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Binding of 3H-melatonin to calmodulin
Life Sciences, Vol. 53, pp. 201-207 Printed in the USA
Pergamon Press
B I N D I N G OF 3H-MELATONIN TO C A L M O D U L I N G. Benltez-King, L. Huerto-Delgadillo, and F. Ant6n-Tay* Instituto M e x i c a n o de Psiquiatria, D e p a r t a m e n t o de Neurofarmacologla, DIC. * U n i v e r s i d a d Aut6noma M e t r o p o l i t a n a - I z t a p a l a p a , D e p a r t a m e n t o de Biologla de la Reproducci6n, DCBS.
(Received in final form April 27, 1993) Summary Studies in m e l a t o n i n m e c h a n i s m of action have s u g g e s t e d that one of them could be the binding of the h o r m o n e to calmodulin. We assessed c a l m o d u l i n - m e l a t o n i n b i n d i n g by c o m b i n i n g liposome incorporation of c a l m o d u l i n with sepa r a t i o n of free and bound 3H-Melatonin by a rapid u l t r a f i l t r a t i o n method. Specific binding to c a l m o d u l i n was saturable, reversible, Ca++-dependent, ligand selective, and showed high affinity. Saturation as well as a s s o c i a t i o n - d i s s o c i a t i o n studies revealed that 3H-Melatonin b i n d s to a single site on the calmodulin m o l e c u l e with a Kd of 188 pM and a total binding capacity Bmax of 35 p M / u g of calmodulin. D i s p l a c e m e n t experiments showed that the r e l a t i v e order of potency of some compounds for i n h i b i t i o n of 3 H - M e l a t o n i n was as follows: M e l a t o n i n > 6 - c h l o r o m e l a t o n i n > 6 - h y d r o x y m e l a t o n i n > luzindole > trifluoperazine. The r e s u l t s explain our p r e v i o u s l y r e p o r t e d m e l a t o n i n e f f e c t s such as cytoskeletal rearrangements, i n h i b i t i o n of c a l m o d u l i n d e p e n d e n t p h o s p h o d i e s t e r a s e a c t i v i t y as well as the m o d i f i c a t i o n of Ca++-calmodulin e l e c t r o p h o r e t i c mobility. The high affinity of m e l a t o n i n b i n d i n g to c a l m o d u l i n suggests that the hormone is able to m o d u l a t e cell a c t i v i t y by intracellularly binding to c a l m o d u l i n at p h y s i o l o g i c a l l y ranges. M e l a t o n i n - c a l m o d u l i n b i n d i n g could m o d u l a t e m a n y intracellular Ca ++ functions and thus, the s e t - p o i n t for cell activity will follow the r h y t h m i c c i r c u l a t i n g levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well p r e s e r v e d compounds, their interaction may r e p r e s e n t a primary mechanism for both the r e g u l a t i o n and the s y n c h r o n i z a t i o n of cell physiology. Melatonin, c o n s i d e r e d to be the main endocrine signal from the pineal gland, appears to synchronize many p h y s i o l o g i c a l r h y t h m s to the p h o t o p e r i o d (i). It has been reported that m e l a t o n i n (MEL) *Correspondence: G. Benltez-King, Instituto M e x i c a n o de Psiquiatrla, D e p a r t a m e n t o de Neurofarmacolog[a, Calzada M ~ x i c o - X o c h i m i l c o No 101, Colonia San L o r e n z o Huipulco, CP 14370 M~xico D.F. M~xico. ~-3~5/93 ~.~ +.~ Cop~ight©1993PergamonPressLtd Allrightsrese~ed.
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t a r g e t cells have specific plasma membrane receptors t h r o u g h which the h o r m o n e acts. For review see (2). However, there is evidence that 3H-MEL binds to m i t o c h o n d r i a l and nuclear membranes, as well as to c y t o s o l i c p r o t e i n s (3-6), and thus may m o d i f y cell p h y s i o l o g y t h r o u g h other mechanisms. Recently we have suggested that such a m e c h a n i s m could be the binding of MEL to c a l m o d u l i n (CAM), and we r e p o r t e d that both CaM cell concentration (7) and c o m p a r t m e n t a l i z a tion (8) in M D C K and NIE-II5 cells were m o d i f i e d by 10 -9 M MEL. Moreover, M E L inhibited CaM-dependent~ p h o s p h o d i e s t e r a s e a c t i v i t y i n vitro (7), and when either MEL or ~H-MEL was p r e i n c u b a t e d with CaM and s e p a r a t e d by gel electrophoresis, c o m i g r a t i o n of CaM with the r a d i o a c t i v i t y as well as m o d i f i c a t i o n of the Ca++-CaM relative m o b i l i t y were observed (7). Therefore, it seemed w o r t h w h i l e to study C a M - M E L b i n d i n g directly. C a l m o d u l i n is a Ca ++ binding protein that m e d i a t e s the main i n t r a c e l l u l a r r e g u l a t o r y effects of Ca ++ (9). Binding of C a ++ ions to CaM