein in the HPM and PPM were compared. These data are shown in Fig. 5. The 90 kDa protein was found to be primarily localized in the highly purified membranes. This protein was weakly and incon-
143
Fig. 5. Plasma membrane localization of the 90 kDa protein. Partially purified membranes were fractionated on a continuous sucrose gradient. The plasma membrane fraction and the mitochondria-rich fraction form bands in t|~e gradieat, and nuclei and debris form a pellet. Each of these fractions and the starting membranes were stained for the PTH-sensitive 90 kDa protein.
sistently stained in the partially purified membranes. Also, neither the mitochondria, nor the pellet from the gradient (primarily nuclei and debris) appeared to contain this protein. Of interest, it was found that the 51 and the 54 kDa species were both found in all the fractions examined, and appeared to be most concentrated in the mitochondrial fraction. We studied the effect of the RSL antiserum on the biological activity of PTH in the renal membrane bioassay. Purified membranes were incubated with the antiserum overnight, and the membranes were then pelleted, washed, and examined for their responsiveness to PTH in comparison to membranes incubated with control rabbit sera. The results of these experiments are shown in Fig. 6. It was found that preincubation with this antiserum consistently resulted in a significant reduction in the biological activity of the membranes, across the entire range of hormone concentration examined. This was the case for both the 1-34 fragment and the native hormone. While relatively small, this effect was very reproducible. However, no antagonistic activity was observed when the hormone was preincubated with the antiserum, (,r when several other PTH antisera were used in place of the RSL. This antagonistic effect of the RSL antiserum also could not be demonstrated in partially purified membranes. We examined whether the antagonist activity in the RSL antiserum was due to the IgG 2 class of
antibodies by use of Protein A-Sepharose. Following adsorption, the bound IgGs were eluted from the Sepharose and both the original column eluent and the eluted IgG were tested for antagonist activity. The results of this experiment are shown in Fig. 7. While some antagonistic activity w ~ recovered in the IgG 2 fraction, the majority of the activity was not associated with this fraction. Thus, it appears that the antagonist activity is due to a mixture of types of immunoglobulins in this serum. The kinetic data from the antagonist experiments are summarized in Table 1. It appears that the presence of the antibodies exerted its most
20 ~
0 11111,
Basal octiv|ty = 0 for both I
(S.E.M, = I
2~g) I
8O'
|.. N
20. O. 100. 80.
it
20. 0
0.1
Baeal activity = 0 for both (S.E.M. (2~) I
1,0
I
I0.0
I00,0
(Hormone) nM Fig. 6. Effect of membrane incubation with the RSL antiserum on the dose-response curve to PTH. Highly purified membranes were incubated overnight with the RSL antiserum (closed circles) or normal rabbit serum (open circles). The membranes were then washed twice with sucrose-TrisEDTA, and finally resuspended for assay; top panel = native hormone response; middle panel = 1 - 3 4 response; bottom panel = preincubation of native PTH with the RSL antiserum overnight.
144
TABLE 1 100-
EFFECT OF RSL ANTIBODIES ON THE KINETIC CONSTANTS FOP. PTH-ACTIVATED ADENYLYL CYCLASE
80-
Sample assayed
80.
g
40.
E
20.
"6
0
.E
o
Antiserum
I
I
1-84 1-34 1-84 1-34
100. 80.
66. 40. 20. 0.I
Kinetic constants from membranes treated with
I
I
I
1.0
I0.0
I00.0
(C) c (C) (E) c (E)
Protein A-Sepharose eluant
KH a
Vnm b
KH
Vmax
660 840 790 1330
100 100 86 80
600 700 700 900
100 100 66 64
a Nanomolar hormone concentration for half-maximal activation determined from Lineweaver-Burke plots. b Expressed as the percent of the control. Calculated by subtraction of basal activity. c C are the control experiments (exposed to control sera) and E are the experimental (exposed to the RSL antiserum or the purified IgO 2 fraction as described in Figs. 6 and 7).
(Hormone) nM
Fig. 7. Effect of purified 18(3 from the RSL antiserum on the dose-response curve for PTH. The RSL antiserum was passed through a Protein A-Sepharose column, and the effluent collected. The antibodies were then eluted with dilute acid. A control Sepharose column was washed with phosphate-buffered saline and the effluent from this column was collected. The membranes were preincubated with the two column effluents, or with the eluted antibodies as described in Fig. 6. The top panel shows the response to native hormone, and the bottom panel the response to the 1-34 fragment. Symbols used: open circles, eluant from control column; closed circles, eluant from Protein A column; trianBJes, 18Gs eluted from Protein A column.
reproducible effec~ on the Vmax for the hormonestimulated activity, rather than on K H, although the KH for the 1-34 fragment was increased slightly in both experiments. This suggests the possibility that total blockage of hormone action might be achieved with adequate amounts of this antiserum. Detection of a 150 kDa putative PTH-binding protein Weinshank and Luben (1985) described a 150 kDa protein which may be related to the PTH receptor system in bone. As shown in Fig. 8, one of our PTH antisera (Rb 6) immunostained a protein of this mass, and this staining was prevented by PTH. However, as shown, oxidized
PTH also prevented staining of this protein, while the 1-34 fragment of PTH had no apparent effect. Fig. 9 illustrate~ the specificity and concentration dependency of the PTH competitive effect. Within the error of these measurements, the oxidized hormone was not different from the native hormone in its ability to prevent immunostalning, and the 1-34 fragment had ire effect at arty concentration examined.
i
ir
Fig, 8. Staining of 150 kDa protein by Rb 6 antiserum and the effect of PTH on immunostaining. The blots on the left were stained in the absence ( - ) and presence ( + ) of native PTH. The blots in the center were stained in the absence and presence of oxidized PTH, and those on the right in the absence and presence of the 1-34 fragment of PTH.
