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Hippocalcin: a calcium-binding protein of the EF-hand superfamily dominantly expressed in the hippocampus
291
Neuroscience Research, 17 (1993) 291-295 © 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0168-0102/93/$06.00 NSR 00670
Update article
Hippocalcin: a calcium-binding protein of the EF-hand superfamily dominantly expressed in the hippocampus Ken Takamatsu and Tetsuya Noguchi Department of Physiology, Toho University School of Medicine, Ohta-ku, Tokyo 143, Japan (Received 11 June 1993; accepted 1 July 1993)
Key words: Hippocalcin; Ca2+-binding protein; EF-hand; Brain; Hippocampus; Myristoylation; Membrane association Summary Hippocalcin is a recently identified Ca2+-binding protein with three EF-hand structures, dominantly expressed in the hippocampal pyramidal layer. The complete amino acid sequence of hippocalcin deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22 574 daltons, and has a striking sequence homology to those of visinin, recoverin, S-modulin, neurocalcins and neural visinin-like proteins. Hippocalcin binds 3 mol of Ca 2÷ per mol of protein at submicromolar Ca 2+ levels, and associates the plasma membrane in a Ca2+-dependent manner. Hippocalcin is myristoylated at its NH2-terminal glycine residue, and this modification is a key event in terms of its membrane-association property.
Introduction The calcium ion is a major second messenger whose intracellular receptors include a number of structurally related Ca2+-binding proteins (Heizmann and Hunziker, 1991). These Ca2+-binding proteins transduce the Ca 2÷ messages into a variety of physiological responses. A new family of intracellular Ca2+-binding proteins with three EF-hand structures, referred to as the recoverin family, has been found in the retina (Yamagata et al., 1990; Dizhoor et al., 1991; Kawamura and Murakami, 1991; McGinnis et al., 1992) and in the brain (Takamatsu et al., 1992a; Hidaka and Okazaki, 1993; Kajimoto et al., 1993). Retina-derived members of the recoverin family are distributed in the photoreceptor cells, and regulate photo-signal transduction
Correspondence to: Dr. K. Takamatsu, Department of Physiology, Toho University School of Medicine, 5-21-16, Ohmori-nishi, Ohta-ku, Tokyo 143, Japan. Tel.: 81-3-3762-4151, ext. 2342; Fax: 81-3-37628225.
systems via prolonging the light activation of cyclic GMP phosphodiesterase in a Ca2+-sensitive manner (Kawamura, 1993; Gray-Keller et al., 1993). Although there is no evidence concerning the physiological function of brain-derived members of the recoverin family, their structural similarity to retina-derived members leads us to postulate that they might have similar functions and act as Ca2+-sensitive regulators in signal transduction systems in the brain. We recently identified a novel member of this family, and found dominant expression of it in the hippocampus by Northern blot and immunoblot analyses, designating it hippocalcin (Kobayashi et al., 1992).
Distribution of hippocalcin In situ hybridization analysis revealed that hippocalcin mRNA was expressed abundantly in the pyramidal cells of the hippocampus; however, moderate-to-weak signals could be detected in the Purkinje cells of the
292 cerebellum, the d e n t a t e g r a n u l e cells a n d pyramidal cells of cerebral cortex layers II to VI a n d weak signals in the large n e u r o n a l cells of the c a u d a t e - p u t a m e n (Saitoh et al., 1993). T h e distribution of hippocalcin i m m u n o r e a c t i v i t y closely m a t c h e d that of m R N A . In most cell types, hippocalcin i m m u n o r e a c t i v i t y was localized in the cytoplasm a n d plasma m e m b r a n e of cell bodies a n d dendrites, suggesting the involvement of hippocalcin in postsynaptic n e u r a l functions. T h e dist r i b u t i o n of hippocalcin is very different from those of other m e m b e r s of the b r a i n - d e r i v e d recoverin family, such as P23k ( T a k a m a t s u et al., 1992b), n e u r o c a l c i n ( H i d a k a and Okazaki, 1993) a n d n e u r a l visinin-like proteins (Kajimoto et al., 1993). Since each of these proteins seems to exist a n d work in a different p o r t i o n in the brain, hippocalcin probably has an i m p o r t a n t role in the n e u r a l functions in the h i p p o c a m p u s .
Primary structure of hippocalcin T h e complete a m i n o acid s e q u e n c e of hippocalcin d e d u c e d from the c D N A is c o m p o s e d of 195 residues, has a calculated m o l e c u l a r mass of 22574 daltons, a n d has a striking s e q u e n c e homology to those of r e t i n a - d e rived m e m b e r s (such as visinin, recoverin a n d S-rnodulin; 4 0 - 5 0 % ) a n d b r a i n - d e r i v e d m e m b e r s (such as n e u r o c a l c i n s a n d n e u r a l visinin-like proteins; 8 0 - 9 0 % ) of the recoverin family (Kobayashi et al., 1992). In t w o - d i m e n s i o n a l gel electrophoresis, hippocalcin migrated as a single spot, m o l e c u l a r mass 23 kDa a n d isoelectric point 5.2 u n d e r CaZ+-free conditions, a n d as a double spot, m o l e c u l a r mass 21 kDa a n d 5.3 u n d e r CaZ+-loaded conditions, indicating that hippocalcin shows C a 2 + - d e p e n d e n t c o n f o r m a t i o n a l changes.
