Aug 6, 1979 - Rosalia C. Mercado-Simmen,$. fj Gillian D. Bryant-Greenwood,f (1 and Frederick C. Greenwood**. From the + Department of Biochemistry and ...
J U U R N A L O F BIOLOGICAL CHEMISTRY Val. 2S5. No X, Issue of Aprd 25. pp 061i-:lfi23, 1980 Prrnted in L1.S A THF,
Characterization of the Binding of 1251-Relaxin to Rat Uterus* (Received for publication, August 6, 1979)
Rosalia C. Mercado-Simmen,$. fj Gillian D. Bryant-Greenwood,f (1 and Frederick C. Greenwood**
+
From the Department of Biochemistry a n d Biophysics, University of Hawaii, the 1Department of Anatomy a n d Reproductive Biology, University of Hawaii, a n d the * * Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822
The binding of ‘z51-relaxinto rat uteruswas charac- and growth of the mammary glands of the same animals (9). in these actionscould be acute and terized and investigated in different physiological The common denominator states. The binding activity was localized in the mem- long term effects on muscle contractility and collagen biosynbrane-enriched particulate fraction. The binding reac- thesis, respectively, mediated by relaxin acting on smooth tion was time- and temperature-dependent,reversible, muscle cells either directly through specific binding compoand of high affinity (Ka= lo9 to 1 0 ” / ~ ) .Bound 1251-nents in target tissues orindirectly by causing the release of relaxin was displaced by native relaxin but not by a other hormones. number of other polypeptide hormones at low concenMore recently, our laboratory has reported that relaxin has trations (0.2 to 10.0 PM). specific binding components in various tissues of mouse, rat, The dissociation of ‘251-relaxinfrom its binding dis- and guinea pig (10). The binding of l”1”relaxin to mouse played neither first orderkinetics nor an enhancement uterine tissue, fractionated mammarytissue from 15- and 20when incubated with 30 nM unlabeled relaxin, and suggests heterogeneity of binding sites rather than the day pregnant rats, andguinea pig pubic symphysis andcervix presence of negative cooperativity among sites. Scat- homogenates was time- and temperature-dependent, and spechard analysis of the binding data was also consistent cific for relaxin. This study is an extension of the earlier study on relaxin with two classesof binding sites. Binding was facilitated by Mn2+> Mg2+> Ca2’ at all binding sites, and is the firstdetailed characterization of the concentrations tested and was absent in buffer alone nature of labeled relaxin binding to plasma membrane-enisolated from a well known relaxin or in buffer with EDTA. At longer incubation times, riched particulate fractions A comparison of the binding Mn2+inactivated the hormone and increased the bind- target tissue, the rat uterus. ing components’ elution into themedium. These cation activity of the uterus at different physiological and experieffects in addition to membrane-enriched fraction-me- mental states of the rat isalso presented. diated hormone proteolysis were responsible for the MATERIALSAND METHODS limited steady state binding of ‘261-relaxinover a 60min time course. This section of the manuscript and partsof “Results” are presented The number of relaxin binding sites varies for differ- in the miniprint supplement at the end of the paper.’ ent physiological and experimental states of the rat, RESULTS with no concomitant change in the apparent association constant K,. Estrogen administrationenhanced the Time Course of Binding-The time course of binding of 125 uterine binding activity to relaxin. I-relaxin to membrane-enriched particulate uterine fracThe results of this study indicate that the binding tions from estrogen-primed ratsa t 27°C and 4°C is shown in components for labeled relaxin in the rat uterus may Fig. 1. Maximal binding was achieved within 5 min of incurepresentthe cellular receptors necessary for the bation a t either temperature. Incubation at 27°C resulted in expression of the biological effects of relaxin in the a 2- to %fold increase in ‘”I-hormone bound, relative to that uterus. bound at 4°C. A limited steady state wasobserved for 25 min at thetwo temperatures; on continued incubation for 30 more min, the amount of radioactivity remaining bound at 27°C Relaxin is a 6300-dalton peptide hormone considered to be decreased to 25% of the maximal value. A similar net loss of of ovarian origin and important only in pregnancy (1, 2). Its particulate-bound radioactivity was observed at 4”C, but this chemical structure has been completely characterized (3-5); occurred a t a slower rate than at the higher temperature. No however, itsexact biological function is not clear. It has binding was observed with membrane-enriched fractionsisomultiple biological effects on various tissues: inhibition of the lated from ratleg muscles. contraction of the uteri of rats and guinea pigs (2, 6, 7); Dissociation of Labeled Relaxin Binding-The dissocialengthening of the pubic symphysis of mice and guinea pigs tion of labeled relaxin from its binding to rat uterine mem(8); dilatation of the cervix of mice, guinea pigs, and rats (1); brane-enriched fractionswas studied as a function of time and temperature. ‘251-Relaxinwas permitted to bind to the mem* This work is supported by Grant H.D. 06633 from the National Institutes of Health. A portion of this work will be submitted by R. M. S.in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry at the University of Hawaii. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 East-West Center Grantee. 11 Research Career Development Awardee, H. D. 70516.
