0013-7227/90/1261-0672$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

Heat shock proteins bind calcitonin. Richard C. Dana, William J. Welch and Leonard J. Deftos. Department of Medicine, University of California and the San Diego Veterans Administration Medical Center, La Jolla, California and the Departments of Medicine and Physiology, University of California, San Francisco, California Abstract: We have demonstrated two heat shock proteins (HSP's) in the human placenta that specifically bind calcitonin. Binding specificity was shown by ligand-affinity chromatography and by competitive binding studies. The HSP's were identified by Western analysis and by amino acid sequence. Our observations are consistent with the hypothesis that HSP's may function by binding and interacting with cellular proteins and peptides during their biogenesis. This interaction may both depend upon and produce conf ormational changes in these ligands during their intracellular processing. Additionally, HSP-peptide hormone interactions may confound studies designed to investigate classical receptorhormone interactions. Introduction Binding to hormone receptors and proteins are among the many reported interactions of the heat-shock protein family (HSP's) (1-3). These interactions may represent a biologically important role for HSP's in cellular physiology and regulation. For steroid hormone receptors, HSP's have been reported to bind with the glucocorticoid and progesterone receptor and to effect their affinity for ligand (2,3). HSP's have also been recently reported to bind to several proteins and peptides derived from fulllength molecules (4-7). These interactions may represent the binding of HSP's moeities to proteins that are in unstable configuration during biosynthesis. During our studies of the calcitonin receptor in the human placenta, we observed that specific binding occurred between calcitonin and HSP's. We here report the details of our observations and consider their implications for studies of peptide hormone receptors and the function of HSP's. Materials and Methods Microsomal membranes were prepared from human placentas and stored at -70°C until use (8,9). Membrane proteins were solubilized with 7.5mM 3-[(3-cholamidopropyl) dimethylammonio] 1-propane sulfonate (CHAPS) for 60 minutes at 4°C at 6-10 mg/ml in 50mM Tris-HCl, pH 6.8, lmM MnCl2 0.5mM Na2EDTA, 0.5mM EGTA, 0.2 mg/ml bacitracin, 5 ug/ml aprotinin, 2 ug/ml leupeptin, 0.5 ug/ml pepstatin, lmM benzamidine, lmM sodium metavanadate, O.lmM phenylmethyl sulfonylfluoride (PMSF). Solubilized proteins were separated from membranes by centrifugation at 45,000 xg for 30 minutes at 4°C. Affinity columns were prepared by coupling synthetic salmon calcitonin (sCT) (Sandoz) to Affigel-10 (BioRad) (10). For affinity chromatography, solubilized membrane proteins were incubated with sCT-Affigel in 50 ml conical polypropylene tubes containing 5 ml of gel per 150 mg of protein in 50 ml of 50mM Tris-HCl, pH 6.8, 40mM NaCl, lmM MnCl, O.lmM PMSF, 1 ug/ml leupeptin, 0.5 ug/ml pepstatin, 2mM bacitracin, 3.75mM CHAPS, 2mM dithiothreitol. After

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overnight incubation with end-over-end mixing at 4°C, the gel was poured into a 1 x 30 cm column and washed with 20 volumes of 50mM Tris-HCl, pH 6.8, lmM MgCl,, O.lmM PMSF, 5OOmM NaCl, and 2 column volumes of distilled H2O. Proteins were eluted from the matrix with O.5M ammonium hydroxide or sCT(lOug/ml), frozen in liquid nitrogen, and lyophilized. ATP-Sepharose (Sigma) chromatography was used to further characterize the proteins eluted from the affinity column (11). SDS-PAGE studies (Figure l and 2) were conducted in a preelectrophoresed Laemmli system with 8% acrylamide gels (12). For Western analysis and

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42 Figure 1. sCT-Affigel selects proteins from human placental membranes that include two with Mr's of =83 and =70Kd (arrows) (SDSPAGE). Lane 1: molecular weight standards (BioRad); Lane 2: 200ug of microsomal membrane proteins; Lane 3: proteins eluted from sCT-Affigel with 0.5 M NH4OH.