exposes specific h y d r o p h o b i c domains that p a r t i c i p a t e in t a r g e t p r o t e i n interactions or in the binding of specific m o l e c u l e s (i0). Even though the most common method for a s s e s s i n g the k i n e t i c c o n s t a n t s of ligand binding to CaM is t h r o u g h e q u i l i b r i u m d i a l y s i s (ii), these constants are difficult to obtain due to the very low s i g n a l / n o i s e ratio of the method. R e c e n t l y we a s s e s s e d the CaM-MEL binding by combining liposome i n c o r p o r a t i o n of CaM with s e p a r a t i o n of free and bound 3H-MEL by a rapid u l t r a f i l t r a t i o n method (12). We now report the stereoselective, high affinity, saturable, reversible, Ca÷+-dependent 3H-MEL binding to CaM. Material
and Methods
C a l m o d u l i n was obtained from frozen sheep brains and p u r i f i e d by h y d r o p h o b i c c h r o m a t o g r a p h y according to G o p a l a k r i s h n a and A n d e r s o n (13). It was d i a l y z e d three times during 24 hrs each, against 4 liters of 50 mM tris pH 7.6 buffer, using a seamless c e l l u l o s e tubing with a 12000 d. molecular weight cut-off. D i a l y z e d CaM was lyophilized and resuspended in the same buffer to desired c o n c e n t r a t i o n s . E l e c t r o p h o r e t i c mobility and b i o l o g i c a l activity w e r e c h e c k e d against commercial CaM (14). 3H-MEL (83.3 Ci/mMole) and 125I-CaM (87.1 uCi/mg) were from NEN-Dupont. N i t r o c e l l u l o s e filters w e r e from Amersham. CaM and other reagents were from Sigma. L u z i n d o l e was a generous gift of Dr. M.L. Dubocovich. B i n d i n g Assay: CaM was incorporated into liposomes by s o n i c a t i o n with p h o s p h a t i d y l choline in a 1:30 ratio during 40 m i n u t e s at room temperature (15). Binding assays were r o u t i n e l y p e r f o r m e d in t r i p l i c a t e in a total volume of i00 ul. Unless o t h e r w i s e specified, CaM (i ug) and .140 pM of 3H-MEL were incubated in 50 mM tris-HCl, 1 mM CaCI 2 buffer during 1 hour at 2 ° . MEL and r e l a t e d compounds were d i s s o l v e d in 20 mM tris pH 7.6, 20% glycerol; final glycerol c o n c e n t r a t i o n was 2 %. For some experiments ~H-MEL was d i l u t e d with cold M E L to 1 Ci/mMole. After the incubation period, bound and free 3H-MEL were s e p a r a t e d by rapid vacuum filtration (total time of 5 sec) t h r o u g h a 0.45 uM pore n i t r o c e l l u l o s e filter p r e s o a k e d in a 10% p o l y e t h y l e n e i m i n e , 50 mM tris-HCl, pH 7.6 buffer (12). Filters were w a s h e d 4 times with 4 ml of cold assay buffer, dried, and their r a d i o a c t i v i t y counted. N o n - s p e c i f i c binding was m e a s u r e d with 10 -4 M melatonin. Efficiency of the filtration system with respect to CaM e n t r a p m e n t was assessed using 125I-CaM d i l u t e d with CaM to the final c o n c e n t r a t i o n employed in the assays.
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Statistical Analysis.Data from saturation experiments were c a l c u l a t e d b y a E n z f i t t e r program. D a t a f r o m a s s o c i a t i o n e x p e r i m e n t s were treated as p s e u d o first order reactions and dissociation experiments, as first order reactions. Both were calculated with the Sigma plot program. Ki v a l u e s w e r e c a l c u l a t e d according to C h e u n g a n d P r u s o f f (16) f r o m d e t e r m i n e d It50 values. Results
E s t i m a t e s of 125I-CaM e n t r a p m e n t w e r e in t h e r a n g e of 90 to 96 % a n d w e r e u s e d to c o r r e c t for s y s t e m e f f i c i e n c y . ~H-MEL binding to l i p o s o m e s a n d to nitrocellulose f i l t e r s in a b s e n c e of C a M w a s s i m i l a r to n o n - s p e c i f i c binding. In a t y p i c a l b i n d i n g a s s a y 60% of t o t a l 3 H - M E L b i n d i n g w a s specific. T h e b i n d i n g of 3 H - M E L to C a M w a s t e m p e r a t u r e i n d e p e n d e n t , w i t h an o p t i m a l p H of 7.6 (Data n o t shown). S p e c i f i c b i n d i n g w a s l i n e a r w i t h C a M c o n c e n t r a t i o n up to 2 u g (Fig i).
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Specific 3HMelatonin b i n d i n g to calmodulin as a function of p r o t e i n concentration. 3H-MEL was incubated with CaM at different p r o t e i n concentrations as described. Nonspecific bindi%%_~as assessed using M melatonin. Data points represent the mean + SEM of two experiments done in triplicate.
