- Email: [email protected]
Interaction of cysteine proteases with calciotropic hormone receptors
Comparative Biochemistry and Physiology Part C 128 Ž2001. 247᎐254
Interaction of cysteine proteases with calciotropic hormone receptors M. Fouchereau-PeronU Station de Biologie Marine, Museum ´ National d’Histoire Naturelle, BP 225. 29182 Concarneau Cedex, France Received 25 January 2000; received in revised form 10 November 2000; accepted 17 November 2000
Abstract The effect of two cysteine proteases: papain and a cathepsin L-like enzyme purified from the oesophagus of Nephrops nor¨ egicus ŽNCP. was studied on the specific binding of calcitonin ŽCT. and calcitonin gene related peptide ŽCGRP. to rat kidney and liver membranes, respectively. In addition, the response of adenylyl cyclase to increasing concentrations of these two enzymes was investigated. Each protease inhibited the initial CGRP and CT binding to rat liver and kidney membranes, respectively, in a manner not significantly different from that obtained in the presence of the unlabeled standard. The adenylyl cyclase activity in rat liver membranes was increased by the addition of each enzyme. The response was higher with papain that induced a fivefold increase of enzyme activity at a 4-grml enzyme concentration. In rat kidney membranes, the magnitude of the response was identical with both enzymes. In contrast with NCP, papain induced a biphasic response. Leupeptin and E 64 , two specific inhibitors of cysteine proteases, reversed the observed effects. Trypsin induced an inhibition of the liver membrane adenylyl cyclase activity and an activation in rat kidney membranes at low protease concentration. Thus, cysteine proteases are able to act, in vitro, at the receptor level in target organs specific for calciotropic hormones. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Calcitonin; Calcitonin gene related peptide; Cathepsin; Cyclic AMP; Papain; Radioreceptorassays; Trypsin
1. Introduction Cysteine proteinases belonging to the papain superfamily comprise a large family of enzymes characterised by an essential cysteine residue in their active site ŽBarrett and Kirsche, 1981.. Among them, cathepsin L has been thought to be the most important endopeptidase involved in intralysosomal protein degradation because it has
U
Tel.: q33-298-97-06-59; fax: q33-298-97-81-24. E-mail address: [email protected] ŽM. Fouchereau-Peron..
the most powerful endopeptidase activity among the other lysosomal peptidases. In addition to cathepsin B, cathepsin L is the best characterised enzyme and is implicated in numerous physiological and pathological function. Some of these processes include tumour invasion ŽDuffy, 1992. and metastasis, bone resorption ŽDelaisse ´ and Vaes, 1992. and rheumatoid arthritis ŽGay et al., 1993.. Previous studies from our laboratory have demonstrated the presence of a cathepsin L-like enzyme in the crustacean oesophagus. The enzyme showed 60% homology with human cathep-
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 2 0 0 - 3
248
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
sin L ŽArlot-Bonnemains et al., 1996.. This proteinase was purified on the basis of its cross-reactivity in radioimmunoassays and radioreceptorassays specific for the calciotropic hormones: calcitonin ŽCT. and calcitonin gene related peptide ŽCGRP.. In addition, the ability of calciotropic hormones to stimulate adenylyl cyclase activity in rat kidney and liver membranes, respectively, was used to purify the CT and CGRP-like immunoreactive molecules. Finally, we demonstrated that the purified molecules were able to induce, in vivo, the same biological effect as calcitonin, i.e. an hypocalcemic and hypophosphatemic response ŽArlot-Bonnemains et al., 1996.. These effects were observed both using the cysteine protease isolated from the oesophagus of Nephrops nor¨ egicus ŽArlot-Bonnemains et al., 1996. and that previously purified from the digestive juice of Homarus americanus ŽLaycock et al., 1989.. Proteolytic activation of adenylyl cyclase has been reported with various proteases and in several biological systems including liver membranes ŽLacombe et al., 1977; Koji and Terayama, 1980. rat kidney cells ŽPinkett and Anderson, 1980., murine lymphoma cells ŽStengel et al., 1980. and human heart ŽCros et al., 1987.. Our studies were performed in order to evaluate the interaction of the crustacean cysteine protease ŽNCP. and of commercial papain on the CGRP andror CT binding to their specific receptors and on adenylyl cyclase activity in rat liver and kidney membranes. In addition, inhibitors were used to analyse the specificity of the observed responses.
2. Material and methods Human-CGRP Ž1᎐37. ŽhCGRP. and 125 I-radiolabelled hCGRP Ž2000 Cirmmole. were obtained from Bachem and Amersham, respectively. Synthetic salmon calcitonin ŽsCT.Ž4000 IUrml. was purchased from Sigma. 125 I-labelled sCTŽ2000 Cirmmole. was obtained from Amersham. Male Wistar rats were supplied by Janvier breeding, Le Genest sur Isle, France. The enzyme inhibitor Antagosan ŽAprotinin, 10 000 KIUrml. was kindly donated by Hoechst Laboratories, France. Bovine serum albumin was from Sigma and was inactivated before use. Papain Ž18 Urmg protein., trypsin Ž11 000 Urmg protein., leupeptin, pep-
statin, E 64 and soybean trypsin inhibitor were obtained from Sigma. The crustacean cysteine protease was purified according to previously published method ŽArlot-Bonnemains et al., 1996.. All other chemicals were of reagent grade. 2.1. Receptor binding assay Receptor binding ability of both enzymes was performed using rat kidney membranes and rat liver membranes for CT and CGRP, respectively. Rat kidney membranes were prepared according to the technique described by Fitzpatrick et al. Ž1969.. The method described by Neville Ž1968. was used to obtain rat liver membranes. Incubations, in a 400-l final volume, were performed at 22⬚C during 1 or 4 h for CGRP and CT radioreceptorassay, respectively ŽArlot-Bonnemains et al., 1983; Yamaguchi et al., 1988a.. Data were expressed as specific binding, that was obtained by subtracting from the total binding the amount of radioactivity associated to the membranes in the presence of 0.3 and 2 grml of unlabelled CGRP and CT, respectively. Receptor binding ability of each enzyme was determined in triplicate and at multiple dilutions. The logit᎐log transformation was used to calculate the quantity of protein Žng. inducing a 50% inhibition of the initial binding. 2.2. cAMP production assay CGRP and CT-like biological activity of NCP, papain and trypsin was tested by their capacity to stimulate adenylyl cyclase in rat liver and kidney membranes, respectively. Cyclic AMP production in tissue membranes was determined by measuring the synthesis of cAMP from non-radioactive ATP. The assay was initiated by the addition of the membranes Ž12 or 50 g for liver and kidney membrane proteins, respectively. to prewarmed assay tubes. It was performed as previously described ŽFouchereau-Peron et al., 1990. except that the CGRP stimulated adenylyl cyclase activity was measured in the presence of 100 M GTP ŽYamaguchi et al., 1988b.. Incubations, in a 50-l final volume, were for 15 min at 37⬚C and for 30 min at 30⬚C for kidney and liver membranes, respectively. cAMP was quantified using the w 3 Hx radioreceptor assay kit from Amersham. Data were expressed as the amount of cAMP Žpmoles. synthesised per min and per mg of protein.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