145 0
e 14'
-1 I
0~1
1.0
10.0
100.0
(peptlde) (nM) Fig. 9. Concentration dependence for reduction of immunostaining the 150 kDa protein by PTH, oxidize,:i PTH and 1-34 PTH. Intensity of staining was quantified by densitometer scanning, and the difference between the staining observed in the presence and absence of each peptide was calculated.
The competition for staining of the 150 kDa protein could be due to the PTH antibodies in the antiserum, if this protein has epitopes which resemble PTH. To examine this possibility, the PTH antibodies were removed from the serum by affinity chromatography and the eluted serum examined for their ability to stain the membrane proteins. Removal of PTH antibodies was found to greatly reduce the staining of both the 54 and the 150 kDa proteins (not shown).
Reproducibility of generation of antisera After obtaining the results described above, we immunized eight additional rabbits with PTH in efforts to obtain new antisera. However, none of the rabbits generated antibodies against the 90 kDa protein and only one produced antibodies against the 150 kDa species. Thus, definition of reproducible conditions for production of such antisera requires further study, but adequate amounts of antisera against the 150 kDa protein are now available to allow purification and characterization of this protein. Discussion This paper further describes our studies of the interactions of FTH antisera with blotted kidney membrane proteins. In the present work and in our prior publications (Chuang et al., 1987; Laethem and Zull, 1988) we describe three separate phenomena: one, a protein which requires
the presence of PTH for its staining, and which has now been shown to be the fl-subunit of the mitochondrial ATPase; two, a 90 kDa protein whose staining is prevented by native FTH at low concentrations, and by the amino terminal fragment of the hormone; and three, two proteins with masses of 54 and 150 kDa whose staining is either prevented or reduced by intact PTH but not by the amino terminal fragment of the hormone. The results with the proteins described in this paper are discussed below, starting with the 90 kDa species.
The 90 kDa protein Of the proteins we have examined, this species appears most likely to be related to the receptor system for PTH. It is localized in the plasma membrane fraction, and the competition between PTH and the antibodies in the RSL serum suggests that the hormone binds this protein on the blots. The antagonist activity of the RSL serum is consistent with the suggestion that antibodies in this serum do block PTH binding to a receptor related to adenylyl cyclase activation. An alternative explanation for the effects of PTH on immunostaining this protein does exist, i.e. the 90 kDa protein has structural similarities to PTH which lead to binding the PTH antibodies. In this case, F f H and the 90 kDa protein would compete for binding the antibodies, producing results similar to those we have obtained. However, the specificity of the competitive effect argues against this possibility. First, we found that the antiserum does not contain antibodies against the 1-34 PTH fragment, but this peptide is an effective competitor for staining of the 90 kDa protein. Second, the 35-84 PTH fragment is ineffective as a competitor for staining the 90 kDa protein, while its xffinity for the PTH antibodies in the serum is equal to that of the native hormone. 'Third, as shown in Fig. 3, PTH prevents antibody staining of the 90 kDa protein when the hormone is preincubated with the blots, and the blots are washed prior to exposure to the antibody. This experimental design eliminates the possibility that FTH could form complexes with the antibodies prior to exposure to the membrane proteins, and greatly reduces the likelihood of formation of such complexes during subsequent steps of the assay.