T h e e f f e c t of Ca ++ c o n c e n t r a t i o n s on s p e c i f i c 3 H - M E L b i n d i n g w a s tested. As shown in Fig 2., 3H-MEL b i n d i n g dependence on C a M activation by Ca ++ w a s o b s e r v e d . Ca ++ c h e l a t i o n w i t h E G T A (20 mM) prevented specific b i n d i n g of 3H-MEL to CaM. As e x p e c t e d , nons p e c i f i c b i n d i n g of 3 H - M E L w a s n o t a l t e r e d by e i t h e r Ca ++ or EGTA. D u e to M E L h y d r o p h o b i c structure it c o u l d be e x p e c t e d t h a t the hormone would bind non-specifically to d i v e r s e p r o t e i n s ; thus we s t u d i e d Ca ++ d e p e n d e n c e of 3H-MEL b i n d i n g to 1 u g e a c h of s e r u m b o v i n e a l b u m i n , e g g albumin, t u b u l i n a n d c r e a t i n e p h o s p h o k i n a s e a n d no m o d i f i c a t i o n s in t h e a m o u n t of 3H-MEL b o u n d w a s o b s e r v e d w i t h or w i t h o u t 1 m M of Ca ++ . Saturation experiments showed that CaM-3H-MEL binding exhibited a s a t u r a b l e s p e c i f i c c o m p o n e n t and a l i n e a r n o n - s p e c i f i c c o m p o n e n t (Fig 3A). S c a t c h a r d isotherm plots (17) w e r e l i n e a r (Fig 3B). Binding parameters from the Scatchard plot w e r e Kd = 175 p M a n d B m a x = 40 p M / u g CaM. A n a v e r a g e of e i g h t e x p e r i m e n t s y i e l d e d a Kd = 188 ± 30 p M a n d B m a x = 35 ± 4 p M / u g CaM. Calculated Hill c o e f f i c i e n t w a s of 0.94 + 0.014. Time course studies confirmed reversibility of b i n d i n g . We
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e m p l o y e d a 1 hr incubation time, since e q u i l i b r i u m was a c h i e v e d well prior to this p e r i o d (Fig 4). Analysis of the a s s o c i a t i o n d i s s o c i a t i o n data from three experiments yielded an e s t i m a t e of the a s s o c i a t i o n rate constant (KI) of 4.5 + 1 x i015 M -I min -I and the d i s s o c i a t i o n rate constant (K_l) of --0.387 ± .069 min -I The e q u i l i b r i u m d i s s o c i a t i o n rate constant (Kd) e s t i m a t e d under none q u i l i b r i u m c o n d i t i o n s from the ratio K_I/K 1 was 102 ± i0 pM. From the d i s s o c i a t i o n constant calculated above a TI/2 of 2.02 ± 0.17 min was obtained.
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Calcium dependence of 3H-Melatonin b i n d i n g to calmodulin. CaM was incubated w i t h 3H-MEL as d e s c r i b e d with increasing concen trations of CaCI 2 in the absence ( O ) and p r e s e n c e (O) of 20 mM of EGTA. N o n - s p e c i f i c binding was assessed using 10 -4 M MEL. Data points r e p r e s e n t the mean + SEM of four experiments performed in triplicate.
D i s p l a c e m e n t studies revealed that the binding of 3H-MEL to CaM showed ligand selectivity. Besides the MEL analogs, we also tested a well k n o w n CaM antagonist, trifluoperazine (ii), and a MEL antagonist, luzindole (18). The Ki values for MEL, 6 - c h l o r o m e l a tonin, 6-hydroxymelatonin, luzindole and t r i f l u o p e r a z i n e were 0.193 0.050, 2.3 ± i.i, 7.1 + 2, 2743 ± 415 and > 5000 nM, respectively. I n c o n s i s t e n t results were obtained with: serotonin, Na c e t y l s e r o t o n i n and N - a c e t y l t r i p t a m i n e possible b e c a u s e they are quite h y d r o p h i l i c compounds. Discussion
A m o n g other p r o t e i n s examined, only CaM p o s s e s s e d high affinity ++ ++ Ca - d e p e n d e n t binding sites for MEL. Upon CaM a c t i v a t i o n by Ca , 3H-MEL b i n d i n g to the protein fulfills the major c r i t e r i a for considering this protein as a receptor: it was saturable, reversible, p r o t e i n dependent and showed ligand selectivity. 3H-MEL w o u l d appear to bind to a single class of sites, since we o b s e r v e d a linear S c a t c h a r d plot and a 0.94 ± 0.014 Hill coefficient. There is a d i r e c t r e l a t i o n s h i p between h y d r o p h o b i c i t y and both binding affinity and activity of w e l l - k n o w n CaM a n t a g o n i s t s such as phenothiazines and n a p h t h a l e n s u l f o n a m i d e s (9,11). Thus, it is p r o b a b l e that the MEL 5-methoxy group, which makes the h o r m o n e very hydrophobic, may interact with an exposed h y d r o p h o b i c b i n d i n g site of CaM. Also, a direct relationship between phenothiazines, CaM affinity, and p o t e n c y to inhibit CaM d e p e n d e n t enzymes has been r e p o r t e d (9,11). There is a similar r e l a t i o n s h i p for MEL binding
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(Kd = 188 pM) and its inhibitory effect on CaM-dependent p h o s p h o d i e s t e r a s e activity (IC50 = 1 nM) as r e p o r t e d (7). Therefore, we p r o p o s e the binding of MEL to CaM as the m e c h a n i s m t h r o u g h