’
Portions of this paper (including “Materials and Methods,” some sections of “Results,” Figs. 2,5 to7 , 9 to 10, and Tables I1 and 111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journalof Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document 79M-1576, cite author(s), andincludea check or money order for $1.80 per set of photocopies.
3617
Uterus Ratl2'I-Relaxin to Binding
3618
brane-enriched fractionsfor 20 min at 27°C. The supernatant was drained off the tube,and 1.0 ml of the Tris-HC1buffer (50 mM, pH 7.4)was added. The particulate suspensions were then incubated at 4"C, 27"C, and 37"C, andthe loss of particulate-bound radioactivity was measured at varying time intervals. Fig. 2 shows that the dissociation was not a simple first order process at all three temperatures. At 27"C, the decrease in bound radioactivity occurred with a half-life of 15 min. The half-time dissociation at 27°C is comparable to that at 4"C, but is 2-fold higher than at37°C. Addition of Mn2+to a final concentration of 1.5 mM did not enhance the half-time dissociation at 27°C (Fig. 3). In order to determine whether negative cooperative interactions of labeled relaxin binding sites in uterine membraneenriched fractions occur, the dissociation of bound labeled hormone was followed in the presence and absence of native relaxin (Fig. 3). At 27"C, the addition of30nM unlabeled relaxin did not enhance the dissociation rate of the labeled hormone from its binding to uterine membrane-enriched fractions. The results contrast with those reportedfor insulin (18) and suggest the absence of negative cooperativity among relaxin binding sites. Specificity of Binding-The specificity of the particulateassociated binding components in the rat uterus for labeled relaxin was investigated using unlabeled relaxin and a number of other peptide hormones. Mixtures of a fixed concentration of '"I-relaxin and varying quantities of the compound tested were permitted to bind to the membrane-enriched particulate fractions for 20 min at 27°C. The results of this experiment (Fig. 4) indicatedthat theamount of unlabeled relaxin needed to inhibit 50%of the binding of the labeled relaxin to the membrane particulate fractions was 150 times less than the most potent inhibitor. Porcine proinsulin inhibited 50% of labeled relaxin binding at 80ng,while bovine proinsulin exhibited the same activity at 300 ng. Insulin, considered to have a three-dimensional organization similar to relaxin (5) was only one-twentieth as effective as its prohormone in binding to the specific relaxin-binding components. Insulinlike growth factor with a similar structure as proinsulin was 50%less potent than porcine proinsulin. C-peptide at 20 and 50 ng did not influence the binding of relaxin to theparticulate fractions. Bovine growth hormoneand prolactin exhibited 50% inhibition of labeled relaxin binding only in the microgram range. Synthetic a and p chains of relaxin have negligible inhibitory activitiescompared to the intact, unlabeled relaxin. However, when the two syntheticchains were recombined in
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INCUBATION TIME (rnin)
FIG. 1. The time course of specific binding of "%relaxin to rat uterine plasma membrane-enriched particulate fractions at 27°C (A) and 4°C (W).Each tube contained 30 PM labeled relaxin and 150 pg of particulate protein. Incubation was in 50 mM Tris-HC1, pH 7.4,1.56mM Mn", 0.1%bovine y-globulin.Percent specific binding is the ratio of specific binding, obtained as the difference between bound radioactivity in the presence and absence of unlabeled relaxin, to the total counts added per incubation tube. Nonspecific binding slowly increased with incubation time, with maximum value reaching 2 to 3%of the total radioactivity bound per tube. Eachpoint is a mean of three determinations.