TABLE 1 DISTRIBUTION OF HIPPOCALCIN MRNA AND IMMUNOREACTIVITY IN RAT BRAIN (MODIFIED FROM SAITOH ET AL., 1993) Area
mRNA
Immunoreactivity
Olfactory bulb Cerebral cortex Layer I Layer 11 Layers III and IV Layers V and VI Hippocampus CA1 and2 CA3 CA4 Dentate gyrus Subiculum Entorhinal cortex Septal nuclei Caudate-putamen Fornix Mamillary nuclei Thalamus Hypothalamus Anterior part Posterior part Ventral part Amygdaloid complex Midbrain Cerebellum Molecular layer Purkinje cell layer Granule cell layer Deep cerebellar nuclei Internal capsulecerebral peduncle Pons-medulla Spinal cord
-
-
-++ + +
++ + +
+++ +++ + + + + -
++ +++ ++ + + + ± :k -
-
+ -
++ -
++ -
-
+ -
Intensities of the signals are expressed as follows: + + +, most intense: + +, intense; +, moderate; +, weak; - , not detected.
Calcium-binding activity T h e predicted a m i n o acid s e q u e n c e of hippocalcin contains three putative CaZ+-binding d o m a i n s of the E F - h a n d structure. This structural f e a t u r e is c o m m o n to all m e m b e r s of the recoverin family. Blots with 45Ca d e m o n s t r a t e d that m a n y m e m b e r s of the recoverin family, including hippocalcin, b i n d Ca 2+, indicating that these p r o t e i n s specifically b i n d Ca 2 + at submicromolar or lower c o n c e n t r a t i o n s in solution. I n d e e d , P23k, a b r a i n - d e r i v e d m e m b e r of this family, has b e e n r e p o r t e d to have two CaZ+-binding sites with dissociation constants of 0 . 2 / ~ M a n d 1 3 / z M ( T a k a m a t s u et al., 1992a). T r y p t o p h a n fluorescence spectroscopy of recoverin indicated that recoverin c o n t a i n s at least two
Ca2+-binding sites f u n c t i o n i n g at s u b m i c r o m o l a r Ca 2+ levels (Dizhoor et al., 1991). T h e third of the three E F - h a n d d o m a i n s does n o t seem to satisfy the structural criteria of a high-affinity CaZ+-binding site. However, all three fusion p r o t e i n s of each E F - h a n d d o m a i n with m a l t o s e - b i n d i n g p r o t e i n showed CaZ+-binding activity on 4SCa-blot analysis, indicating that hippocalcin may have the ability to b i n d 3 mol of Ca 2+ per mol of p r o t e i n at s u b m i c r o m o l a r Ca 2+ levels (Kobayashi et al., 1993). Since s e q u e n t i a l b i n d i n g of Ca 2+ to hippocalcin causes c o n f o r m a t i o n a l changes a n d the t h r e e - d i m e n sional c o n f o r m a t i o n of a p r o t e i n m o l e c u l e is crucial to its Ca2+-binding affinity, the Ca2+-binding p r o p e r t y of
293 native hippocalcin might differ from that of the fusion proteins.
not known whether NH2-terminal acyl heterogeneity is limited to photoreceptor proteins or is common to the members of recoverin family.