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automated Edman degradation (UCSD Protein Sequencing Facility), the gels were electroblotted onto Immobilon PVDF (Millipore). Binding studies (Figure 3) with purified BiP and "sI-sCT were conducted as previously described for the hormone (13). BiP was purified from HeLa cells as previously described (11); the preparation contained 90% BiP according to gel electrophoresis and no other HSP protein family members were present. Results Our results indicate that calcitonin binds to the HSP's with specificity and nanomolar affinity: The elution of sCT-Affigel with 0.5 M NH40H resulted in only several protein bands from a complex mixture of applied proteins (Figure 1 ) . Two high molecular weight bands, subsequently identified as HSP's, eluted with sCT (Figure 2 ) . These two bands were also demonstrated by Western analysis to stain with antisera to BiP and hsc70 (11,14) (Figure 2 ) . The proteins eluted with sCT were found to bind ATP-Sepharose, as do several members of the hsp 70 family (Figure 2) (11). Edman sequencing of these two bands revealed that they had the following sequence identity with 18 of 19 amino acids of the hsc70/BiP members of the HSP Family of proteins: E E E D K K E D V G - V V V I D L G - Y (16). Control insulin-Affigel columns and blank Affigel columns did not reveal any comparable findings. In solution binding studies, purified BiP demonstrated specific binding of 1J5I-sCT that could be displaced with nanomolar concentrations of unlabelled sCT (Figure 3 ) . Thus, by amino acid sequence studies, calcitonin- and ATP-binding studies, and Western analyses with antibodies specific to HSP's, the two bands that bind calcitonin were identified as members of the HSP family. Discussion We have observed that caloitonin binds to members of the HSP family, BiP and hsc70, with nanomolar affinity (Figure 1-3). Our observations are consistent with the emerging hypothesis that HSP's exhibit transient interactions with moeities of cellular proteins that are not in their thermodynamically stable conformation (4-7,17). This binding may serve to stabilize the proteins as they are being processed and modified according to the structural requisites of different cellular compartments. Such HSP binding has been reported for a variety of proteins during their biosynthesis (4-7). our studies demonstrate a related phenomenon for the peptide hormone, calcitonin. We observed that HSP's bind to this peptide hormone with specificity and nanomolar affinity (Figures 1-3). Since calcitonin is processed from a precursor molecule and has a hydrophobic region, it may have the requisite biochemical and processing characteristics for HSP binding (18). Thus, the HSP's may facilitate the conformational changes of nascent chains of calcitonin precursor molecules as they are being biosynthesized, cleaved, and transported within the cell (4-7,18). HSP binding could maintain the protein in its most favorable configuration during such biosynthetic processing and intracellular translocation.

Figure 2. Calcitonin binding proteins (arrows as in Figure l) are HSP's (SDS-PAGE). Lane 1: Coomassie blue stain of sCT (lOug/ml) eluate from sCT-Affigel. Lane 2: Western analysis of NH40H eluate from sCT-Affigel (Figure 1, Lane 3) with BiP antiserum and, Lane 3, with HSP70 antiserum (11,17); Lane 4: Coomassie blue stain of 0.5 M NH«OH eluate subsequently eluted from 1 ml of ATP-Sepharose (Sigma) (11). 400-

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Figure 3. Binding of 125 I-sCT by BiP. Purified B i P , 0.05 ng/tube (0.5 m l ) , was incubated with " 5 I-sCT (10,000cpm) in the presence and absence of the indicated concentrations of unlabelled sCT for 18 hours at 4°C in PBS, pH 7.5 (5mM MnCl 2 , 20 uM A T P ) . The BiP-sCT complex was precipitated with rabbit antiserum to BiP (11,17) using a doubleantibody system or with 2 0 % PEG ( 1 3 ) ; both methods gave indistinguishable results. Increasing amounts of unlabelled sCT produced a progressive displacement of BiP-sCT binding with 5 0 % displacement produced by 0.5nM sCT. Control peptides (Insulin, PTHrP and CGRP) produced no displacement.