* 1.5mM Mna in TRIS ~ 1 .mM 5 Mn2" 30 nM Relaxinin TRlS
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25
30
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Dissociation (min.) Time
FIG. 3. The dissociation profile of '251-relaxinfrom its binding to rat uterine plasma membrane-enriched fractions at 27 "C in 50 m~ Tris buffer, 1.5 mM Mn2+, pH7.4, in thepresence (). and absence (A)of unlabeled relaxin (final concentration = 30 m. Steady state binding conditions were as described under "Materials and Methods." Total specific binding was measured at zero dissociation time, and was 5 to 8% of total radioactivity added per tube.
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FIG. 4. Competitive inhibition of '251-relaxinbinding (30 pm) to rat uterine membrane-enriched preparations (150 to 200 pg of protein) by unlabeled relaxin and other polypeptides. Labeled relaxin and the corresponding polypeptide were incubated together in Tris buffer, 1.5 mM Mn2' at 27°C for 20 min. At the end of the incubation period, bound was separated from unbound radioactivity as described under "Materials and Methods." B/Bo is the ratio of bound labeled relaxin in the presence and absence of the unlabeled relaxin; (@- - U) combined added polypeptides: (A-A) porcine proinsulin; synthetic (Y and p chains of relaxin; (W) (o"--o) insulin-like growth factor; (0-- -0) bovine proinsulin; (W - -D) C-peptide; (M synthetic ) a chain; (0-- -0)porcine insulin; (A-- -A)synthetic p chain. Each point is the mean of at least three determinations. GH, growth hormone; PRL, prolactin.
equimolar concentrations without aperiod of incubation, and the mixture added to the binding component assay system, the resulting inhibitory activity was only 20 times less than the native, intact hormone (Fig. 4). Integrity of theLabeled Hormone in the Incubation Buffer-The net loss of particulate-bound radioactivity when the incubations were carried out at either 4°C or 27°C led us to examine the '"I-labeled relaxin remaining in the supernatants after thebinding assay. Table I shows the results of the study. The precipitability with trichloroacetic acid of the labeled hormone in the supernatantsobtained from the binding assays at 27°C did not change with incubation timesup to
3619
'251-Relaxin Binding to Rat Uterus 60 min. Incontrast,the immunoreactivity of the labeled hormone with rabbit antibody to porcine relaxin, although constant until30-min incubation time, dropped by almost 50% at 60-min incubation. There was little if any decrease in the immunoreactivity at 4°C (Table I). When the incubates at 27°C were fractionated on Sephadex G-25, the appearance of a salt peak co-eluting with Na'"'I was observed in the 30-min incubates. The peak, which possiblyrepresents low molecular weight hormonal fragments, increased in sizeupon60-min incubation (data notshown). These fragments were nonetheless precipitated by trichloroacetic acid. It should be noted that immunoprecipitability, although more sensitive than trichloroacetic acid precipitability as anindex of integrity, does not necessarily equate to receptor-binding ability. It was assumed that alteration of the hormone is dependent on the presence of membranes in the assay system. This was tested by incubating aliquots of the labeled hormone in TrisHC1 buffer (with and without 1.56 mM Mn") in the presence of membrane-enriched particulate fractions at 27°C for different incubation times up to 60 min. In the absence of membranes, there was a decrease in the immunoreactivity of labeled relaxin with time in Mn2+-containingbuffer. With Mn'+, the immunoreactivity of the hormone at 60 min fell by 25% that at zero incubation time, with no change in trichloroacetic acid precipitability. This was not observedin the metal ion-free buffer where the immunoreactivity of the hormone remained constant over the 60-min incubation. However, the loss of immunoreactivity associated with Mn" was less than thatobserved in the presence of membrane-enriched particulate fractions in the assay system, where a 55%decrease in immunoreactivity relative to that at zero incubation time was observed in binding studies run with Mn2+in the buffer. Bovine y-globulin at a concentration of 0.1 mg/tube did not inhibit relaxin alteration by the membrane-enriched fractions. The protease inhibitors phenylmethylsulfonyl fluoride, iodoacetamide, and trypsin inhibitor at concentrations of 5 mg/ml each partially inhibited the process (Table I), while at the same time diminishing the specific binding of 1251-relaxin. Stability of Membrane-enriched Particulate Fraction-associatedBinding Componentsfor Labeled Relaxin-The