146
The properties of the RSL antiserum were highly specific. No other PTH antiserum was found to stain the 90 and 105 kDa proteins, and no other antiserum investigated possessed antagonist activity against the native hormone and its 1-34 fragment. While it appears that this activity is the result of PTH immunization, only a portion of the antagonist activity was shown to be due to IgG. The nature of the majority of the activity may be due to other immunoglobulins in the antiserum. The limited amount of this antiserum prevented extensive testing of the antagonist effect, but the data which were obtained (Table 1, Fig. 7) indicate that the antibodies are blocking PTH access to the receptor activating adenylyl cyclase. If adequate amounts of these antibodies were available, total blockage of PTH action might well be observed. Using photoaffinity labeling methods, Wright et al. (1987) also noted a 90 kDa protein (95 kDa with attached ligand) in cloned bone cells which appears to specifically bind the 1-34 domain of bovine IrrH. In addition, others have found a protein with a mass near 80 kDa in canine renal membranes and cloned bone cells (Nissenson et al., 198"/; $higeno et al., 1988). The molecular weights of these proteins are similar, but not identical. However, estimation of mass on polyacrylamide gels is subject to some error, and the various proteins have been obtained from different species. Also, cross-li~ed proteins may run anomalously on polyacrylamide gels. Thus, it is possible that all these proteins are similar to the 90 kDa species we find in the bovine membranes. The specificity and affinity of the protein for native PTH require comment. The apparent KD for I ~ H binding to the blotted protein is below 10 -9 M. This is similar to binding constants determined for putative PTH receptors by radioligand binding studies. Oxidation of the methionines in PTH (residues 8 and 18) reduces the affinity significantly, as is expected for a biologically active receptor (Frelinger and zun, 1986). Also, deletion of segments of the amino terminal domain of PTH reduces the binding, and the 35-84 fragment does not appear to have any affinity for the 90 kDa protein. These results parallel the biological activity and affinity of amino terminal deleted forms of PTH in other
assays. However, the observation that the 1-34 fragment is less effective as a competitor than is the native hormone is unexpected, since th_e 1-34 fragment appears to be as potent as native hormone in most bioassays. Also since the antagonist experiments show that the antibodies are equally effective against 1-34 and the native hormone, the lower effectiveness of the 1-34 PTH fragment in these competition studies is surprising. Since the binding studies are done on blotted proteins, this feature may be the result of the conformation of this protein on the blots; other interpretations are also possible but only speculative at the present time. A protein with a mass of 105 kDa which appears to bind PTH was also observed in the highly purified membranes. The specificity of this protein appears to differ from that of the 90 kDa molecule. Specifically, the 1-34 fragment was not as effective a competitor for this protein while the carboxyl terminal fragments were more effective than with the 90 kDa species (Fig. 3). Whether these two proteins are related (e.g. as forms of receptor processed to different extents) or are totally different in structure and activity will be interesting to examine.
The 150 kDa protein This protein is similar in mass to a putative PTH receptor described by Weinshank and Luben (1985). Our results suggest that rather than binding PTH, this protein may bind the antibodies against PTH. Thus, the interactions of this protein with antibodies in the PTH antisera are prevented by native hormone, and by oxidized forms of the hormone, but not by the biologically active amino terminal fragment of PTH. Furthermore, removal of PTH antibodies ~y affinity chromatography also removed the antibodies which react with this protein. Both this protein, and the 54 kDa species described here and earlier (Chuang et al., 1987), may contain epitopes related to the carboxyl terminal portions of PTH. Alternatively, it is possible that these proteins specifically bind the carboxyl terminal domain of PTH. Isolation and further characterization of these interesting proteins will provide definitive answers to these possible interpretahons.
t47
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M., Nyreida, K. and Arnaud, C.D. (1987) Biochemistry 26, 1874-1878. Schechter, Y., Maron, R., Elias, D. and Cohen, I.R. (1982) Science 316, 542-544. Schechter, Y., Elias, R., Maron, R. and Cohen, I.R. (1984) J. Biol. Chem. 259, 6411-6415. Schreiber, A., Couroud, O., Andre, C., Vray, B. and Strossberg, A.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7385-7389. Schwabe, M., Prinder, G.L. and Faltynek, C.R. (1988) Eur. J. Immunol. 18, 2009-2014. Scge, K. and Peterson, P.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2443-2447. Shigeno, C., Hiraki, Y., Westerberg, D.P., Potts, Jr., J.T. and Segre, G.V. (1988) J. Biol. Chem. 264, 3864-3870. ~trossberg, A.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7585-7390. Wasserman, N.H., Penn, A.H., Freimuth, P.l., Wentzel, S., Cleveland, W. and Erlanger, B.F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4810-4815. Weinshank, R.L. and Luben, R.A. (1985) Eur. J. Biochem. 153, 179-188. Wein~hank, R.L., Cain, C.D., Vasquez, N.P. and Luben, R.A. (1985) Mt~l. Cell. Endocrinol. 41, 237-246. Wright, B., Tyler, G.A., O'Brien, R., Caporale, L.H. and Rosenblatt, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 26-30. Zull, J.E. and Chuar,g, J.C. (1985) J. Biol. Chem. 260, 1608-1615. Zull, J.E., Chuang, J. and Maibon, C.C. (1977) J. Biol. Chem. 252, 1071-1078.
139
MOLCEL 02169
Examination of parathyroid hormone antisera for the presence of receptor antibodies James E. Zull, J a c i n t a C h u a n g a n d Susan K. Smith Department of Biology, Case Western Reserve University, ~'leveland, OH 44106, U.S.A.