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S a t u r a t i o n of 3H-Melatonin binding to calmodulin. A) CaM (i u~) was incubated with increasing c o n c e n t r a t i o n s of H-MEL as d e s c r i b e d in methods. 3H-MEL, bound in the p r e s e n c e of 10 -4 M MEL (non-specific binding) ( ), s u b t r a c t e d from total binding (0) in order to determine specific binding (O)B) Specific binding r e p l o t t e d by the Scatchard method. Data points r e p r e s e n t the m e a n of t r i p l i c a t e d e t e r m i n a t i o n s from a single experiment. w h i c h this h o r m o n e inhibits p h o s p h o d i e s t e r a s e activation. Also, the data p r e s e n t e d here suggest that p r e v i o u s l y r e p o r t e d c y t o s k e l e t a l r e a r r a n g e m e n t s (19) as well as the m o d i f i c a t i o n of CaM Ca ++ shift by MEL (7) are due to MEL binding to CaM. M o r e o v e r it is possible, that some of the r e p o r t e d nuclear, m i t o c h o n d r i a l and c y t o s o l i c 3H-MEL b i n d i n g (3-6) could be due to the p r e s e n c e of CaM at these sites. The p i c o m o l a r affinity of MEL binding to CaM w o u l d make it possible for the hormone to modulate cell activity by intracellularly binding to the protein at physiological c o n c e n t r a t i o n ranges. By contrast, the known CaM antagonists, be they p e p t i d e s such as melittin, mastoparans, dynorphin, endorphin, or drugs, such as trifluoperazine, W7 or calmidazolium, bind to CaM in the m i c r o m o l a r range (20-22). Due to its high lipophilicity, MEL may have access to many cell types and to i n t r a c e l l u l a r CaM as
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well. However, MEL cell responses, will depend, not only on the a c c e s s i b i l i t y of the hormone to the cell, but also on the t o p o l o g y of CaM, the t a r g e t enzymes and substrates, local Ca 2+ t r a n s i e n t s and the g e n e t i c a l l y r e g u l a t e d CaM compartmentalization. Thus, both cellular specificity and specific cellular responses for MEL must be considered. MEL
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Fig. 4 A s s o c i a t i o n and d i s s o c i a t i o n of 3H-Melatonin b i n d i n g to calmodulin. CaM (i ug) was incubated with 140 pM of 3H-MEL, and the amount of total 3H-MEL b i n d i n g was d e t e r m i n e d as a function of incubation time. At 60 min of incubation, 10 -4 M of MEL was added to the i n c u b a t i o n mixture, and the decline in total binding with time was determined. Non-specific binding was estimated by p r o c e s s i n g identical incubates in the p r e s e n c e of 10 -4 M MEL. Data points represent the mean of triplicate determinations from a single experiment. Two further e x p e r i m e n t s y i e l d e d similar results. CaM is an ubiquitous Ca ++ binding protein that relays i n t r a c e l l u l a r Ca ++ signal to many important p h y s i o l o g i c a l enzymes (9). Since M E L antagonizes Ca ++ activated CaM, MEL target cell a c t i v i t y will follow the rhythmic circulating levels of the pineal h o r m o n e and thus, MEL should be considered as a r h e o s t a t i c h o r m o n e (23) . CaM and MEL are p h y l o g e n e t i c a l l y well p r e s e r v e d structures that are p r e s e n t even in primitive organisms (24,25), and since their i n t e r a c t i o n could modulate many intracellular C a + + functions, it is t e m p t i n g to p o s t u l a t e that this interaction r e p r e s e n t s a p r i m a r y m e c h a n i s m for both the regulation and the s y n c h r o n i z a t i o n of cell physiology.
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Acknowledgments We thank J.L. C h ~ v e z by t e c h n i c a l a s s i s t a n c e . S u p p o r t e d b y g r a n t s f r o m C o n s e j o N a c i o n a l de C i e n c i a y T e c n o l o g i a (No 0 8 6 6 N 9 1 1 0 ) a n d f r o m D I G I C S A .
in p a r t
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
R.J. R E I T E R , E n d o c r i n e Rev. 12 1 5 1 - 1 8 0 (1991). B. S T A N K O V , F. F R A S C H I N I , a n d R.J. R E I T E R , B r a i n Res. Rev. i_66 2 4 5 - 2 5 6 (1991). D.P. C A R D I N A L I , M.I. V A C A S a n d E. E S T E V E Z BOYER, E n d o c r i n o l o g y 103 4 3 7 - 4 4 1 (1979). L.P. N I L E S , Y.W. WONG, R.K. M I S H R A a n d G.M. BROWN, Eur. J. P h a r m a c o l . 5_55 2 1 9 - 2 2 1 (1979). M. C O H E N , D. R O S E L L E , B. CHABNER, T.J. S C H M I D T a n d M. L I P P M A N , N a t u r e 274 8 9 4 - 8 9 5 (1978). F. A N T O N - T A Y , C. F O R R A Y a n d B.G. O R T E G A - C O R O N A , J. P i n e a l Res. 1 2 5 - 1 3 3 (1988). G. B E N I T E Z - K I N G , L. H U E R T O - D E L G A D I L L O a n d F. A N T O N - T A Y , B r a i n Res. 5 5 Z 2 8 9 - 2 9 2 (1991). G. B E N I T E Z - K I N G , L. H U E R T O - D E L G A D I L L O a n d F. A N T O N - T A Y , Proc. N e u r o s c i . Soc. 1_/7 1193 Abstr. 