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FIG. 8. Loss of membrane-enriched fraction-binding activity following preincubation of the particulate fractions at 27°C (A) and at 4 ° C (m in 50 m~ Tris buffer, pH 7.4, and at 27°C (0)in buffer containing 1.56 m~ MnZ+. Label used for binding is 1 PM, '251-relaxin.Also illustrated in the figureis the specific Iz5Irelaxin-binding activity of the supernatants from the particulate preincubations at the above-stated conditions. The soluble binding component in the supernatant representing the material released into the medium during incubation of the particulate fractions, was polyethylene glycol precipitated together with its bound labeled hormone, as described under "Materials and Methods." One hundred percent on the left vertical axis represents the specific binding of the control, unincubated membrane-enriched particulate fractions relative to those of the particulate fractions incubated at different times prior to the binding assay; the right vertical axis represents the ratio of specific binding in each tube to the total counts added per tube.
decrease in the binding of labeled relaxin to membrane-enriched particulate fractions with time could also be due to inactivation of particulate-associated binding components. Accordingly, the effect of preincubation of the membraneenriched fractions on their subsequent ability to bind labeled hormone was studied. Membrane-enriched particulate fractions were incubated at 27°C or 4°C for up to 60 min in TrisHC1 buffer, pH 7.4. The particulate fractions at each incubation time were pelleted and were allowedto bind the iodinated relaxin for 5 min at 27°C. Particulate-bound radioactivity for each preincubation time interval was measured as the percentage bound relative to zero time membrane-enriched particulate fraction incubation. As shown in Fig. 8, the particulate fraction's ability to bind the labeled relaxin decreased with increasing preincubation of membrane-enriched fractions TABLEI prior to thebinding assay. At 4"C, the decrease was lessrapid Per cent immunoprecipitability and percent trichloroacetic acid than that of 27°C. precipitability of labeled relaxin in supernatants during binding In addition, the soluble binding activity in the medium from assays Aliquots from supernatants were incubated overnight at 4°C with the membrane-enriched particulate fraction preincubations 100 pJ of rabbit antiserum to porcine relaxin. After 24 h, goat anti- was measured to determine whether the loss of particulate rabbit y-globulin (1:8 dilution inbarbitonebuffer)and NRS (1:20 binding activity is due to the elution of binding component dilution in barbitone buffer, pH 8.6) were added, mixed gently, and into the medium. At 4°Cand 27"C, the decrease in particulateincubated overnight. The precipitate was centrifuged at 2000 X g for bound radioactivity was accompanied by an increase in soluble 20 min and counted. Trichloroacetic acid precipitates of the superbinding activity as measured by polyethylene glycol precipinatants were obtained with 10%trichloroacetic acid tation of the complex formed between labeled hormone and ImmunoTrichloroacetic acid ImmunoImmunoreactivity,b its solubilizedbinding component. The amount of soluble Incubation time Precipitabil- reactivity? reactivity,* 27"C, probinding activity at 4°Cwaslower thanthatat 27"C, in tease inhibi4OC ity," 27°C 27"c agreement with the observed slower decrease in particulatetors bound radioactivity at thelower temperature. The increase in min % % % % soluble binding activity at 27°C within 30-min incubation and 1. 