(Received 31 May 1989; accepted 16 August 1989)
Key words: Parathyroid hormone antiserum; Receptor antibody; (Bovine kidney)
Summary Proteins from bovine kidney membranes were separated by denaturating polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper. The blots were immunostained with parathyroid hormone (PTH) antisera, and the effect of the presence of PTH on immunostaining was determined. Immunostaining of membrane proteins by two specific antisera was altered by PTH. With one antiserum, the immunostaining of two specific proteins (apparent mass 90 and 105 kDa) was prevented by PTH. With the second antiserum the immunostaining of a 150 kDa protein was prevented by the hormone. These effects ,sere strongest with the 90 and 150 kDa proteins and these were investigated further. Antibody binding was prevented either by co-incubation or by preincubation of the blots with P'rH, followed by washing and subsequent exposure to the anti,era. Concentrations of PTH as low as 1 nM prevented antibody binding to the 90 kD, species, but somewha~ higher PTH conceatrations were required with the 150 kDa protein. Oxidation of the PTH methionir, e residues in the amino terminal segment of PTH, and deletion of the first nine residues in the hormone greatly reduced the competition with the 90 kDa protein, but had no effect on immunostaining of the 150 kDa species. The 35-84 fragment of PTH was not a competitor for the 90 kDa species, while the 1-34 fragment was ineffective with the 150 kDa protein. The antiserum labeling the 90 kDa species was also found to possess antagonist activity in activation of renal membrane adenylyl cyclase by PTH and by the biologically active 1-34 fragment of the hormone. The antiserum against the 150 kDa species did not have antagonist activity in this assay. It is concluded that the 90 kDa protein identified in this study may be a receptor related to the functions of the amino terminal domain of PTH, while the 150 kDa protein may be either a specific binding protein for the carboxy terminal portions of the hormone, or a protein with homology with PTH which cross-reacts with the PTH antisen~m. While the study shows that P'I'H antisera may contain anti-idiotypic antibodies against putative PTH receptors, more work is required to define reproducible conditions for generation of such antibodies.
Introduction Address for correspondence: James E. Zull, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. Supported by NIH Grant No. AM-28247.
Putative parathyroid hormone (PTH) receptors have been identified in plasma membrane frac-
0303-7207/89./$03.50 © 1989 Elsevier Scientific Publishers Ireland, Ltd.
14o tions and cells from a variety of tissues (Coltrera et al., 1981; Draper et al., 1982; Goldring et al., 1984; Weinshank et al., 1985a,b; Nissenson et al., 1987; Wright et ,xl., 1987; Shigeno et al., 1988). Cross-linking studies have led to identification of an 85 kDa species, which appears to be linked to a guanosine triphosphate (GTP) binding protein (Nissenson et al., '1987). This protein is thought to be associated with the activation of target cell adenylyl cyclase. Other studies which utilized cross-linking approaches have also identified possible PTH receptor species with a mass of 80-90 kDa (Brennan and Levine, 1987; Wright et al., 1987). A putative receptor with a mass of 150 kDa has also been identified in bone cell membranes (Weinshank and Luben, 1985). These potential PTH receptors are all specific for the amino terminal domain of PTH, which has known biological activities. Membrane and cellular binding of carboxyl terminal fragments of PTH has also been detected, implying the existence of receptors for proteins which may specifically bind this portion of the PTH molecule (McKee and Murray, 1985; Rao and Murray, 1985). However, a biological function for these receptors has not been identified. In a previous study we utilized PTH antisera to detect PTH binding proteins on nitrocellulose blots of denaturing polyacrylamide gels (Chuang et al., 1987). This 'ligand blotting' approach (Gershoni et al., 1983; Schwabe et al., 1988) led to the identification of the/~.subunit of the mitochondrial ATPase as a specific PTH binding protein (Laethem and ZuU, 1988). In that work we also observed a 54 kDa protein which was stained with the PTH antisera, but whose staining was reduced by the hormone, i.e. it appeared that the hormone and the antibodies were competing for binding to this protein on the blots. These findings suggested the possibility that PTH antisera might contain antibodies against PTH binding proteins. Indeed, with other hormones and cell surface ligands, receptor antibodies have been shown to be present in ligand antisera (Flier et al., 1975; Sege and Peterson, 1978; Schreiber et al., 1980; Schechter et al., 1982; Wasserman et al., 1982; Cleveland et al., 1985). These antibodies arise through an anti-idiotypic pathway (Jerne et al., 1982; Schechter et al., 1982,
1984; Elias et al., 1984) and in several of these cases, direct efforts to obtain antibodies against ligand receptors by immunization with ligands proved successful. Here we describe additional studies of the interactions of antibodies in PTH antisera with isolated kidney membrane proteins on nitrocellulose blots. This work has identified three proteins which either bind PTH on such blots, or bind antibodies against PTH. In one case, an antiserum was found to contain antagonist activity in the renal membrane adenylyl cyclase assay for PTH. While these results indicate that anti-receptor antibodies may be obtained by immunization with PTH, most PTH antisera were found not to be antagonistic to the action of the hormone and not to contain antibodies against PTH binding proteins. Thus, more study of conditions necessary for generation of these antibodies is required. Methods and materials
Parathyroid hormone and related peptides Bovine PTH free of oxidized forms was pu~fled from defatted glandular tissue as described (Frelinger and Zull, 1984). Oxidized forms of the hormone were prepared by hydrogen peroxide oxidation and separation on high performance liquid chromatography (HPLC). The 9-84 and 19-84 fragments were obtained by CNBr cleavage of PTH and its oxidized forms (Frelingor and Zull, 1986). The 35-84 fragment of PTH was obtained by cathepsin D digestion (Zull and Chuang, 1985). The 1-34 PTH fragment was purchased from Bachem (Torrence, CA, U.S.A.). Antisera The antisera used for the work were raised in our laboratory by immunization of rabbits and guinea pigs with highly purified bovine PTH, or were kindly provided by Dr. L. Mallette (Houston Medical Center, HoL)ston, TX, U.S.A.) Membrane preparations Isolated bovine kidney membrane fractions were prepared as described in earlier work (Zull et al., 1977). Two types of preparation are used: a partially purified preparation which has not been separated on sucrose gradients (PPM), and a highly
141
purified preparation which is separated on a linear sucrose gradient (HPM).