4 7 4 . 1 3 (1991). J.C. S T O C L E T , D. G E R A R D , M.C. K I L H O F F E R , C. L U G N I E R , R. M I L L E R a n d P. S C H A E F F E R , Progr. N e u r o b i o l . 29 3 2 1 - 3 6 4 (1987). Y.S. BABU, J.S. SACK, T.V. G R E E N H O U G H , C.E. BUGG, A.R. M E A N S a n d W.J. COOK, N a t u r e 315 3 7 - 4 0 (1985). R.M. L E V I N a n d B. WEISS, Mol. P h a r m a c o l . 1/3 6 9 0 - 6 9 7 (1977). P.F. M O R G A N , J. P A T E L a n d P.J. M A R A N G O S , B i o c h e m . P h a r m a c o l . 36 4 2 5 7 - 4 2 6 2 (1987). R. G O P A L A K R I S H N A a n d W.B. A N D E R S O N , B i o c h e m . B i o p h y s . Res. Comm. 104 8 3 0 - 8 3 6 (1982). D. H U L E N , A. BARON, J. S A L I S B U R Y a n d M. C L A R K E , C e l l Mot. C y t o s k e l . 18 1 1 3 - 1 2 2 (1991). M.M. T A M K U N , J.A. T A L V E N H E I M O aND W.A. C A T T E R A L L , J. Biol. Chem. 259 1 6 7 6 - 1 6 8 8 (1984). Y. C H E N G a n d W.H. P R U S O F F , B i o c h e m . P h a r m a c o l . 2-2 3 0 9 9 - 3 1 0 8 (1973). G. S C A T C H A R D , Ann. N.Y. Acad. Sci. 5!i 6 6 0 - 6 6 3 (1949). M.L. D U B O C O V I C H , J. P h a r m a c o l . Exp. Ther. 246 9 0 2 - 9 1 0 (1988). G. B E N I T E Z - K I N G , L. H U E R T O - D E L G A D I L L O a n d F. A N T O N - T A Y . J. P i n e a l Res. 9 2 0 9 - 2 2 0 (1990). D.A. M A L E N C I K a n d S.R. A N D E R S O N , B i o c h e m i s t r y 2_!1 3 4 8 0 - 3 4 8 6 (1982). H. H I D A K A , M. A S A N O a n d T. TANAKA, Molec. P h a r m a c . 20 5 7 1 - 5 7 8 (1981) . K. G I E T Z E N , B i o c h e m . J. 216 6 1 1 - 6 1 6 (1983). N. M R O S O V S K Y , R h e o s t a s i s 28-32, O x f o r d U n i v e r s i t y P r e s s N.Y. (1990). Y. I I D A , J . M o l e c . B i o l . 159 167-177 ( 1 9 8 2 ) . I. B A L Z E R , a n d R. H A R D E L A N D , S c i e n c e 253 7 9 5 - 7 9 7 (1991).
Pergamon Press
B I N D I N G OF 3H-MELATONIN TO C A L M O D U L I N G. Benltez-King, L. Huerto-Delgadillo, and F. Ant6n-Tay* Instituto M e x i c a n o de Psiquiatria, D e p a r t a m e n t o de Neurofarmacologla, DIC. * U n i v e r s i d a d Aut6noma M e t r o p o l i t a n a - I z t a p a l a p a , D e p a r t a m e n t o de Biologla de la Reproducci6n, DCBS.
(Received in final form April 27, 1993) Summary Studies in m e l a t o n i n m e c h a n i s m of action have s u g g e s t e d that one of them could be the binding of the h o r m o n e to calmodulin. We assessed c a l m o d u l i n - m e l a t o n i n b i n d i n g by c o m b i n i n g liposome incorporation of c a l m o d u l i n with sepa r a t i o n of free and bound 3H-Melatonin by a rapid u l t r a f i l t r a t i o n method. Specific binding to c a l m o d u l i n was saturable, reversible, Ca++-dependent, ligand selective, and showed high affinity. Saturation as well as a s s o c i a t i o n - d i s s o c i a t i o n studies revealed that 3H-Melatonin b i n d s to a single site on the calmodulin m o l e c u l e with a Kd of 188 pM and a total binding capacity Bmax of 35 p M / u g of calmodulin. D i s p l a c e m e n t experiments showed that the r e l a t i v e order of potency of some compounds for i n h i b i t i o n of 3 H - M e l a t o n i n was as follows: M e l a t o n i n > 6 - c h l o r o m e l a t o n i n > 6 - h y d r o x y m e l a t o n i n > luzindole > trifluoperazine. The r e s u l t s explain our p r e v i o u s l y r e p o r t e d m e l a t o n i n e f f e c t s such as cytoskeletal rearrangements, i n h i b i t i o n of c a l m o d u l i n d e p e n d e n t p h o s p h o d i e s t e r a s e a c t i v i t y as well as the m o d i f i c a t i o n of Ca++-calmodulin e l e c t r o p h o r e t i c mobility. The high affinity of m e l a t o n i n b i n d i n g to c a l m o d u l i n suggests that the hormone is able to m o d u l a t e cell a c t i v i t y by intracellularly binding to c a l m o d u l i n at p h y s i o l o g i c a l l y ranges. M e l a t o n i n - c a l m o d u l i n b i n d i n g could m o d u l a t e m a n y intracellular Ca ++ functions and thus, the s e t - p o i n t for cell activity will follow the r h y t h m i c c i r c u l a t i n g levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well p r e s e r v e d compounds, their interaction may r e p r e s e n t a primary mechanism for both the r e g u l a t i o n and the s y n c h r o n i z a t i o n of cell physiology. Melatonin, c o n s i d e r e d to be the main endocrine signal from the pineal gland, appears to synchronize many p h y s i o l o g i c a l r h y t h m s to the p h o t o p e r i o d (i). It has been reported that m e l a t o n i n (MEL) *Correspondence: G. Benltez-King, Instituto M e x i c a n o de Psiquiatrla, D e p a r t a m e n t o de Neurofarmacolog[a, Calzada M ~ x i c o - X o c h i m i l c o No 101, Colonia San L o r e n z o Huipulco, CP 14370 M~xico D.F. M~xico. ~-3~5/93 ~.~ +.~ Cop~ight©1993PergamonPressLtd Allrightsrese~ed.