0 32.2 77.0 32.1 32.2 correspondingly at 4 ° C within 60 min, coincided with the 2. 31.6 2 77.4 32.1 32.5 3. 5 77.0 28.9 34.7 30.5 decreasing particulate-associated binding activity observed at 76.6 29.0 4.10 33.9 these time periods at thetwo temperatures (Fig. 8). 5. 3027.5 76.3 29.8 33.3 Preincubation of membrane-enriched particulate fractions 6. 45 76.7 13.8 32.9 in buffer containing Mn2+ (1.56mM) at 27°C enhanced the 7. 60 76.1 14.9 30.8 28.0 decrease in the binding capacity of the particulate fractions a Calculated as the ratio of counts in pellet (after precipitation of supernatant to a final trichloroacetic acid concentration of 10%) to by 20% relative to preincubation in Tris-HC1 alone (Fig. 8). Whether the enhancing effect of MnZ+was due to anincrease the total counts in supernatants. Calculated as the ratio of bound labeled porcine relaxin to rabbit in the elution of the binding component into the medium anti-porcine relaxin antibody, to the total counts in supernatants. cannot be determined. The amount of soluble binding activity
3620
'251-Relanin Binding to Rat Uterus
TABLEIV Binding affinity andnumber of high affinity binding sites in the uterine plasma membrane-enriched fraction duringvarious physiological and experimental states of the rat
inhibition of spontaneous contraction, and perhaps chronically to changesin collagen metabolism. The binding of 1 ng/ml of relaxin, which is similar in magnitude (4 ng/ml), observed for detectable relaxationof rat uterus contractions spontaneously High affinity (KO)x in vitro is readily detectable. Thedegree of ligand specificity Experimental state binding sites 10"" was indicated by theinhibitory effects on labeledrelaxin pmol/mg proM" binding of a variety of polypeptide hormones related in evotern lutionary terms (5);these inhibited only at high concentra1. Cyclic, non-estrogen-primed tions. Proinsulin > insulin-likegrowth factor > insulin in2. Cyclic, estrogen-primed 194 f 29' 3.9 3. Ovariectomized hibited over a concentration range 100- to 1000-fold greater 4. Ovariectomized, estrogen-primed 115 -t 4ab 7.7 than either unlabeled, native relaxin or the combined syn5. Pregnancy states thetic a and ,l3 chains (equimolar concentrations). Thus, the 7' 1.2 a.5-daypregnant uterine-binding component clearly distinguishes between re12 f 5' 0.78 b.IO-day pregnant laxin and other hormones related or unrelated to it. and is 151 f 16' 1.39 c.17-day pregnant most likely a component of the relaxin receptor system. 31 f 10' 2.47 d.19-day pregnant 16' 4.8 More studies are needed to identify other receptors for e.20-day pregnant 4? 10.4 f. I-day post-parturn growth factors in the uterus and their relationship with putative relaxin receptors since individualresults from the ligand " No observable specific binding. Mean of at least three determinations. specificity studies were unexpected. The inhibitory activity on One determination. labeled relaxin binding of proinsulin and insulin-like growth factor was greater than insulin, despite the higher degree of decreased with incubation time until,at periods greater than similarity in the three-dimensionalorganization of insulin and 30 min, the amount of binding activity was much less than relaxin (5). The effect of proinsulin, however, appears to be that in the preincubation medium containing no cation(Fig. specific on the basis of two observations. First, insulin-like 8). growth factor whose biological effects resemble insulin (23) Number of Relaxin High Affinity Binding Sites andAs- but whose proposed structure has been described as "mini" sociation Constants K, Under Different Physiological a n d proinsulin (24), has an inhibitory effect comparable to the Experimental States of the Rat-The concentration as well prohormone. In addition, proinsulin but not insulin inhibited as theassociation constants of the high affinity binding com- the labeledrelaxin-anti-relaxin immunoassay (25), and has ponents in the uteruswere investigated in rats underdifferent more effect on the relaxation of rat uterine muscle in uitro.2 physiological and experimental states. The results are shown The effect of Mn2+ on thebinding