249
2.3. Other methods The criteria to judge labelled hormone degradation were the following: Labelled hormones Ž0.2 and 0.05 nM sCT and hCGRP, respectively. were incubated, in a 400-l volume, in the presence or in the absence of 0.4 g of papain for 4 or 1 h at 20⬚C, respectively. Then 3 g of E 64 was added and the degradation of labelled hormones measured by precipitation of radioactivity with 8% trichloracetic acid in 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% Žwrv. albumin or adsorption to 6 mg silica ŽQUSO G32. in 0.1 M sodium acetate buffer, pH 7.5 ŽCuatrecasas, 1971.. The effect of cysteine proteases on the calciotropic hormone receptors in rat liver and kidney membranes was tested as follows: 1.5 ml of membranes containing 0.9 mg of proteins were incubated in the absence or in the presence of a papain concentration inducing a 50% inhibition in the displacement curves Ž1 and 2 g, respectively. for 15 min at 37⬚C in Hepes Tris 50 mM buffer ŽpH 7.5. containing 2% Žwrv. albumin and 1000 KIU aprotininrml. The reaction was stopped by addition of 18 g E 64 and membranes were washed three times with 10 ml of the same buffer by centrifugation for 15 min at 27 000 = g. Membranes were resuspended in 1.5 ml of Hepes Tris 55 mM ŽpH 7.5., and the hormone binding performed as described in the receptor binding assay section. Cathepsin L activities were measured using NCBZ-Phe-Arg-MCA as substrate at pH 5.5 ŽBarrett and Kirsche, 1981. and accounted for 2.04 and 1.82 mUrg of protein for NCP and papain, respectively. Protein measurement was performed using the bicinchoninic acid protein assay and bovine serum albumin as the standard ŽSmith et al., 1985.. Statistical analysis were performed using ANOVA followed by the Duncan multiple range test.
3. Results 3.1. Effect of cysteine proteases on the CGRP and CT radioreceptorassay The effect of increasing concentrations of crustacean cysteine protease ŽNCP. and papain was
Fig. 1. Effect of increasing concentrations of unlabeled homologous peptide, NCP and papain on the binding of 125 ICGRP and 125 I-CT to rat liver Žleft panel. and kidney Žright panel. membranes, respectively. The logit transformation of the percentage of the initial binding Ž BrB0 = 100. was plotted against the logarithm of the protein concentration. The equations of the regression lines were y s y2.34y 1.50 x; y s 6.67 y 1.56 x; y s 6.8᎐1.20 x for human CGRP, NCP, papain Žleft panel. and y s 0.612y 0.71 x; y s 4.16y 0.79 x; y s 4.78y 0.77x for salmon CT, NCP, papain Žright panel.. Correlation coefficients for the different lines are r 2 s 0.997, 0.971 and 0.995 for unlabeled CGRP, NCP and papain, respectively Žleft panel. and 0.820, 0.978 and 0.751 for salmon CT, NCP and papain, respectively Žright panel..
tested on the CGRP binding to rat liver membranes ŽFig. 1, left panel. and it appeared that these two enzymes inhibited the CGRP binding in a dose-dependent manner. The logit log dose response curves indicated a good parallelism between the effect obtained with human CGRP standard and the NCP. Fifty percent inhibition of the initial CGRP binding was observed with 81 " 25 and 214 " 41 ng of NCP and papain, respectively; that is a fivefold lower concentration of NCP. When the effect of these two enzymes was analysed on the CT binding to kidney membranes ŽFig. 1, right panel. no significant difference was observed in the displacement curves obtained with NCP or papain. Fifty percent inhibition was observed with 288 " 48 and 361 " 76 ng of proteins from NCP and papain, respectively. Control experiments demonstrated no effect of these proteases on the labelled hormone degradation. In the presence of 0.4 g of papain the degradation of labelled CT and CGRP accounted for 16 and 10%, respectively ŽTable 1.. Similarly, no degradation was observed at the membrane receptor level: the initial binding was not affected by incubation in the presence of papain and the ED50 of displacement curves was identical after incubation of membranes in the absence or in the presence of papain ŽTable 1.. We analysed the effect of different inhibitors
250
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Table 1 Effect of papain on the labelled hormone and membrane receptor degradation Hormone bindingb
% Degradeda
125 125
I-CT I-CGRP
QUSO Ž n s 6.
TCA Ž n s 6.
BrTr0.1 mg of proteins
ED50 Žngrml.
No papain Ž n s 3.
qPapain Ž n s 3.
No papain Ž n s 3.
qPapain Ž n s 3.
16.2" 1.8 10.2" 1.2
15 " 0.8 5.4" 1.2
1.26" 0.29 13.7" 1.6
0.95 " 0.31 13.4" 1.4
1.6" 0.4 0.23" 0.06
1.4" 0.3 0.22" 0.05
a 125
I. labelled CT or CGRP were incubated , in a 0.4-ml volume, in the absence or in the presence of 0.4 g papain and incubated for 4 and 1 h at 20⬚C, respectively. Then, 0.1 ml aliquot was analysed by QUSO separation or TCA precipitation as described under Section 2. b Membranes Ž0.9 mg. were incubated in the absence and in the presence of 2 Žliver membranes. and 3 Žkidney membranes. g of papain and processed as described in Section 2. The binding was further determined in the standard conditions Ž1 or 4 h of incubation at 20⬚C for CGRP and CT binding, respectively..
on the binding interaction. Leupeptin is a specific inhibitor of serine and cysteine proteases. Pepstatin is specific for aspartic proteases, E 64 for papain, cathepsin B and L, soybean trypsin inhibitor inhibits trypsin and has no effect on metallo, cysteine and aspartic proteases. No effect was observed with pepstatin Ž10 grml. or soybean trypsin inhibitor Ž20 grml. on the displacement of initial CGRP binding induced by NCP or papain. In contrast, the effect of these two enzymes was abolished in the presence of 5 grml leupeptin or 3.5 grml E 64 ŽFig. 2, left panel.. Similarly, no effect of 10 grml pepstatin and 20 grml soybean trypsin inhibitor was observed on the displacement of CT binding by these two cysteine proteases ŽFig. 2, right panel.. However, the addition of the trypsin inhibitor induced an increase of the CT binding inhibition induced by NCP. The effect was reversed when the incubation was performed in the presence of 5 grml leupeptin and 3.5 grml E 64 .