Electrophoresis, electrobiotting, and immunostaining These methods were described earlier (Chuang et al., 1987). The specificity and sensitivity of the antisera used were also determined on dot-blots as described.
Experiments and results In earlier work we used several PTH antisera to demonstrate direct PTH binding to blotted membrane proteins, i.e. PTH was required for immunostaining of this protein on the blots (Chuang et al., 1987). In those studies, however, we also noted the presence of a protein which was stained with the antiserum, and whose staining was reduced by PTH, Therefore, in further studies with immunostaining of blotted membranes, we looked for proteins whose immunostaining might either be enhanced or prevented by PTH. This involved examination of 26 antisera in all. Each antiserum was used to stain blotted kidney membrane proteins in the presence and absence of PTH.
Detection and properties of a 90 kDa PTH binding protein With several of the antisera we were able to detect staining of the/3-subunit of the mitochondrial ATPase which contaminates most membrane preparations (Laethem and Zull, 1988). This staining occurred only in the presence of PTH. However, in one serum (RSL) we observed strong staining of two additional proteins which was inhibited by PTH. This result and its specificity are shown in Fig. 1. As indicated, the mass of these two proteins is 90 and 105 kDa, and the staining of these proteins was inhibited by the hormone and some of its fragments. At the concentrations of peptides used in this experiment, native hormone, oxidized hormone, the 1-34 fragment, and the 9-84 fragment all reduced the staining of both proteins, although the 1-34 fragment was not as effective as native PTH with the 105 kDa species. On the other hand, the 19-84 and 35-84 fragments were much less effective in preventing staining of the 90 kDa species, but still prevented most of the staining of the 105 kDa protein.
Fig. 1. The staining of the 90 and 105 kDa proteins by the RSL antiserum and the inhibition of staining by PTH and its fragments. Individual nitrocellulose strips from blots of highly purified membranes were incubated with the RSL antiserum in the presence of 100 nM concentrations of the following peptides: lane C, no peptide; lane D, native PTH; lane E, oxidized PTH; lane F, 1-34 PTH peptide, lane G, 9-84 PTH peptide; lane H, 19-84 PTH peptide; and lane I, 35-84 PTH peptide. The blots were then developed as described in the Methods section (first antibody dilution •l/100). Lane A shows the molecular weight markers and lane B shows the proteins stained with Amido Black.
As with most of our PTH antisera, this serum contained antibodies directed primarily against regions of the PTH molecule which are carboxyterminal to residue 34. This is shown in Fig. 2. Under identical conditions, no detectable reaction with the 1-34 fragment was observed at amounts as high as 1/zg on the blots, while as little as 1 ng of the 35-84 fragment could be detected. Other studies (Frelinger, 1986) established that the hormone oxidized at methionines 8 and 18, and the 19-84 and 9-84 fragments of the' hormone all react as well as the 35-84 fragment with RSL. Dilution of the RSL antiserum showed that the staining of the 90 kDa protein was considerably stronger than that of the 105 kDa species. Since only limited amounts of this serum were available, it was used at dilutions which led only to staining of the 90 kDa protein for additional studies. Fig. 3 shows that the inhibition of antibody binding could be observed by preincubation of the blots with PTH, followed by washing and then exposure to the antiserum. If the, blots were first exposed to the antiserum, then washed and exposed to PTH the effect of the hormone was significantly reduced. However, in this protocol the staining was
142
1°°t 80
60,
\
40,
1
20. (~ 0.5
I 1 .o (Peptide)
1 o.o nM
1oo.o
Fig. 4. The effect of different conceW.-ations of competing peptides on staining the 90 kDa protein. The strips were co-incubated with the antiserum and the indicated concentration of competing peptides as described in Fig. 2. The intensity of staining of the 90 kDa band was then determined by densitometry. Fig. 2. Specificity of the RSL antiserum for the carboxy terminal segment of PTH. The 1-34 and 35-84 fragments of the hormone were applied to the blots in the amounts indicated. The blots were then developed with first and second antibody as described (Chuang et al., 1987).
generally quite light because of the additional washes involved following exposure to the antiserum.
-, 97K
9OK"
- , 68K
" 43K
ABODEF
G
H
Fig. 3. Effect of preincubation with PTH or with RSL on the immunostaining of the 90 kDa protein. Strips A and B were incubated with the antiserum for 2 h, then either washed with buffer for 2 h (A), or incubated with 100 nM PTH for 2 h (B). Strips C and D were incubated with buffer for 2 h (C) or with 100 nM PTH for 2 h (D), then washed with buffer for 2 h, then exposed to the antiserum for 2 h. Strips E and F were treated as in Fig. 2, i.e. co-incubation of antiserum and buffer (E) or antiserum and PTH (F) for 2 h. Lanes G and H show the total protein staining and the molecular weight markers, respectively (first antibody dilutions were 1/1000).