of '"1-relaxin to uterine in TableIV. No specific binding activity was exhibited by the binding components appears tobe a specific phenomenon. A uterus in the absenceof estrogen priming or afterovariectomy. similar effect has been observed in a different system where Binding was, however, generated upon estrogen administra- high concentrations of Mn2+ (2 to8 mM) had inhibitory effect tion to the cyclic and ovariectomized rats. The number of on the binding of ~H]hydroxybenzylproterenolto P-adrenerhigh affinitysites was low at the early stages of pregnancy but gic receptor (26). The similarities in the activity profiles at began to rise in midpregnancy, and reached a peak at 17-day early incubation period ofMn2',Mg'+, and Ca2+ a t varying pregnancy. At later stages, there was a marked decrease in concentrations, plus the finding that Mg' and Caz+ can mimic the binding capacity which continued until post partum.The the manganese effect but only to a limited extent, discount affinity constantsKOfor the hormonewere very similarunder the effects of the ions on the basis of ionic strengths alone. In the different physiological states examined. addition, Mg'+ has no effect, on the binding profile of Mn2+ when the 2 ions are added in equal concentrations. The role DISCUSSION of Mn2+ is not clear at present; however, its effect may be In this study, we have examined the properties of labeled relatedto "inducing conformationalchanges in thememrelaxin binding to rat uterine membrane-enriched particulate branes of the target cells" (27),or increasingaffinity and fractions, using known properties of other receptor systems asconcentration of relaxin binding sites (28), or both. In solubicriteria for specific hormone-receptor interactions (17,20,21). lized membrane-enriched particulate preparations, Mn" reWe have used iodinated relaxin to measure the binding of ducedthe binding of the labeled hormonetoits binding the hormone t,o uterine particulate fractions. Iodination does components; the solubilized preparations were more responnot alter relaxin's binding affinity for, or its capacity to bind sive in binding labeled relaxin in the absence of the cation.' to,theuterineplasmamembrane-enriched fractions. The In this study,we have shown that thelimited steady state number of highaffinitybinding sitesandthe association exhibited by labeled relaxin to rat uterineplasma membraneconstant of theparticulatefractions for the iodinated enriched fractionsis a consequence of three specific processes, hormone agreed with those values obtained for the unlabeled namely proteolysis of labeled relaxin mediated by the memrelaxin. Hence, it is reasonable to ascribe the properties de- brane-enriched fraction,Mn2'-induced inactivation of the horscribed for the interactions between iodinated relaxin and its mone, and the solubilization of the binding component into membrane-enriched fraction-associatedbinding component to the medium. The relaxin-degrading activity associated with thosebetweenthese binding componentsandthenative, the membrane-enriched particulate fraction could be a conunlabeled hormone. sequence of cell disruption in the process of particulate isolaOur findings show that "'I-relaxin was bound specifically tion or of the presence of proteolytic enzymes either related to rat uterine particulatefractions. The specificity is 2-fold in or unrelated to the binding components.The internalevidence that the observedbinding activity wastissue- and ligand- suggests more likely that the proteases associated with the specific. Plasmamembrane-enrichedparticulatefractions particuIat,e fractions are contaminants or are integral parts of fromrat leg muscles, a non-relaxin target tissue, did not the membranes, and unrelated to the binding components. exhibit any specific binding; this contrasts markedly with the This distinction between the ability of the membrane-enhighly specific binding found in the uterus, a tissue which Unpublished observations from our laboratory. respondsacutelyto relaxin in uiuo andin vitro (22) by
‘251-Relaxin Binding
to Rat Uterus
3621