3.2. Effect of NCP and papain on the adenylyl cyclase acti¨ ity CGRP and CT induce an increase of adenylyl cyclase activity in their respective target tissues ŽFischer and Born, 1987.. We analysed the effect of NCP and papain, two cysteine proteases, on the basal adenylyl cyclase activity in rat liver and kidney membranes . In rat liver membranes ŽFig. 3, left panel., increasing concentrations of NCP induced an increase of adenylyl cyclase activity. A 150% activation was observed with 14.4" 4.6 grml Ži.e. 60 " 19 grmg of membrane proteins.. Adenylyl cyclase responded more sensitively to papain as 150% activation above the control level was already observed with 0.66" 0.4 grml. The maximum response is observed at 4 grml. The adenylyl cyclase activity remained constant above 4 grml and a significant decrease was observed for a papain concentration of 24 grml. Control
Fig. 2. Effect of inhibitors on the specific CGRP and CT binding to rat liver Žleft panel. and kidney Žright panel. membranes. In the left panel, the CGRP binding was measured in the presence of 156 or 781 ngrml of proteins from NCP and papain, respectively, and in the absence or the presence of various inhibitors. In the right panel, the effect of various inhibitors on the CT binding was determined in the presence of 0.6 or 1 grml of NCP or papain, respectively. Results were expressed as the percentage of initial binding BrB0 where B0 represents the hormone binding in the absence of enzymes and inhibitors. Data are the mean " S.E.M. of three different experiments. U : Significant when compared with control value Žno added inhibitor. P- 0.05.
Fig. 3. Effect of increasing concentrations of NCP or papain on the basal adenylate cyclase activity in liver Žleft panel. and kidney Žright panel. membranes, respectively. Liver and kidney membranes were incubated in the presence of NCP and papain for 30 min at 30⬚C Žliver membranes. and 15 min at 37⬚C Žkidney membranes. and the synthesis of cAMP from non radioactive ATP was quantified using a commercial kit. Each point represents the mean " S.E.M. of triplicate determinations in a single experiment. Similar results were observed in three other individual experiments. U Significant when compared with the basal value. P- 0.05.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254 251
252
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Table 2 Effect of human CGRP 8-37 Ž10y6 M. on the papain stimulated adenylate cyclase activity in rat liver membranes Papain, g
0 6 13
Adenylyl cyclase activity, % stimulated No CGRP 8-37
q CGRP 8-37
100 209 " 9 257 " 11
100 163 " 13 157 " 18a
a
Significant when compared to the control Žno CGRP 8-37. value.
experiments demonstrated that this effect of papain was decreased when adenylyl cyclase was measured in the presence of the CGRP receptor antagonist, human CGRP8-37 ŽTable 2. suggesting an interaction of papain at the hormone receptor level. In rat kidney membranes ŽFig. 3, right panel. the effect of these two enzymes was quite different. NCP induced an increase of the adenylyl cyclase activity. 150% activation was observed at 5.6" 1.6 g of proteinrml Ži.e. 1.5 grmg of membrane protein.. There was a progressive increase of the control adenylyl cyclase activity: the maximum was reached with 20 g of proteinrml. Papain induced also an activation of the kidney adenylyl cyclase activity. A sharp increase was observed, followed by a progressive decrease of the activity that returned to the basal value. 150% activation was observed for a protein concentration of 0.9" 0.08 grml. In contrast to liver membranes, there was no significant difference in the maximal activity obtained with each enzyme. The effect of various inhibitors to suppress the stimulated activities was analysed. In liver and kidney membranes ŽFig. 4. only leupeptin and E 64
induced an inhibition of the adenylyl cyclase activities. No significant effect was observed with pepstatin and soybean trypsin inhibitor. A significant inhibition of papain stimulated adenylyl cyclase activity was obtained only in the presence of the two cysteine protease inhibitors; leupeptin and E 64 . To demonstrate the specificity of the cysteine protease effect on adenylyl cyclase activity, the effect of trypsin, a serine protease, was measured in the same experimental conditions ŽFig. 5.. The protease effect was related to the membrane preparation. In liver membranes, the behaviour of trypsin was different from that obtained with papain and NCP ŽFig. 3, left panel.: a progressive decrease of the basal adenylyl cyclase activity was observed, with 50% inhibition at a trypsin concentration of 5 grml. In rat kidney membranes, trypsin induced an increase of the basal adenylyl cyclase activity at concentrations of 1 and 2 grml. In this aspect, the response is similar to that observed with papain ŽFig. 3, right panel.. However, at higher concentrations Ž16 grml., we observed an inhibition of the adenylyl cyclase activity that represents a twofold decrease of the basal activity.
4. Discussion Increasing evidence indicates that some proteases, especially endopeptidases, that have been traditionally considered to participate principally in the degradation of extracellular proteins, are also signalling molecules regulating multiple cellular function by activating specific receptors ŽDery et al., 1998.. In the present study, we de-
Fig. 4. Effect of inhibitors on the enzyme stimulated adenylate cyclase activity in rat liver Žleft panel. and kidney Žright panel. membranes. Rat liver membranes were incubated in the presence of 40 or 8 grml of NCP and papain, respectively, with and without different inhibitors. Rat kidney membranes were similarly incubated in the presence of 20 or 2.7 grml of NCP and papain, respectively. Results are expressed as percent of the basal activity measured in the different experimental conditions. Data are the mean " S.E.M. of triplicate determinations in a single experiment. Similar results were obtained in two other individual experiments. U Significant when compared with the control Žno added inhibitor.. P- 0.05.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Fig. 5. Effect of increasing concentrations of trypsin on the adenylyl cyclase activity in rat kidney and liver membranes. Experimental conditions were as described in the legend to Fig. 3. Data are the mean " S.E.M. of triplicate determinations obtained in a single experiment. Similar results were obtained in four other individual experiments. U Significant when compared to the basal value Žno added trypsin.. P- 0.05.