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t a r g e t cells have specific plasma membrane receptors t h r o u g h which the h o r m o n e acts. For review see (2). However, there is evidence that 3H-MEL binds to m i t o c h o n d r i a l and nuclear membranes, as well as to c y t o s o l i c p r o t e i n s (3-6), and thus may m o d i f y cell p h y s i o l o g y t h r o u g h other mechanisms. Recently we have suggested that such a m e c h a n i s m could be the binding of MEL to c a l m o d u l i n (CAM), and we r e p o r t e d that both CaM cell concentration (7) and c o m p a r t m e n t a l i z a tion (8) in M D C K and NIE-II5 cells were m o d i f i e d by 10 -9 M MEL. Moreover, M E L inhibited CaM-dependent~ p h o s p h o d i e s t e r a s e a c t i v i t y i n vitro (7), and when either MEL or ~H-MEL was p r e i n c u b a t e d with CaM and s e p a r a t e d by gel electrophoresis, c o m i g r a t i o n of CaM with the r a d i o a c t i v i t y as well as m o d i f i c a t i o n of the Ca++-CaM relative m o b i l i t y were observed (7). Therefore, it seemed w o r t h w h i l e to study C a M - M E L b i n d i n g directly. C a l m o d u l i n is a Ca ++ binding protein that m e d i a t e s the main i n t r a c e l l u l a r r e g u l a t o r y effects of Ca ++ (9). Binding of C a ++ ions to CaM exposes specific h y d r o p h o b i c domains that p a r t i c i p a t e in t a r g e t p r o t e i n interactions or in the binding of specific m o l e c u l e s (i0). Even though the most common method for a s s e s s i n g the k i n e t i c c o n s t a n t s of ligand binding to CaM is t h r o u g h e q u i l i b r i u m d i a l y s i s (ii), these constants are difficult to obtain due to the very low s i g n a l / n o i s e ratio of the method. R e c e n t l y we a s s e s s e d the CaM-MEL binding by combining liposome i n c o r p o r a t i o n of CaM with s e p a r a t i o n of free and bound 3H-MEL by a rapid u l t r a f i l t r a t i o n method (12). We now report the stereoselective, high affinity, saturable, reversible, Ca÷+-dependent 3H-MEL binding to CaM. Material
and Methods
C a l m o d u l i n was obtained from frozen sheep brains and p u r i f i e d by h y d r o p h o b i c c h r o m a t o g r a p h y according to G o p a l a k r i s h n a and A n d e r s o n (13). It was d i a l y z e d three times during 24 hrs each, against 4 liters of 50 mM tris pH 7.6 buffer, using a seamless c e l l u l o s e tubing with a 12000 d. molecular weight cut-off. D i a l y z e d CaM was lyophilized and resuspended in the same buffer to desired c o n c e n t r a t i o n s . E l e c t r o p h o r e t i c mobility and b i o l o g i c a l activity w e r e c h e c k e d against commercial CaM (14). 3H-MEL (83.3 Ci/mMole) and 125I-CaM (87.1 uCi/mg) were from NEN-Dupont. N i t r o c e l l u l o s e filters w e r e from Amersham. CaM and other reagents were from Sigma. L u z i n d o l e was a generous gift of Dr. M.L. Dubocovich. B i n d i n g Assay: CaM was incorporated into liposomes by s o n i c a t i o n with p h o s p h a t i d y l choline in a 1:30 ratio during 40 m i n u t e s at room temperature (15). Binding assays were r o u t i n e l y p e r f o r m e d in t r i p l i c a t e in a total volume of i00 ul. Unless o t h e r w i s e specified, CaM (i ug) and .140 pM of 3H-MEL were incubated in 50 mM tris-HCl, 1 mM CaCI 2 buffer during 1 hour at 2 ° . MEL and r e l a t e d compounds were d i s s o l v e d in 20 mM tris pH 7.6, 20% glycerol; final glycerol c o n c e n t r a t i o n was 2 %. For some experiments ~H-MEL was d i l u t e d with cold M E L to 1 Ci/mMole. After the incubation period, bound and free 3H-MEL were s e p a r a t e d by rapid vacuum filtration (total time of 5 sec) t h r o u g h a 0.45 uM pore n i t r o c e l l u l o s e filter p r e s o a k e d in a 10% p o l y e t h y l e n e i m i n e , 50 mM tris-HCl, pH 7.6 buffer (12). Filters were w a s h e d 4 times with 4 ml of cold assay buffer, dried, and their r a d i o a c t i v i t y counted. N o n - s p e c i f i c binding was m e a s u r e d with 10 -4 M melatonin. Efficiency of the filtration system with respect to CaM e n t r a p m e n t was assessed using 125I-CaM d i l u t e d with CaM to the final c o n c e n t r a t i o n employed in the assays.