The involvementof estrogen in enhancing thesensitivity of riched fraction to degrade and to bind is also seen in the known release of radioactive material from binding which at longer the targettissue to relaxin in vivo is consistent with the dissociation times is relatively more intact. Degradation of effect of estrogen in increasingthe sensitivity of the rat uterus relaxin appears to occur via a limited proteolysis step since in vitro to relaxin and with changes in relaxin receptor conthe radioactivity peak eluting with Na’*’I and observable at centrations after estrogen treatment.However, a comparison longer incubation times is only a small fraction of the total of results between the nonpregnant and pregnant uteri suggests that relaxin binding sites and tissue sensitivity are not protein peak (data not shown). The decrease in relaxin immunoreactivity in aqueoussolu- related solely to circulating estrogen levels. The results of the present investigation indicate that the tions independentof the presence of membranes suggests the involvement ofMn’+ in relaxin inactivation. A number of relaxin-binding components found in the plasma membraneof the rat uterus may be related polypeptide hormones hasbeen reported to be susceptible to enriched particulate fractions ion-induced aggregation (29). More recently, ithas been to specific relaxin receptors initiating the inhibition of the shown thatZn”, Mn2+, orCa2’ at 1mM can precipitaterelaxin uterine contractile response to relaxin observed in vivo or in at a concentration of 10 mg/ml, possibly through cross-linkage vitro. Although no direct correlation has been attempted at specific relaxin binding with anybiological of the dicarboxylic groups of 2 relaxin molecules.:’ Chelation this stage to relate of Mn2+ toglutamic acid or aspartic acid residues of relaxin response, the indirect evidence accumulated in this study is investigations. could also result in the alteration of the hormone’s three- an encouragement to further dimensional organization. In contrast, the negative effect of Acknowledgments-We thank Dr. Kevin Catt for his critical review Mn2+ on relaxin immunoreactivity in the presence of memof the manuscript, Dr. Walter Morishige for stimulating discussions brane particulate fractions may be related toa Mn”-activated and advice, Dr. Wayne Chamley for hishelp at the start of this protease witha substrate specificity for relaxin. Further stud- project, and Frank Simmen for helpful discussions. ies will be needed toclarify the mechanism of Mn2+-induced relaxin inactivation. On the other hand, the elution of memREFERENCES brane-associated binding components into the medium,even 1. Hisaw, F. L. (1926) Proc. Soc. Exp. Biol. Med. 23,661-663 in the absence of any ion, is not an uncommon membrane 2. Hall, K. (1960) J . Reprod. Fertil. 1, 368-384 event (30). T h e result suggests that partsof the relaxin recep3. Schwabe, C., McDonald, J . K., and Steinetz, B. G. (1976) Biochem. Biophys. Res. Cornmun. 70,397-405 tor systeminvolved in the recognition of and interaction with 4. Schwabe, C., McDonald, J. K., and Steinetz, B. G. (1977) Biothe hormone, areloosely attached to theplasma membranes chem. Biophys. Res. Cornmun. 75, 503-510 instead of embedded in the lipoprotein bilayer. 5. James, R.,Niall, H., Kwok, S., and Bryant-Greenwood, G. D . T h e results of our dissociation studies and Scatchard anal(1977) Nature 267,544-546 ysis are both consistent with two classes of binding sites. In 6. Porter, D. G. (1971) J. Reprod. Fertil. 26,251 the dissociation studies, thepresence of an initial rapid phase 7. Sawyer, W. H., Frieden, E. H., and Martin, A. S. (1953) Am. J . Physiol. 172,547 followed byaslowerdissociating componentseemsto be 8. Steinetz, B. G., Beach, V. L., Kroc, R. I., Stasilli, N. R., Mussbaum, common to many peptide hormones and may also represent R. E., Nemith, R. J., and Dunn, R. K. (1960) Endocrinology increasing “tightness” of binding with time. The curvilinear 67, 102 Scatchard plot obtained using plasmamembrane-enriched 9. Harness, J. R., and Anderson, R. R. (1975) Proc. SOC.Exp. Bid. fractions from rats a t all physiological and experimental states Med. 148,933 examined, suggests thespecificity to uterine fractions of two 10. McMurtry, J., Kwok, S., and Bryant-Greenwood, G. D. (1978) J . classes of binding sites. Moreover, the finding discounts the Reprod. Fertil. 