monstrate that two enzymes from the papain family: papain and a cathepsin L-like enzyme isolated from the oesophagus of Nephrops nor¨ egicus were able to interact both with the CT and CGRP receptors and to activate the adenylyl cyclase activity in rat kidney and liver membranes. No significant difference was observed in the effect of these two enzymes on the displacement of the initial CGRP and CT binding to rat liver and kidney membranes. The effect observed is specific of cysteine proteases as only E 64 and leupeptin inhibited the protease-induced displacement of labelled hormones binding to their specific receptors. Control experiments demonstrated that in the presence of papain no degradation of either labelled hormone or membrane receptors occurred. Both enzymes stimulated adenylyl cyclase activity in the two membranes preparations. In contrast with previous reports ŽKoji and Terayama, 1980. showing a small effect of papain on adenylyl cyclase activity in rat liver membranes, our study demonstrates an important effect of this protease on cAMP production. Moreover, the adenylyl cyclase activity was not further modified by increasing the papain concentration from 4 to 18 grml. Similarly, NCP induces a progressive but smaller increase of the basal adenylyl cyclase activity. In contrast in rat kidney membranes, papain induced a biphasic response, similar to the re-
253
sponse obtained on lymphoma cell membranes ŽStengel et al., 1980.. The effect obtained with NCP remained constant as a function of enzyme concentration. The use of specific inhibitors demonstrated that papain and NCP effect was reversed by the same specific cysteine protease inhibitors in rat liver and kidney membranes. Trypsin, a serine protease, induced an inhibition of liver membrane adenylyl cyclase activity. The effect observed in rat kidney membranes was similar to that obtained with papain at low enzyme concentrations. In accordance with other studies performed with different cysteine proteases ŽLacombe et al., 1977; Koji and Terayama, 1980; Pinkett and Anderson, 1980; Stengel et al., 1980; Cros et al., 1987. our data demonstrate that the enzyme behaviour differs according to the enzyme and the membrane preparation. This suggests that the existence of different multiple protease activating sites on the receptor cyclase complex recognising specific proteases. Moreover, using CGRP8-37, a specific antagonist of CGRP action, we demonstrated that the effect observed on the rat liver membrane adenylyl cyclase activity was receptor mediated. Thus, cysteine proteases are able to act, in vitro, at the receptor level in target organs specific for calciotropic hormones. This effect is also observed in vivo as we previously demonstrated that crustacean cysteine proteases induced the same biological effect as CT, that is hypocalcemia and hypophosphatemia in young rats ŽArlot-Bonnemains et al., 1996.. Cysteine proteases are known as important enzymes for bone turnover and are mainly involved in the degradation of bone collagen by osteoclasts ŽEverts et al., 1992.. If these enzymes are able to interact at the membrane level and to influence the action of calciotropic hormones, they could be of high physiological importance in CT andror CGRP action at the cellular level.
Acknowledgements We are grateful to Y. Le Gal for critical comments and to Mrs C. Michel for her expert technical assistance. This study was supported by a grant in research from INSERM and Academie ´ des sciences.
254
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
References Arlot-Bonnemains, Y., Fouchereau-Peron, M., Moukhtar, M.S.and, Milhaud, G., 1983. Characterization of target organs for calcitonin in lower and higher vertebrates. Comp. Biochem. Physiol. 76A, 377᎐380. Arlot-Bonnemains, Y, Milhaud, G., Fouchereau-Peron, M., 1996. A lobster cysteine protease with immunoreactivity and activities of calcitonin and CGRP. C. R. Acad. Sci. Paris 319, 975᎐982. Barrett, A.J., Kirsche, H., 1981. Cathepsin B, cathepsin H and cathepsin L. Meth. Enzymol. 80, 535᎐561. Cros, G.H., Chaptal, P.A., Serrano, J.J., 1987. The effects of trypsin, plasmin, chymotrypsin, and thrombin on human heart adenylate cyclase activity. Res. Commun. Chem. Pathol. Pharmacol. 57, 277᎐280. Cuatrecasas, P., 1971. Perturbation of the insulin receptor of isolated fat cells with proteolytic enzymes. J. Biol. Chem. 246, 6522᎐6531. Delaisse, ´ J.M., Vaes, G., 1992. Mechanisms of mineral solubilisation and matrix degradation in osteoclastic bone resorption. In Rifkin, B.R. and Gay, C.V. ŽEds. Biology and physiology of the osteoclast, pp. 289᎐314. Boca Raton: CRC Press. Dery, O., Corvera, C.U., Steinhoff, M., Bunnett, N.W., 1998. Proteinase activated receptors: novel mechanisms of signaling by serine proteases. Am. J. Physiol. 274, C1429᎐C1452. Duffy, M.J., 1992. The role of proteolytic enzymes in cancer invasion and metastasis. Clin. Exp. Metastasis 10, 145᎐155. Everts, V., Delaisse, J.M., Korper, W., Niehof, A., Vaes, G., Beertsen, W., 1992. Degradation of collagen in the bone resorbing compartment underlying the osteoclast involves both cysteine proteases and matrix metalloproteinases. J. Cell. Physiol. 150, 221᎐231. Fischer, J.A., Born, W., 1987. Calcitonin gene products: evolution, expression and biological targets. Bone Miner. 2, 347᎐359. Fitzpatrick, D.F, Davenport, G.R., Forte, L., Landon, E.J., 1969. Characterization of plasma membrane proteins in mammalian kidney. I- preparation of a membrane fraction and separation of the protein. J. Biol. Chem. 244, 3561᎐3569.
Fouchereau-Peron, M., Arlot-Bonnemains, Y., Milhaud, G., Moukhtar, M.S., 1990. Calcitonin gene related peptide stimulates adenylyl cyclase activity in trout gill cell membranes. Biochem. Biophys. Res. Commun. 172, 582᎐587. Gay, S., Gay, R.E., Koopman, W.J., 1993. Molecular and cellular mechanisms of joint destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction. Ann. Rheum. Dis. 52, S39᎐S47. Koji, T., Terayama, H., 1980. In vivo and in vitro effects of proteases upon adenylyl cyclase activity of liver cells and plasma membranes. Biochim. Biophys. Acta 633, 10᎐21. Lacombe, M.L., Stengel, D., Hanoune, J., 1977. Proteolytic activation of adenylate cyclase from rat liver plasma membranes. FEBS Lett. 77, 159᎐163. Laycock, M.V., Hirama, T., Hasnain, S., Watson, D., Storer, A.C., 1989. Purification and characterization of a digestive cysteine proteinase from the American lobster Ž Homarus americanus.. Biochem. J. 263, 439᎐444. Neville, D.M.J., 1968. Isolation of an organ specific protein antigen from cell surface membrane rat liver. Biochim. Biophys. Acta 154, 540᎐552. Pinkett, M.O., Anderson, W.B., 1980. Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase. Arch. Biochem. Biophys. 200, 261᎐268. Smith, P.K., Krohn, R.I., Hermanson, G.T. et al., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76᎐85. Stengel, D., Lad, P.M., Nielsen, T.B., Rodbell, M., Hanoune, J., 1980. Proteolysis activates adenylate cyclase activity in rat liver and AC lymphoma cell independently of the guanine nucleotide regulatory site. FEBS Lett. 115, 260᎐264. Yamaguchi, A., Chiba, T., Okimura, Y. et al., 1988a. Receptors for calcitonin gene related peptide on rat liver plasma membranes. Biochem. Biophys. Res. Commun. 152, 383᎐392. Yamaguchi, A., Chiba, T., Yamatani, T. et al., 1988b. Calcitonin gene related peptide stimulates adenylate cyclase activation via a guanine nucleotide dependent process in rat liver plasma membranes. Endocrinology 123, 2591᎐2597.