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Statistical Analysis.Data from saturation experiments were c a l c u l a t e d b y a E n z f i t t e r program. D a t a f r o m a s s o c i a t i o n e x p e r i m e n t s were treated as p s e u d o first order reactions and dissociation experiments, as first order reactions. Both were calculated with the Sigma plot program. Ki v a l u e s w e r e c a l c u l a t e d according to C h e u n g a n d P r u s o f f (16) f r o m d e t e r m i n e d It50 values. Results
E s t i m a t e s of 125I-CaM e n t r a p m e n t w e r e in t h e r a n g e of 90 to 96 % a n d w e r e u s e d to c o r r e c t for s y s t e m e f f i c i e n c y . ~H-MEL binding to l i p o s o m e s a n d to nitrocellulose f i l t e r s in a b s e n c e of C a M w a s s i m i l a r to n o n - s p e c i f i c binding. In a t y p i c a l b i n d i n g a s s a y 60% of t o t a l 3 H - M E L b i n d i n g w a s specific. T h e b i n d i n g of 3 H - M E L to C a M w a s t e m p e r a t u r e i n d e p e n d e n t , w i t h an o p t i m a l p H of 7.6 (Data n o t shown). S p e c i f i c b i n d i n g w a s l i n e a r w i t h C a M c o n c e n t r a t i o n up to 2 u g (Fig i).
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Specific 3HMelatonin b i n d i n g to calmodulin as a function of p r o t e i n concentration. 3H-MEL was incubated with CaM at different p r o t e i n concentrations as described. Nonspecific bindi%%_~as assessed using M melatonin. Data points represent the mean + SEM of two experiments done in triplicate.
T h e e f f e c t of Ca ++ c o n c e n t r a t i o n s on s p e c i f i c 3 H - M E L b i n d i n g w a s tested. As shown in Fig 2., 3H-MEL b i n d i n g dependence on C a M activation by Ca ++ w a s o b s e r v e d . Ca ++ c h e l a t i o n w i t h E G T A (20 mM) prevented specific b i n d i n g of 3H-MEL to CaM. As e x p e c t e d , nons p e c i f i c b i n d i n g of 3 H - M E L w a s n o t a l t e r e d by e i t h e r Ca ++ or EGTA. D u e to M E L h y d r o p h o b i c structure it c o u l d be e x p e c t e d t h a t the hormone would bind non-specifically to d i v e r s e p r o t e i n s ; thus we s t u d i e d Ca ++ d e p e n d e n c e of 3H-MEL b i n d i n g to 1 u g e a c h of s e r u m b o v i n e a l b u m i n , e g g albumin, t u b u l i n a n d c r e a t i n e p h o s p h o k i n a s e a n d no m o d i f i c a t i o n s in t h e a m o u n t of 3H-MEL b o u n d w a s o b s e r v e d w i t h or w i t h o u t 1 m M of Ca ++ . Saturation experiments showed that CaM-3H-MEL binding exhibited a s a t u r a b l e s p e c i f i c c o m p o n e n t and a l i n e a r n o n - s p e c i f i c c o m p o n e n t (Fig 3A). S c a t c h a r d isotherm plots (17) w e r e l i n e a r (Fig 3B). Binding parameters from the Scatchard plot w e r e Kd = 175 p M a n d B m a x = 40 p M / u g CaM. A n a v e r a g e of e i g h t e x p e r i m e n t s y i e l d e d a Kd = 188 ± 30 p M a n d B m a x = 35 ± 4 p M / u g CaM. Calculated Hill c o e f f i c i e n t w a s of 0.94 + 0.014. Time course studies confirmed reversibility of b i n d i n g . We
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e m p l o y e d a 1 hr incubation time, since e q u i l i b r i u m was a c h i e v e d well prior to this p e r i o d (Fig 4). Analysis of the a s s o c i a t i o n d i s s o c i a t i o n data from three experiments yielded an e s t i m a t e of the a s s o c i a t i o n rate constant (KI) of 4.5 + 1 x i015 M -I min -I and the d i s s o c i a t i o n rate constant (K_l) of --0.387 ± .069 min -I The e q u i l i b r i u m d i s s o c i a t i o n rate constant (Kd) e s t i m a t e d under none q u i l i b r i u m c o n d i t i o n s from the ratio K_I/K 1 was 102 ± i0 pM. From the d i s s o c i a t i o n constant calculated above a TI/2 of 2.02 ± 0.17 min was obtained.
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Calcium dependence of 3H-Melatonin b i n d i n g to calmodulin. CaM was incubated w i t h 3H-MEL as d e s c r i b e d with increasing concen trations of CaCI 2 in the absence ( O ) and p r e s e n c e (O) of 20 mM of EGTA. N o n - s p e c i f i c binding was assessed using 10 -4 M MEL. Data points r e p r e s e n t the mean + SEM of four experiments performed in triplicate.