53,209-216 11. Shenvood, 0. D., and O’Byrne, E. M. (1974) Arch.Biochem. low affinity binding sites as mere “damaged” high affinity Biophys. 160, 185-196 binding components. The data in Table IV indicate that the number but not the12. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J . 133, 529539 affinity of the binding components for relaxin is affected by 13. Kwok, S. C. M., McMurtry, J.P., and Bryant, G. D.(1975) the physiological and experimental state of the animal. This Proceedings of the International Symposium on Growth Horresult is consistent with those reported for several polypeptide mone and Related Peptides, Milan, 414-421, pp. Excepta Medica, Amsterdam hormones at estrous and different pregnancy states (31, 32). A relationship between the number of relaxin binding sites 14. ODea, K.,and Meyer, P. (1975) Eur.J . Pharmacol. 30,260-267 and serum levels of the hormone was evident during preg- 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J . (1951) J . Biol. Chem. 193,265-275 nancy. At early and midterm, the number of high affinity 16. Song, C. S., and Bodansky, 0. (1967) J . Biol. Chem. 242,694-699 relaxin sites increases in parallel to the increase in relaxin 17. Cuatrecasas, P. (1972) Proc. Natl. Acad. Sci. U. S. A . 69,318-322 levels measured in the serum and ovarian extracts from the 18. DeMeyts, P., Roth, J., Neville, D. M., Jr., Gavin, J . R., 111, and Lesniak, M. A. (1965) Biochem. Biophys. Res. Commun. 55(1), rat (33, 34). At late term, the “surge” of relaxin shown by 154-161 these authors is consistent in time with the decrease in the 19. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-673 number of binding sites shown here. Thisloss in the binding M. S., and Swartz, T. L. (1973) J. B i d . Chem. 248(18), components may be real or apparent, an actual reductionin 20. Soloff, 6471-6478 number or an increase in receptor occupancy due toincreased 21. Carpenter, G., Lembach, K. J . , Morrison, M. M., and Cohen, S. relaxin secretion. At present, we could not distinguish between (1975) J . B i d . Chem. 250(11),4297-4304 the two; however, preliminary experiments where the number 22. Vinquist, N . (1959) Acta Endocrinol. Suppl. 46, 15-31 of relaxin binding sites was determined asa function of time 23. Froesh, E. R., Zapf, J., Menli, C., Mader, M., Waldvogel, M., Kaufmann, U., and Morrel, B. (1975) Adu. Metab. Disord. 8, after injection in vivo of physiological doses (10”’ M) of 211-235 unlabeled relaxin’ point to an actual reduction in the number 24. Blundell, T. L., Bedarkar, S., Rinderknecht, E., and Humbel, R. of binding sites as responsible for the loss of binding activity E. (1978) Proc. Natl. Acad. Sci.U. S. A . 75(1), 180-184 a t or near term. Studies from others indicate that hormones 25. Bryant, G. D., and Stelmasiak, T. (1975) Endocrinol. Res.Cornmun. 1,415-433 regulate their own receptor number (32); this may be true for 26. Williams, L. T., MuUikin, D., and Lefkowitz, R. K. (1978) J . B i d . relaxin as well.
’ N. Isaacs, personal communications.
Chem. 253(1), 2984-2989 27. Bentley, P. J. (1965) J. Endocrinol. 32, 215-219
3622
'251-Relaxin Binding to Rat Uterus
28. Pearlmutter, A. F., and Soloff, M. S. (1979) J. Biol. Chem. 254(10), 3899-3906 29. Wahlborg, A., and Frieden, E. (1965) Arch. Biochem. Biophys. 111, 702-712 30. Gavin, J. R., 111, Buell, D. N., andRoth, J . (1972) Science 178, 168-169 31. Feherty, P., Robertson, D. M., Waynforth, H. B., and Kellie, A.
E. (1970) Biochem. J. 120,837-844 32. Catt, K. J., and Dufau, M. L. (1977) Annu. Reu. Physiol. 39,529557 33. Anderson, L. L., Bast, J. P., and Melamby, R. M. (1975) J. Endocrinology 59, 371-376 34. Sherwood, 0.D., and Crenekovic, V. E. (1979) Endocrinology 104(4), 893-897
1
Ion Concentrotlon In mM
'251-Reluxin Binding to Rut Uterus
3623
g :j
60
8 10
"0
L m
w
30 20
5
10
15
20
25 30 35 40 45 50 55 60
INCUBATION TIME I man1
2
4
6 B I0 12 1 4 1 FRACTION NUMBER
6
88 20