Interaction of cysteine proteases with calciotropic hormone receptors M. Fouchereau-PeronU Station de Biologie Marine, Museum ´ National d’Histoire Naturelle, BP 225. 29182 Concarneau Cedex, France Received 25 January 2000; received in revised form 10 November 2000; accepted 17 November 2000
Abstract The effect of two cysteine proteases: papain and a cathepsin L-like enzyme purified from the oesophagus of Nephrops nor¨ egicus ŽNCP. was studied on the specific binding of calcitonin ŽCT. and calcitonin gene related peptide ŽCGRP. to rat kidney and liver membranes, respectively. In addition, the response of adenylyl cyclase to increasing concentrations of these two enzymes was investigated. Each protease inhibited the initial CGRP and CT binding to rat liver and kidney membranes, respectively, in a manner not significantly different from that obtained in the presence of the unlabeled standard. The adenylyl cyclase activity in rat liver membranes was increased by the addition of each enzyme. The response was higher with papain that induced a fivefold increase of enzyme activity at a 4-grml enzyme concentration. In rat kidney membranes, the magnitude of the response was identical with both enzymes. In contrast with NCP, papain induced a biphasic response. Leupeptin and E 64 , two specific inhibitors of cysteine proteases, reversed the observed effects. Trypsin induced an inhibition of the liver membrane adenylyl cyclase activity and an activation in rat kidney membranes at low protease concentration. Thus, cysteine proteases are able to act, in vitro, at the receptor level in target organs specific for calciotropic hormones. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Calcitonin; Calcitonin gene related peptide; Cathepsin; Cyclic AMP; Papain; Radioreceptorassays; Trypsin
1. Introduction Cysteine proteinases belonging to the papain superfamily comprise a large family of enzymes characterised by an essential cysteine residue in their active site ŽBarrett and Kirsche, 1981.. Among them, cathepsin L has been thought to be the most important endopeptidase involved in intralysosomal protein degradation because it has
U
Tel.: q33-298-97-06-59; fax: q33-298-97-81-24. E-mail address: [email protected] ŽM. Fouchereau-Peron..
the most powerful endopeptidase activity among the other lysosomal peptidases. In addition to cathepsin B, cathepsin L is the best characterised enzyme and is implicated in numerous physiological and pathological function. Some of these processes include tumour invasion ŽDuffy, 1992. and metastasis, bone resorption ŽDelaisse ´ and Vaes, 1992. and rheumatoid arthritis ŽGay et al., 1993.. Previous studies from our laboratory have demonstrated the presence of a cathepsin L-like enzyme in the crustacean oesophagus. The enzyme showed 60% homology with human cathep-
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 2 0 0 - 3
248
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
sin L ŽArlot-Bonnemains et al., 1996.. This proteinase was purified on the basis of its cross-reactivity in radioimmunoassays and radioreceptorassays specific for the calciotropic hormones: calcitonin ŽCT. and calcitonin gene related peptide ŽCGRP.. In addition, the ability of calciotropic hormones to stimulate adenylyl cyclase activity in rat kidney and liver membranes, respectively, was used to purify the CT and CGRP-like immunoreactive molecules. Finally, we demonstrated that the purified molecules were able to induce, in vivo, the same biological effect as calcitonin, i.e. an hypocalcemic and hypophosphatemic response ŽArlot-Bonnemains et al., 1996.. These effects were observed both using the cysteine protease isolated from the oesophagus of Nephrops nor¨ egicus ŽArlot-Bonnemains et al., 1996. and that previously purified from the digestive juice of Homarus americanus ŽLaycock et al., 1989.. Proteolytic activation of adenylyl cyclase has been reported with various proteases and in several biological systems including liver membranes ŽLacombe et al., 1977; Koji and Terayama, 1980. rat kidney cells ŽPinkett and Anderson, 1980., murine lymphoma cells ŽStengel et al., 1980. and human heart ŽCros et al., 1987.. Our studies were performed in order to evaluate the interaction of the crustacean cysteine protease ŽNCP. and of commercial papain on the CGRP andror CT binding to their specific receptors and on adenylyl cyclase activity in rat liver and kidney membranes. In addition, inhibitors were used to analyse the specificity of the observed responses.
2. Material and methods Human-CGRP Ž1᎐37. ŽhCGRP. and 125 I-radiolabelled hCGRP Ž2000 Cirmmole. were obtained from Bachem and Amersham, respectively. Synthetic salmon calcitonin ŽsCT.Ž4000 IUrml. was purchased from Sigma. 125 I-labelled sCTŽ2000 Cirmmole. was obtained from Amersham. Male Wistar rats were supplied by Janvier breeding, Le Genest sur Isle, France. The enzyme inhibitor Antagosan ŽAprotinin, 10 000 KIUrml. was kindly donated by Hoechst Laboratories, France. Bovine serum albumin was from Sigma and was inactivated before use. Papain Ž18 Urmg protein., trypsin Ž11 000 Urmg protein., leupeptin, pep-
statin, E 64 and soybean trypsin inhibitor were obtained from Sigma. The crustacean cysteine protease was purified according to previously published method ŽArlot-Bonnemains et al., 1996.. All other chemicals were of reagent grade. 2.1. Receptor binding assay Receptor binding ability of both enzymes was performed using rat kidney membranes and rat liver membranes for CT and CGRP, respectively. Rat kidney membranes were prepared according to the technique described by Fitzpatrick et al. Ž1969.. The method described by Neville Ž1968. was used to obtain rat liver membranes. Incubations, in a 400-l final volume, were performed at 22⬚C during 1 or 4 h for CGRP and CT radioreceptorassay, respectively ŽArlot-Bonnemains et al., 1983; Yamaguchi et al., 1988a.. Data were expressed as specific binding, that was obtained by subtracting from the total binding the amount of radioactivity associated to the membranes in the presence of 0.3 and 2 grml of unlabelled CGRP and CT, respectively. Receptor binding ability of each enzyme was determined in triplicate and at multiple dilutions. The logit᎐log transformation was used to calculate the quantity of protein Žng. inducing a 50% inhibition of the initial binding. 2.2. cAMP production assay CGRP and CT-like biological activity of NCP, papain and trypsin was tested by their capacity to stimulate adenylyl cyclase in rat liver and kidney membranes, respectively. Cyclic AMP production in tissue membranes was determined by measuring the synthesis of cAMP from non-radioactive ATP. The assay was initiated by the addition of the membranes Ž12 or 50 g for liver and kidney membrane proteins, respectively. to prewarmed assay tubes. It was performed as previously described ŽFouchereau-Peron et al., 1990. except that the CGRP stimulated adenylyl cyclase activity was measured in the presence of 100 M GTP ŽYamaguchi et al., 1988b.. Incubations, in a 50-l final volume, were for 15 min at 37⬚C and for 30 min at 30⬚C for kidney and liver membranes, respectively. cAMP was quantified using the w 3 Hx radioreceptor assay kit from Amersham. Data were expressed as the amount of cAMP Žpmoles. synthesised per min and per mg of protein.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