The effects of different concentrations of PTH and its fragments on staining of the 90 kDa species are presented in Fig. 4. This study clearly shows that the competitive effect of PTH is greatest with the native hormone. However, unexpectedly, the 1-34 fragments was not as effective as the oxidized form or its 9-84 fragment. On the other hand, the 35-84 fragment had no effect over the concentration range investigated. In additional studies we attempted to demonstrate binding of iodinated PTH directly to the blotted 90 kDa protein by autoradiography. However, using this approach, the radioactive hormone was found to bind strongly and non-specifically to the nitrocellulose blots and the background levels of radioactivity were too high to show selective binding to any particular protein. In our earlier work (Chuang et al., 1987), the RSL antiserum was also used but we did not observe the 90 and 105 kDa proteins. However, those studies were conducted with partially purified membranes (PPM), while the experiments described above were conducted with highly purified membranes (HPM). Further examination of the localization of the 90 kDa protein was conducted and the amounts of this protein in the HPM and PPM were compared. These data are shown in Fig. 5. The 90 kDa protein was found to be primarily localized in the highly purified membranes. This protein was weakly and incon-
143
Fig. 5. Plasma membrane localization of the 90 kDa protein. Partially purified membranes were fractionated on a continuous sucrose gradient. The plasma membrane fraction and the mitochondria-rich fraction form bands in t|~e gradieat, and nuclei and debris form a pellet. Each of these fractions and the starting membranes were stained for the PTH-sensitive 90 kDa protein.
sistently stained in the partially purified membranes. Also, neither the mitochondria, nor the pellet from the gradient (primarily nuclei and debris) appeared to contain this protein. Of interest, it was found that the 51 and the 54 kDa species were both found in all the fractions examined, and appeared to be most concentrated in the mitochondrial fraction. We studied the effect of the RSL antiserum on the biological activity of PTH in the renal membrane bioassay. Purified membranes were incubated with the antiserum overnight, and the membranes were then pelleted, washed, and examined for their responsiveness to PTH in comparison to membranes incubated with control rabbit sera. The results of these experiments are shown in Fig. 6. It was found that preincubation with this antiserum consistently resulted in a significant reduction in the biological activity of the membranes, across the entire range of hormone concentration examined. This was the case for both the 1-34 fragment and the native hormone. While relatively small, this effect was very reproducible. However, no antagonistic activity was observed when the hormone was preincubated with the antiserum, (,r when several other PTH antisera were used in place of the RSL. This antagonistic effect of the RSL antiserum also could not be demonstrated in partially purified membranes. We examined whether the antagonist activity in the RSL antiserum was due to the IgG 2 class of
antibodies by use of Protein A-Sepharose. Following adsorption, the bound IgGs were eluted from the Sepharose and both the original column eluent and the eluted IgG were tested for antagonist activity. The results of this experiment are shown in Fig. 7. While some antagonistic activity w ~ recovered in the IgG 2 fraction, the majority of the activity was not associated with this fraction. Thus, it appears that the antagonist activity is due to a mixture of types of immunoglobulins in this serum. The kinetic data from the antagonist experiments are summarized in Table 1. It appears that the presence of the antibodies exerted its most
20 ~
0 11111,
Basal octiv|ty = 0 for both I
(S.E.M, = I
2~g) I
8O'
|.. N
20. O. 100. 80.
it
20. 0
0.1
Baeal activity = 0 for both (S.E.M. (2~) I
1,0
I
I0.0
I00,0
(Hormone) nM Fig. 6. Effect of membrane incubation with the RSL antiserum on the dose-response curve to PTH. Highly purified membranes were incubated overnight with the RSL antiserum (closed circles) or normal rabbit serum (open circles). The membranes were then washed twice with sucrose-TrisEDTA, and finally resuspended for assay; top panel = native hormone response; middle panel = 1 - 3 4 response; bottom panel = preincubation of native PTH with the RSL antiserum overnight.
144
TABLE 1 100-
EFFECT OF RSL ANTIBODIES ON THE KINETIC CONSTANTS FOP. PTH-ACTIVATED ADENYLYL CYCLASE
80-
Sample assayed
80.
g
40.
E
20.
"6
0
.E
o
Antiserum
I
I
1-84 1-34 1-84 1-34
100. 80.
66. 40. 20. 0.I
Kinetic constants from membranes treated with
I
I
I
1.0
I0.0
I00.0
(C) c (C) (E) c (E)
Protein A-Sepharose eluant
KH a
Vnm b
KH
Vmax
660 840 790 1330
100 100 86 80
600 700 700 900
100 100 66 64
a Nanomolar hormone concentration for half-maximal activation determined from Lineweaver-Burke plots. b Expressed as the percent of the control. Calculated by subtraction of basal activity. c C are the control experiments (exposed to control sera) and E are the experimental (exposed to the RSL antiserum or the purified IgO 2 fraction as described in Figs. 6 and 7).