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RAPID COMMUNICATIONS In addition to supporting such a general view of the function of HSP's for calcitonin, our observations have several other implications. They demonstrate that the human placenta produces HSP's. More importantly, they raise some concerns about studies where peptide hormone affinity matrices have been used in an attempt to study and isolate peptide hormQne receptors (9,19). This approach may be complicated by the promiscuous binding characteristics of the HSP's (4-7,9,19). For example, previous studies of the calcitonin receptor may, in fact, be complicated by the affinity of HSP's for calcitonin that we have observed here (19). Indeed, the several species of the calcitonin receptor that have been identified by ligand binding are consistent in size and characterization with the several species of calcitonin-binding HSP's that we have observed in our studies (9,19). Furthermore, HSP may be responsible for the recently reported binding of peptides to cytosolic proteins that were considered to be receptor variants and for some other unexpected observations about putitive receptorhormone interactions (20,21). It is, of course, possible that HSP's may act as receptors for some peptide hormones. It should be noted, however, that a biological response to calcitonin has not been reported for the placenta (9,19). In any case, rigorous identification of receptor species beyond mere binding studies is critical for conclusive functional studies of receptor physiology. These practical and confounding considerations aside, our observations also provide a model for studying the interactions of HSP's with a naturally occurring peptide hormone during its biosynthesis, cellular processing, and secretion (18). Such studies could provide additional insight into the physiological significance of the HSP's and their importance in peptide hormone biogenesis (21). References 1. Mizzen LA, Welch WJ 1988 Characterization of the Thermotolerant Cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression. J Cell Biol 106:1105 2. Bresnick EH, Dalman FC, Sanchez EF, Pratt WB 1989 Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the Lcell glucocorticoid receptor. J Biol Chem 264:4992 3. Radanyi C, Renoir JM, Sabbah M, Baulieu EE 1989 Chick heat-shock protein of Mr=90,000, free or released from progesterone receptor, is in a dimeric form. J Biol Chem 264:2568 4. Flynn GC, Chappell TG, Rothman JE 1989 Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 248:385 5. Kassenbrock CK, Garcia PD, Walter P, Kelley RB 1989 Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333:90 6. Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R 1988 A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332:800

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7. Chirico WJ, Waters MG, Blobel G 1988 70K heat shock related proteins stimulate protein translocation into microsomes. Nature 332:805 8. Jacobs S, Cuatracasas P 1985 Purification of the insulin receptor from human placenta. Methods in Enzymology 109:405 9. Nicholson GC, D'Santos CS, Evans T, Moseley JM, Kemp BE and Martin TJ 1988 Solubilization of functional calcitonin receptors. Biochem J 253:505 10.Scott CD, Baxter RC 1989 Purification and characterization of insulin-like growth factor II receptors. Methods in Enzymology 168:309 11. Welch WJ, Feramisco 1985 Rapid purification of mammalian 70,000 dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol 5:1229-1236. 12. Laemmli U 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 13. Catherwood BD, LJ Deftos 1984 General principles, problems, and interpretation in the radioimmunoassay of calcitonin. Biomedical Pharmacology 38:235 14. Deftos LJ, Murray SS, Burton DW, Parmer RJ, O'Connor DT, Delegeane AM, Mellon PI 1986 A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues. Biochemical and Biophysical Research Communication 137:418 15. Moos M, Jr, Nguyen NY, Liu T-Y 1989 Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J Biol Chem 263:6005 16. Murno S, Pehlham HRB 1989 An Hsp70-like protein in the ER: Identity with the 78 Kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46:291 17. Welch WJ, Feramisco JR 1984 Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem 259:4501 18. Deftos LJ, Roos BA 1989 Medullary thyroid carcinoma and calcitonin gene expression. In: Peck WA (ed) Bone and Mineral Research. Elsevier Science Publishers B.V., Amsterdan, vol 6:267 19. Nicholson GC, D'Santos CS, Evans T, Moseley JM, Kemp BE, Michelangeli VP, Martin TJ 1988 Human placental calcitonin receptors. Biochem J 250:877 20. Omary MB, Kagnoff MF 1989 Identification of nuclear receptors for VIP on a human colonic adenocarcinoma cell lines. Science 238:1578 21. Pelham H 1988 Heat shock proteins: coming in from the cold. Nature 332:776 Correspondence: Dr. L.J. Deftos, San Diego VA Medical Center (111C), 3350 La Jolla Village Drive, San Diego, CA 92161 Acknowledgements: This work was supported by the National Institutes of Health (AM15888, GM33551) and the Veterans Administration. Douglas Burton, Peter Mullen, and Jeanne Ebbets provided invaluable assistance.

Heat shock proteins bind calcitonin.

We have demonstrated two heat shock proteins (HSP's) in the human placenta that specifically bind calcitonin. Binding specificity was shown by ligand-...
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