249
2.3. Other methods The criteria to judge labelled hormone degradation were the following: Labelled hormones Ž0.2 and 0.05 nM sCT and hCGRP, respectively. were incubated, in a 400-l volume, in the presence or in the absence of 0.4 g of papain for 4 or 1 h at 20⬚C, respectively. Then 3 g of E 64 was added and the degradation of labelled hormones measured by precipitation of radioactivity with 8% trichloracetic acid in 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% Žwrv. albumin or adsorption to 6 mg silica ŽQUSO G32. in 0.1 M sodium acetate buffer, pH 7.5 ŽCuatrecasas, 1971.. The effect of cysteine proteases on the calciotropic hormone receptors in rat liver and kidney membranes was tested as follows: 1.5 ml of membranes containing 0.9 mg of proteins were incubated in the absence or in the presence of a papain concentration inducing a 50% inhibition in the displacement curves Ž1 and 2 g, respectively. for 15 min at 37⬚C in Hepes Tris 50 mM buffer ŽpH 7.5. containing 2% Žwrv. albumin and 1000 KIU aprotininrml. The reaction was stopped by addition of 18 g E 64 and membranes were washed three times with 10 ml of the same buffer by centrifugation for 15 min at 27 000 = g. Membranes were resuspended in 1.5 ml of Hepes Tris 55 mM ŽpH 7.5., and the hormone binding performed as described in the receptor binding assay section. Cathepsin L activities were measured using NCBZ-Phe-Arg-MCA as substrate at pH 5.5 ŽBarrett and Kirsche, 1981. and accounted for 2.04 and 1.82 mUrg of protein for NCP and papain, respectively. Protein measurement was performed using the bicinchoninic acid protein assay and bovine serum albumin as the standard ŽSmith et al., 1985.. Statistical analysis were performed using ANOVA followed by the Duncan multiple range test.
3. Results 3.1. Effect of cysteine proteases on the CGRP and CT radioreceptorassay The effect of increasing concentrations of crustacean cysteine protease ŽNCP. and papain was
Fig. 1. Effect of increasing concentrations of unlabeled homologous peptide, NCP and papain on the binding of 125 ICGRP and 125 I-CT to rat liver Žleft panel. and kidney Žright panel. membranes, respectively. The logit transformation of the percentage of the initial binding Ž BrB0 = 100. was plotted against the logarithm of the protein concentration. The equations of the regression lines were y s y2.34y 1.50 x; y s 6.67 y 1.56 x; y s 6.8᎐1.20 x for human CGRP, NCP, papain Žleft panel. and y s 0.612y 0.71 x; y s 4.16y 0.79 x; y s 4.78y 0.77x for salmon CT, NCP, papain Žright panel.. Correlation coefficients for the different lines are r 2 s 0.997, 0.971 and 0.995 for unlabeled CGRP, NCP and papain, respectively Žleft panel. and 0.820, 0.978 and 0.751 for salmon CT, NCP and papain, respectively Žright panel..
tested on the CGRP binding to rat liver membranes ŽFig. 1, left panel. and it appeared that these two enzymes inhibited the CGRP binding in a dose-dependent manner. The logit log dose response curves indicated a good parallelism between the effect obtained with human CGRP standard and the NCP. Fifty percent inhibition of the initial CGRP binding was observed with 81 " 25 and 214 " 41 ng of NCP and papain, respectively; that is a fivefold lower concentration of NCP. When the effect of these two enzymes was analysed on the CT binding to kidney membranes ŽFig. 1, right panel. no significant difference was observed in the displacement curves obtained with NCP or papain. Fifty percent inhibition was observed with 288 " 48 and 361 " 76 ng of proteins from NCP and papain, respectively. Control experiments demonstrated no effect of these proteases on the labelled hormone degradation. In the presence of 0.4 g of papain the degradation of labelled CT and CGRP accounted for 16 and 10%, respectively ŽTable 1.. Similarly, no degradation was observed at the membrane receptor level: the initial binding was not affected by incubation in the presence of papain and the ED50 of displacement curves was identical after incubation of membranes in the absence or in the presence of papain ŽTable 1.. We analysed the effect of different inhibitors
250
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Table 1 Effect of papain on the labelled hormone and membrane receptor degradation Hormone bindingb
% Degradeda
125 125
I-CT I-CGRP
QUSO Ž n s 6.
TCA Ž n s 6.
BrTr0.1 mg of proteins
ED50 Žngrml.
No papain Ž n s 3.
qPapain Ž n s 3.
No papain Ž n s 3.
qPapain Ž n s 3.
16.2" 1.8 10.2" 1.2
15 " 0.8 5.4" 1.2
1.26" 0.29 13.7" 1.6
0.95 " 0.31 13.4" 1.4
1.6" 0.4 0.23" 0.06
1.4" 0.3 0.22" 0.05
a 125
I. labelled CT or CGRP were incubated , in a 0.4-ml volume, in the absence or in the presence of 0.4 g papain and incubated for 4 and 1 h at 20⬚C, respectively. Then, 0.1 ml aliquot was analysed by QUSO separation or TCA precipitation as described under Section 2. b Membranes Ž0.9 mg. were incubated in the absence and in the presence of 2 Žliver membranes. and 3 Žkidney membranes. g of papain and processed as described in Section 2. The binding was further determined in the standard conditions Ž1 or 4 h of incubation at 20⬚C for CGRP and CT binding, respectively..
on the binding interaction. Leupeptin is a specific inhibitor of serine and cysteine proteases. Pepstatin is specific for aspartic proteases, E 64 for papain, cathepsin B and L, soybean trypsin inhibitor inhibits trypsin and has no effect on metallo, cysteine and aspartic proteases. No effect was observed with pepstatin Ž10 grml. or soybean trypsin inhibitor Ž20 grml. on the displacement of initial CGRP binding induced by NCP or papain. In contrast, the effect of these two enzymes was abolished in the presence of 5 grml leupeptin or 3.5 grml E 64 ŽFig. 2, left panel.. Similarly, no effect of 10 grml pepstatin and 20 grml soybean trypsin inhibitor was observed on the displacement of CT binding by these two cysteine proteases ŽFig. 2, right panel.. However, the addition of the trypsin inhibitor induced an increase of the CT binding inhibition induced by NCP. The effect was reversed when the incubation was performed in the presence of 5 grml leupeptin and 3.5 grml E 64 .