(Hormone) nM
Fig. 7. Effect of purified 18(3 from the RSL antiserum on the dose-response curve for PTH. The RSL antiserum was passed through a Protein A-Sepharose column, and the effluent collected. The antibodies were then eluted with dilute acid. A control Sepharose column was washed with phosphate-buffered saline and the effluent from this column was collected. The membranes were preincubated with the two column effluents, or with the eluted antibodies as described in Fig. 6. The top panel shows the response to native hormone, and the bottom panel the response to the 1-34 fragment. Symbols used: open circles, eluant from control column; closed circles, eluant from Protein A column; trianBJes, 18Gs eluted from Protein A column.
reproducible effec~ on the Vmax for the hormonestimulated activity, rather than on K H, although the KH for the 1-34 fragment was increased slightly in both experiments. This suggests the possibility that total blockage of hormone action might be achieved with adequate amounts of this antiserum. Detection of a 150 kDa putative PTH-binding protein Weinshank and Luben (1985) described a 150 kDa protein which may be related to the PTH receptor system in bone. As shown in Fig. 8, one of our PTH antisera (Rb 6) immunostained a protein of this mass, and this staining was prevented by PTH. However, as shown, oxidized
PTH also prevented staining of this protein, while the 1-34 fragment of PTH had no apparent effect. Fig. 9 illustrate~ the specificity and concentration dependency of the PTH competitive effect. Within the error of these measurements, the oxidized hormone was not different from the native hormone in its ability to prevent immunostalning, and the 1-34 fragment had ire effect at arty concentration examined.
i
ir
Fig, 8. Staining of 150 kDa protein by Rb 6 antiserum and the effect of PTH on immunostaining. The blots on the left were stained in the absence ( - ) and presence ( + ) of native PTH. The blots in the center were stained in the absence and presence of oxidized PTH, and those on the right in the absence and presence of the 1-34 fragment of PTH.
145 0
e 14'
-1 I
0~1
1.0
10.0
100.0
(peptlde) (nM) Fig. 9. Concentration dependence for reduction of immunostaining the 150 kDa protein by PTH, oxidize,:i PTH and 1-34 PTH. Intensity of staining was quantified by densitometer scanning, and the difference between the staining observed in the presence and absence of each peptide was calculated.
The competition for staining of the 150 kDa protein could be due to the PTH antibodies in the antiserum, if this protein has epitopes which resemble PTH. To examine this possibility, the PTH antibodies were removed from the serum by affinity chromatography and the eluted serum examined for their ability to stain the membrane proteins. Removal of PTH antibodies was found to greatly reduce the staining of both the 54 and the 150 kDa proteins (not shown).
Reproducibility of generation of antisera After obtaining the results described above, we immunized eight additional rabbits with PTH in efforts to obtain new antisera. However, none of the rabbits generated antibodies against the 90 kDa protein and only one produced antibodies against the 150 kDa species. Thus, definition of reproducible conditions for production of such antisera requires further study, but adequate amounts of antisera against the 150 kDa protein are now available to allow purification and characterization of this protein. Discussion This paper further describes our studies of the interactions of FTH antisera with blotted kidney membrane proteins. In the present work and in our prior publications (Chuang et al., 1987; Laethem and Zull, 1988) we describe three separate phenomena: one, a protein which requires
the presence of PTH for its staining, and which has now been shown to be the fl-subunit of the mitochondrial ATPase; two, a 90 kDa protein whose staining is prevented by native FTH at low concentrations, and by the amino terminal fragment of the hormone; and three, two proteins with masses of 54 and 150 kDa whose staining is either prevented or reduced by intact PTH but not by the amino terminal fragment of the hormone. The results with the proteins described in this paper are discussed below, starting with the 90 kDa species.
The 90 kDa protein Of the proteins we have examined, this species appears most likely to be related to the receptor system for PTH. It is localized in the plasma membrane fraction, and the competition between PTH and the antibodies in the RSL serum suggests that the hormone binds this protein on the blots. The antagonist activity of the RSL serum is consistent with the suggestion that antibodies in this serum do block PTH binding to a receptor related to adenylyl cyclase activation. An alternative explanation for the effects of PTH on immunostaining this protein does exist, i.e. the 90 kDa protein has structural similarities to PTH which lead to binding the PTH antibodies. In this case, F f H and the 90 kDa protein would compete for binding the antibodies, producing results similar to those we have obtained. However, the specificity of the competitive effect argues against this possibility. First, we found that the antiserum does not contain antibodies against the 1-34 PTH fragment, but this peptide is an effective competitor for staining of the 90 kDa protein. Second, the 35-84 PTH fragment is ineffective as a competitor for staining the 90 kDa protein, while its xffinity for the PTH antibodies in the serum is equal to that of the native hormone. 'Third, as shown in Fig. 3, PTH prevents antibody staining of the 90 kDa protein when the hormone is preincubated with the blots, and the blots are washed prior to exposure to the antibody. This experimental design eliminates the possibility that FTH could form complexes with the antibodies prior to exposure to the membrane proteins, and greatly reduces the likelihood of formation of such complexes during subsequent steps of the assay.