D i s p l a c e m e n t studies revealed that the binding of 3H-MEL to CaM showed ligand selectivity. Besides the MEL analogs, we also tested a well k n o w n CaM antagonist, trifluoperazine (ii), and a MEL antagonist, luzindole (18). The Ki values for MEL, 6 - c h l o r o m e l a tonin, 6-hydroxymelatonin, luzindole and t r i f l u o p e r a z i n e were 0.193 0.050, 2.3 ± i.i, 7.1 + 2, 2743 ± 415 and > 5000 nM, respectively. I n c o n s i s t e n t results were obtained with: serotonin, Na c e t y l s e r o t o n i n and N - a c e t y l t r i p t a m i n e possible b e c a u s e they are quite h y d r o p h i l i c compounds. Discussion
A m o n g other p r o t e i n s examined, only CaM p o s s e s s e d high affinity ++ ++ Ca - d e p e n d e n t binding sites for MEL. Upon CaM a c t i v a t i o n by Ca , 3H-MEL b i n d i n g to the protein fulfills the major c r i t e r i a for considering this protein as a receptor: it was saturable, reversible, p r o t e i n dependent and showed ligand selectivity. 3H-MEL w o u l d appear to bind to a single class of sites, since we o b s e r v e d a linear S c a t c h a r d plot and a 0.94 ± 0.014 Hill coefficient. There is a d i r e c t r e l a t i o n s h i p between h y d r o p h o b i c i t y and both binding affinity and activity of w e l l - k n o w n CaM a n t a g o n i s t s such as phenothiazines and n a p h t h a l e n s u l f o n a m i d e s (9,11). Thus, it is p r o b a b l e that the MEL 5-methoxy group, which makes the h o r m o n e very hydrophobic, may interact with an exposed h y d r o p h o b i c b i n d i n g site of CaM. Also, a direct relationship between phenothiazines, CaM affinity, and p o t e n c y to inhibit CaM d e p e n d e n t enzymes has been r e p o r t e d (9,11). There is a similar r e l a t i o n s h i p for MEL binding
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(Kd = 188 pM) and its inhibitory effect on CaM-dependent p h o s p h o d i e s t e r a s e activity (IC50 = 1 nM) as r e p o r t e d (7). Therefore, we p r o p o s e the binding of MEL to CaM as the m e c h a n i s m t h r o u g h
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S a t u r a t i o n of 3H-Melatonin binding to calmodulin. A) CaM (i u~) was incubated with increasing c o n c e n t r a t i o n s of H-MEL as d e s c r i b e d in methods. 3H-MEL, bound in the p r e s e n c e of 10 -4 M MEL (non-specific binding) ( ), s u b t r a c t e d from total binding (0) in order to determine specific binding (O)B) Specific binding r e p l o t t e d by the Scatchard method. Data points r e p r e s e n t the m e a n of t r i p l i c a t e d e t e r m i n a t i o n s from a single experiment. w h i c h this h o r m o n e inhibits p h o s p h o d i e s t e r a s e activation. Also, the data p r e s e n t e d here suggest that p r e v i o u s l y r e p o r t e d c y t o s k e l e t a l r e a r r a n g e m e n t s (19) as well as the m o d i f i c a t i o n of CaM Ca ++ shift by MEL (7) are due to MEL binding to CaM. M o r e o v e r it is possible, that some of the r e p o r t e d nuclear, m i t o c h o n d r i a l and c y t o s o l i c 3H-MEL b i n d i n g (3-6) could be due to the p r e s e n c e of CaM at these sites. The p i c o m o l a r affinity of MEL binding to CaM w o u l d make it possible for the hormone to modulate cell activity by intracellularly binding to the protein at physiological c o n c e n t r a t i o n ranges. By contrast, the known CaM antagonists, be they p e p t i d e s such as melittin, mastoparans, dynorphin, endorphin, or drugs, such as trifluoperazine, W7 or calmidazolium, bind to CaM in the m i c r o m o l a r range (20-22). Due to its high lipophilicity, MEL may have access to many cell types and to i n t r a c e l l u l a r CaM as
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well. However, MEL cell responses, will depend, not only on the a c c e s s i b i l i t y of the hormone to the cell, but also on the t o p o l o g y of CaM, the t a r g e t enzymes and substrates, local Ca 2+ t r a n s i e n t s and the g e n e t i c a l l y r e g u l a t e d CaM compartmentalization. Thus, both cellular specificity and specific cellular responses for MEL must be considered. MEL
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80 90
Time (minutes)
Fig. 4 A s s o c i a t i o n and d i s s o c i a t i o n of 3H-Melatonin b i n d i n g to calmodulin. CaM (i ug) was incubated with 140 pM of 3H-MEL, and the amount of total 3H-MEL b i n d i n g was d e t e r m i n e d as a function of incubation time. At 60 min of incubation, 10 -4 M of MEL was added to the i n c u b a t i o n mixture, and the decline in total binding with time was determined. Non-specific binding was estimated by p r o c e s s i n g identical incubates in the p r e s e n c e of 10 -4 M MEL. Data points represent the mean of triplicate determinations from a single experiment. Two further e x p e r i m e n t s y i e l d e d similar results. CaM is an ubiquitous Ca ++ binding protein that relays i n t r a c e l l u l a r Ca ++ signal to many important p h y s i o l o g i c a l enzymes (9). Since M E L antagonizes Ca ++ activated CaM, MEL target cell a c t i v i t y will follow the rhythmic circulating levels of the pineal h o r m o n e and thus, MEL should be considered as a r h e o s t a t i c h o r m o n e (23) . CaM and MEL are p h y l o g e n e t i c a l l y well p r e s e r v e d structures that are p r e s e n t even in primitive organisms (24,25), and since their i n t e r a c t i o n could modulate many intracellular C a + + functions, it is t e m p t i n g to p o s t u l a t e that this interaction r e p r e s e n t s a p r i m a r y m e c h a n i s m for both the regulation and the s y n c h r o n i z a t i o n of cell physiology.
Vol. 53, No. 3, 1993
Calmodulin-Melatonin Binding
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Acknowledgments We thank J.L. C h ~ v e z by t e c h n i c a l a s s i s t a n c e . S u p p o r t e d b y g r a n t s f r o m C o n s e j o N a c i o n a l de C i e n c i a y T e c n o l o g i a (No 0 8 6 6 N 9 1 1 0 ) a n d f r o m D I G I C S A .
in p a r t
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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