3.2. Effect of NCP and papain on the adenylyl cyclase acti¨ ity CGRP and CT induce an increase of adenylyl cyclase activity in their respective target tissues ŽFischer and Born, 1987.. We analysed the effect of NCP and papain, two cysteine proteases, on the basal adenylyl cyclase activity in rat liver and kidney membranes . In rat liver membranes ŽFig. 3, left panel., increasing concentrations of NCP induced an increase of adenylyl cyclase activity. A 150% activation was observed with 14.4" 4.6 grml Ži.e. 60 " 19 grmg of membrane proteins.. Adenylyl cyclase responded more sensitively to papain as 150% activation above the control level was already observed with 0.66" 0.4 grml. The maximum response is observed at 4 grml. The adenylyl cyclase activity remained constant above 4 grml and a significant decrease was observed for a papain concentration of 24 grml. Control
Fig. 2. Effect of inhibitors on the specific CGRP and CT binding to rat liver Žleft panel. and kidney Žright panel. membranes. In the left panel, the CGRP binding was measured in the presence of 156 or 781 ngrml of proteins from NCP and papain, respectively, and in the absence or the presence of various inhibitors. In the right panel, the effect of various inhibitors on the CT binding was determined in the presence of 0.6 or 1 grml of NCP or papain, respectively. Results were expressed as the percentage of initial binding BrB0 where B0 represents the hormone binding in the absence of enzymes and inhibitors. Data are the mean " S.E.M. of three different experiments. U : Significant when compared with control value Žno added inhibitor. P- 0.05.
Fig. 3. Effect of increasing concentrations of NCP or papain on the basal adenylate cyclase activity in liver Žleft panel. and kidney Žright panel. membranes, respectively. Liver and kidney membranes were incubated in the presence of NCP and papain for 30 min at 30⬚C Žliver membranes. and 15 min at 37⬚C Žkidney membranes. and the synthesis of cAMP from non radioactive ATP was quantified using a commercial kit. Each point represents the mean " S.E.M. of triplicate determinations in a single experiment. Similar results were observed in three other individual experiments. U Significant when compared with the basal value. P- 0.05.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254 251
252
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Table 2 Effect of human CGRP 8-37 Ž10y6 M. on the papain stimulated adenylate cyclase activity in rat liver membranes Papain, g
0 6 13
Adenylyl cyclase activity, % stimulated No CGRP 8-37
q CGRP 8-37
100 209 " 9 257 " 11
100 163 " 13 157 " 18a
a
Significant when compared to the control Žno CGRP 8-37. value.
experiments demonstrated that this effect of papain was decreased when adenylyl cyclase was measured in the presence of the CGRP receptor antagonist, human CGRP8-37 ŽTable 2. suggesting an interaction of papain at the hormone receptor level. In rat kidney membranes ŽFig. 3, right panel. the effect of these two enzymes was quite different. NCP induced an increase of the adenylyl cyclase activity. 150% activation was observed at 5.6" 1.6 g of proteinrml Ži.e. 1.5 grmg of membrane protein.. There was a progressive increase of the control adenylyl cyclase activity: the maximum was reached with 20 g of proteinrml. Papain induced also an activation of the kidney adenylyl cyclase activity. A sharp increase was observed, followed by a progressive decrease of the activity that returned to the basal value. 150% activation was observed for a protein concentration of 0.9" 0.08 grml. In contrast to liver membranes, there was no significant difference in the maximal activity obtained with each enzyme. The effect of various inhibitors to suppress the stimulated activities was analysed. In liver and kidney membranes ŽFig. 4. only leupeptin and E 64
induced an inhibition of the adenylyl cyclase activities. No significant effect was observed with pepstatin and soybean trypsin inhibitor. A significant inhibition of papain stimulated adenylyl cyclase activity was obtained only in the presence of the two cysteine protease inhibitors; leupeptin and E 64 . To demonstrate the specificity of the cysteine protease effect on adenylyl cyclase activity, the effect of trypsin, a serine protease, was measured in the same experimental conditions ŽFig. 5.. The protease effect was related to the membrane preparation. In liver membranes, the behaviour of trypsin was different from that obtained with papain and NCP ŽFig. 3, left panel.: a progressive decrease of the basal adenylyl cyclase activity was observed, with 50% inhibition at a trypsin concentration of 5 grml. In rat kidney membranes, trypsin induced an increase of the basal adenylyl cyclase activity at concentrations of 1 and 2 grml. In this aspect, the response is similar to that observed with papain ŽFig. 3, right panel.. However, at higher concentrations Ž16 grml., we observed an inhibition of the adenylyl cyclase activity that represents a twofold decrease of the basal activity.
4. Discussion Increasing evidence indicates that some proteases, especially endopeptidases, that have been traditionally considered to participate principally in the degradation of extracellular proteins, are also signalling molecules regulating multiple cellular function by activating specific receptors ŽDery et al., 1998.. In the present study, we de-
Fig. 4. Effect of inhibitors on the enzyme stimulated adenylate cyclase activity in rat liver Žleft panel. and kidney Žright panel. membranes. Rat liver membranes were incubated in the presence of 40 or 8 grml of NCP and papain, respectively, with and without different inhibitors. Rat kidney membranes were similarly incubated in the presence of 20 or 2.7 grml of NCP and papain, respectively. Results are expressed as percent of the basal activity measured in the different experimental conditions. Data are the mean " S.E.M. of triplicate determinations in a single experiment. Similar results were obtained in two other individual experiments. U Significant when compared with the control Žno added inhibitor.. P- 0.05.
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
Fig. 5. Effect of increasing concentrations of trypsin on the adenylyl cyclase activity in rat kidney and liver membranes. Experimental conditions were as described in the legend to Fig. 3. Data are the mean " S.E.M. of triplicate determinations obtained in a single experiment. Similar results were obtained in four other individual experiments. U Significant when compared to the basal value Žno added trypsin.. P- 0.05.