146
The properties of the RSL antiserum were highly specific. No other PTH antiserum was found to stain the 90 and 105 kDa proteins, and no other antiserum investigated possessed antagonist activity against the native hormone and its 1-34 fragment. While it appears that this activity is the result of PTH immunization, only a portion of the antagonist activity was shown to be due to IgG. The nature of the majority of the activity may be due to other immunoglobulins in the antiserum. The limited amount of this antiserum prevented extensive testing of the antagonist effect, but the data which were obtained (Table 1, Fig. 7) indicate that the antibodies are blocking PTH access to the receptor activating adenylyl cyclase. If adequate amounts of these antibodies were available, total blockage of PTH action might well be observed. Using photoaffinity labeling methods, Wright et al. (1987) also noted a 90 kDa protein (95 kDa with attached ligand) in cloned bone cells which appears to specifically bind the 1-34 domain of bovine IrrH. In addition, others have found a protein with a mass near 80 kDa in canine renal membranes and cloned bone cells (Nissenson et al., 198"/; $higeno et al., 1988). The molecular weights of these proteins are similar, but not identical. However, estimation of mass on polyacrylamide gels is subject to some error, and the various proteins have been obtained from different species. Also, cross-li~ed proteins may run anomalously on polyacrylamide gels. Thus, it is possible that all these proteins are similar to the 90 kDa species we find in the bovine membranes. The specificity and affinity of the protein for native PTH require comment. The apparent KD for I ~ H binding to the blotted protein is below 10 -9 M. This is similar to binding constants determined for putative PTH receptors by radioligand binding studies. Oxidation of the methionines in PTH (residues 8 and 18) reduces the affinity significantly, as is expected for a biologically active receptor (Frelinger and zun, 1986). Also, deletion of segments of the amino terminal domain of PTH reduces the binding, and the 35-84 fragment does not appear to have any affinity for the 90 kDa protein. These results parallel the biological activity and affinity of amino terminal deleted forms of PTH in other
assays. However, the observation that the 1-34 fragment is less effective as a competitor than is the native hormone is unexpected, since th_e 1-34 fragment appears to be as potent as native hormone in most bioassays. Also since the antagonist experiments show that the antibodies are equally effective against 1-34 and the native hormone, the lower effectiveness of the 1-34 PTH fragment in these competition studies is surprising. Since the binding studies are done on blotted proteins, this feature may be the result of the conformation of this protein on the blots; other interpretations are also possible but only speculative at the present time. A protein with a mass of 105 kDa which appears to bind PTH was also observed in the highly purified membranes. The specificity of this protein appears to differ from that of the 90 kDa molecule. Specifically, the 1-34 fragment was not as effective a competitor for this protein while the carboxyl terminal fragments were more effective than with the 90 kDa species (Fig. 3). Whether these two proteins are related (e.g. as forms of receptor processed to different extents) or are totally different in structure and activity will be interesting to examine.
The 150 kDa protein This protein is similar in mass to a putative PTH receptor described by Weinshank and Luben (1985). Our results suggest that rather than binding PTH, this protein may bind the antibodies against PTH. Thus, the interactions of this protein with antibodies in the PTH antisera are prevented by native hormone, and by oxidized forms of the hormone, but not by the biologically active amino terminal fragment of PTH. Furthermore, removal of PTH antibodies ~y affinity chromatography also removed the antibodies which react with this protein. Both this protein, and the 54 kDa species described here and earlier (Chuang et al., 1987), may contain epitopes related to the carboxyl terminal portions of PTH. Alternatively, it is possible that these proteins specifically bind the carboxyl terminal domain of PTH. Isolation and further characterization of these interesting proteins will provide definitive answers to these possible interpretahons.
t47
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M., Nyreida, K. and Arnaud, C.D. (1987) Biochemistry 26, 1874-1878. Schechter, Y., Maron, R., Elias, D. and Cohen, I.R. (1982) Science 316, 542-544. Schechter, Y., Elias, R., Maron, R. and Cohen, I.R. (1984) J. Biol. Chem. 259, 6411-6415. Schreiber, A., Couroud, O., Andre, C., Vray, B. and Strossberg, A.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7385-7389. Schwabe, M., Prinder, G.L. and Faltynek, C.R. (1988) Eur. J. Immunol. 18, 2009-2014. Scge, K. and Peterson, P.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2443-2447. Shigeno, C., Hiraki, Y., Westerberg, D.P., Potts, Jr., J.T. and Segre, G.V. (1988) J. Biol. Chem. 264, 3864-3870. ~trossberg, A.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7585-7390. Wasserman, N.H., Penn, A.H., Freimuth, P.l., Wentzel, S., Cleveland, W. and Erlanger, B.F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4810-4815. Weinshank, R.L. and Luben, R.A. (1985) Eur. J. Biochem. 153, 179-188. Wein~hank, R.L., Cain, C.D., Vasquez, N.P. and Luben, R.A. (1985) Mt~l. Cell. Endocrinol. 41, 237-246. Wright, B., Tyler, G.A., O'Brien, R., Caporale, L.H. and Rosenblatt, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 26-30. Zull, J.E. and Chuar,g, J.C. (1985) J. Biol. Chem. 260, 1608-1615. Zull, J.E., Chuang, J. and Maibon, C.C. (1977) J. Biol. Chem. 252, 1071-1078.