monstrate that two enzymes from the papain family: papain and a cathepsin L-like enzyme isolated from the oesophagus of Nephrops nor¨ egicus were able to interact both with the CT and CGRP receptors and to activate the adenylyl cyclase activity in rat kidney and liver membranes. No significant difference was observed in the effect of these two enzymes on the displacement of the initial CGRP and CT binding to rat liver and kidney membranes. The effect observed is specific of cysteine proteases as only E 64 and leupeptin inhibited the protease-induced displacement of labelled hormones binding to their specific receptors. Control experiments demonstrated that in the presence of papain no degradation of either labelled hormone or membrane receptors occurred. Both enzymes stimulated adenylyl cyclase activity in the two membranes preparations. In contrast with previous reports ŽKoji and Terayama, 1980. showing a small effect of papain on adenylyl cyclase activity in rat liver membranes, our study demonstrates an important effect of this protease on cAMP production. Moreover, the adenylyl cyclase activity was not further modified by increasing the papain concentration from 4 to 18 grml. Similarly, NCP induces a progressive but smaller increase of the basal adenylyl cyclase activity. In contrast in rat kidney membranes, papain induced a biphasic response, similar to the re-
253
sponse obtained on lymphoma cell membranes ŽStengel et al., 1980.. The effect obtained with NCP remained constant as a function of enzyme concentration. The use of specific inhibitors demonstrated that papain and NCP effect was reversed by the same specific cysteine protease inhibitors in rat liver and kidney membranes. Trypsin, a serine protease, induced an inhibition of liver membrane adenylyl cyclase activity. The effect observed in rat kidney membranes was similar to that obtained with papain at low enzyme concentrations. In accordance with other studies performed with different cysteine proteases ŽLacombe et al., 1977; Koji and Terayama, 1980; Pinkett and Anderson, 1980; Stengel et al., 1980; Cros et al., 1987. our data demonstrate that the enzyme behaviour differs according to the enzyme and the membrane preparation. This suggests that the existence of different multiple protease activating sites on the receptor cyclase complex recognising specific proteases. Moreover, using CGRP8-37, a specific antagonist of CGRP action, we demonstrated that the effect observed on the rat liver membrane adenylyl cyclase activity was receptor mediated. Thus, cysteine proteases are able to act, in vitro, at the receptor level in target organs specific for calciotropic hormones. This effect is also observed in vivo as we previously demonstrated that crustacean cysteine proteases induced the same biological effect as CT, that is hypocalcemia and hypophosphatemia in young rats ŽArlot-Bonnemains et al., 1996.. Cysteine proteases are known as important enzymes for bone turnover and are mainly involved in the degradation of bone collagen by osteoclasts ŽEverts et al., 1992.. If these enzymes are able to interact at the membrane level and to influence the action of calciotropic hormones, they could be of high physiological importance in CT andror CGRP action at the cellular level.
Acknowledgements We are grateful to Y. Le Gal for critical comments and to Mrs C. Michel for her expert technical assistance. This study was supported by a grant in research from INSERM and Academie ´ des sciences.
254
M. Fouchereau-Peron r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 247᎐254
References Arlot-Bonnemains, Y., Fouchereau-Peron, M., Moukhtar, M.S.and, Milhaud, G., 1983. Characterization of target organs for calcitonin in lower and higher vertebrates. Comp. Biochem. Physiol. 76A, 377᎐380. Arlot-Bonnemains, Y, Milhaud, G., Fouchereau-Peron, M., 1996. A lobster cysteine protease with immunoreactivity and activities of calcitonin and CGRP. C. R. Acad. Sci. Paris 319, 975᎐982. Barrett, A.J., Kirsche, H., 1981. Cathepsin B, cathepsin H and cathepsin L. Meth. Enzymol. 80, 535᎐561. Cros, G.H., Chaptal, P.A., Serrano, J.J., 1987. The effects of trypsin, plasmin, chymotrypsin, and thrombin on human heart adenylate cyclase activity. Res. Commun. Chem. Pathol. Pharmacol. 57, 277᎐280. Cuatrecasas, P., 1971. Perturbation of the insulin receptor of isolated fat cells with proteolytic enzymes. J. Biol. Chem. 246, 6522᎐6531. Delaisse, ´ J.M., Vaes, G., 1992. Mechanisms of mineral solubilisation and matrix degradation in osteoclastic bone resorption. In Rifkin, B.R. and Gay, C.V. ŽEds. Biology and physiology of the osteoclast, pp. 289᎐314. Boca Raton: CRC Press. Dery, O., Corvera, C.U., Steinhoff, M., Bunnett, N.W., 1998. Proteinase activated receptors: novel mechanisms of signaling by serine proteases. Am. J. Physiol. 274, C1429᎐C1452. Duffy, M.J., 1992. The role of proteolytic enzymes in cancer invasion and metastasis. Clin. Exp. Metastasis 10, 145᎐155. Everts, V., Delaisse, J.M., Korper, W., Niehof, A., Vaes, G., Beertsen, W., 1992. Degradation of collagen in the bone resorbing compartment underlying the osteoclast involves both cysteine proteases and matrix metalloproteinases. J. Cell. Physiol. 150, 221᎐231. Fischer, J.A., Born, W., 1987. Calcitonin gene products: evolution, expression and biological targets. Bone Miner. 2, 347᎐359. Fitzpatrick, D.F, Davenport, G.R., Forte, L., Landon, E.J., 1969. Characterization of plasma membrane proteins in mammalian kidney. I- preparation of a membrane fraction and separation of the protein. J. Biol. Chem. 244, 3561᎐3569.
Fouchereau-Peron, M., Arlot-Bonnemains, Y., Milhaud, G., Moukhtar, M.S., 1990. Calcitonin gene related peptide stimulates adenylyl cyclase activity in trout gill cell membranes. Biochem. Biophys. Res. Commun. 172, 582᎐587. Gay, S., Gay, R.E., Koopman, W.J., 1993. Molecular and cellular mechanisms of joint destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction. Ann. Rheum. Dis. 52, S39᎐S47. Koji, T., Terayama, H., 1980. In vivo and in vitro effects of proteases upon adenylyl cyclase activity of liver cells and plasma membranes. Biochim. Biophys. Acta 633, 10᎐21. Lacombe, M.L., Stengel, D., Hanoune, J., 1977. Proteolytic activation of adenylate cyclase from rat liver plasma membranes. FEBS Lett. 77, 159᎐163. Laycock, M.V., Hirama, T., Hasnain, S., Watson, D., Storer, A.C., 1989. Purification and characterization of a digestive cysteine proteinase from the American lobster Ž Homarus americanus.. Biochem. J. 263, 439᎐444. Neville, D.M.J., 1968. Isolation of an organ specific protein antigen from cell surface membrane rat liver. Biochim. Biophys. Acta 154, 540᎐552. Pinkett, M.O., Anderson, W.B., 1980. Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase. Arch. Biochem. Biophys. 200, 261᎐268. Smith, P.K., Krohn, R.I., Hermanson, G.T. et al., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76᎐85. Stengel, D., Lad, P.M., Nielsen, T.B., Rodbell, M., Hanoune, J., 1980. Proteolysis activates adenylate cyclase activity in rat liver and AC lymphoma cell independently of the guanine nucleotide regulatory site. FEBS Lett. 115, 260᎐264. Yamaguchi, A., Chiba, T., Okimura, Y. et al., 1988a. Receptors for calcitonin gene related peptide on rat liver plasma membranes. Biochem. Biophys. Res. Commun. 152, 383᎐392. Yamaguchi, A., Chiba, T., Yamatani, T. et al., 1988b. Calcitonin gene related peptide stimulates adenylate cyclase activation via a guanine nucleotide dependent process in rat liver plasma membranes. Endocrinology 123, 2591᎐2597.