N-Myristoylation of hippocalcin Membrane-association property The NH2-terminal sequences of all members of the recoverin family meet the criteria for NHz-terminal myristoylation: a glycine residue at the NH 2 terminus, and a serine or threonine residue at position 5 (Towler et al., 1988). Since rabbit reticulocyte lysate was known to contain N-myristoyl transferase activity, [3H]myristic acid incorporation into hippocalcin was examined in vitro (Kobayashi et al., 1993). 3H-label was associated with newly synthesized hippocalcin and was resistant to 1 M hydroxylamine treatment, suggesting an amide linkage involved in the myristoylation of protein. In the case of mutagenic (Glya-Ala 1) hippocalcin, no 3H-label incorporation could be detected. Since native hippocalcin comigrated precisely with myristoylated recombinant hippocalcin on two-dimensional gels, but not with nonmyristoylated recombinant hippocalcin, it seems likely that native hippocalcin is myristoylated. Recent mass spectrometry findings indicate that NH2-terminal peptides derived from recoverin contain a mixture of saturated and unsaturated aac fatty acids; however, the heterogeneous acylation was not detected in previous analyses of myristoylated proteins, except one other photoreceptor protein, transducin (Dizhoor et al., 1992). Since structural analyses of the NHz-terminal peptides of other members of the recoverin family, including hippocalcin, have not been established, it is
While proteins which undergo myristoylation have diverse functions and are found at various locations within cells, in several cases this modification has been shown to be related to their association with cell membranes (Towler et al., 1988). Although myristoylated hippocalcin is soluble in the Ca 2+-free form, it shows a fairly tight membrane association in the Ca2+-bound form. The myristoylation of hippocalcin is a key event in terms of its Ca2+-dependent membrane-association properties (Kobayashi et al., 1993). Hippocalcin increases hydrophobicity by accepting Ca2+; however, the hydrophobic interaction with phospholipids cannot fully explain the Ca2+-dependent membrane association of hippocalcin. These findings suggest that the NH2-terminal myristic acid on hippocalcin interacts with the lipid bilayer and assists in interactions with other membrane proteins. Recently, it has been reported that myristoylated recoverin, but not the nonmyristoylated form, bound to phosphatidylcholine vesicles as well as rod outer segment membranes in a Ca2+-dependent manner, suggesting that a specific protein receptor for myristoylated recoverin is not required for its Ca2+-dependent membrane association (Zozulya and Stryer, 1992). However, the finding that recoverin binds to a concanavalin A-Sepharose column
Hippocalcin 6-Neurocalcin NVP-1 Recoverin visinin
MGKQNSK-LRPEMLQDLRENTEFSELELQEWYKGFLKDCPTGILNVDEFKKIYANFFPYAMPPKFAEHVF
69
*******_*********************************************************** *******_****--**************************************************** ******************************************************************* **NSR*SA*SR*V**E**AS*RYT*E**SR**E**QR**SD*RIRC***ER**G****NSE*QGY*RQ**
69 69
Hippocalcin 6-Neurocalcin NVP-1 Recoverin Visinin
RTFDTNSDGTIDFREFIIALSVTSGGRLEQKLMWAFSMYDLDGNGYISREEMLEIVQAIYKMVSSV--MK
137 137
*****************************************************************--** ********************************************************************** ****************************************************************** *********************************************************************
70 70
139 140 140
195 MPEDESTPEKRTEKIFRQMDTNNDGKLSLEEFTRGAKSDPSIVRLLQCDPSSAFPVLS Hippocalcin ******************************************************** 193 6-Neurocalcin ************************************************** 191 NVP-1 202 L****N*****A***WGFFGKKD*D**TEK**IE*TLANKE*L**I*FE*QKVKEKLKEKKL Recoverin 192 L****NS*Q**AD*LWAYFNKGEND*IAEG**ID*VMKNDA*M**I*YE*KK visinin Fig. 1. Alignmentof the deduced amino acid sequence of hippocalcin and homologousproteins. Amino acids identicalto hippocalcinare marked by " *2' Hippocalcin (Kobayashiet al., 1992), d-Neurocalcin (Hidaka and Okazaki, 1993), NVP-1; neural visinin-likeprotein-1 (Kajimotoet al., 1993), Recoverin(Dizhoor et al., 1991), Visinin(Yamagataet al., 1990).
294 c o n t a i n i n g i m m o b i l i z e d b l e a c h e d r h o d o p s i n indicates an i n t e r a c t i o n b e t w e e n r e c o v e r i n a n d r h o d o p s i n (Dizh o o r et al., 1991). T h e possibility that t h e N H z - t e r m i nal myristic acid on p r o t e i n s is p r i m a r i l y involved in p r o t e i n - p r o t e i n i n t e r a c t i o n s is s u g g e s t e d by r e c e n t studies on pp60 Src a n d an a s u b u n i t of g u a n i n e nuc l e o t i d e - b i n d i n g p r o t e i n s ( R e s h a n d Ling, 1990; L i n d e r et al., 1991).
Functional aspects R e c o v e r i n was d i s c o v e r e d as t h e activating factor of g u a n y l a t e cyclase to m e d i a t e t h e C a 2 + - d e p e n d e n t stimulation o f cyclic G M P resynthesis d u r i n g t h e recovery of the electrical light r e s p o n s e ( D i z h o o r et al., 1991). R e c e n t l y , r e c o v e r i n has b e e n r e p o r t e d to p r o l o n g the rising p h a s e of t h e light r e s p o n s e , while having little to no effect on the kinetics o f r e s p o n s e recovery, suggesting t h a t it delays t e r m i n a t i o n o f the t r a n s d u c t i o n casc a d e r a t h e r t h a n affect resynthesis o f cyclic G M P and r e c o v e r t h e d a r k c u r r e n t ( G r a y - K e l l e r et al., 1993). S - m o d u l i n , a n o t h e r r e t i n a l m e m b e r of the r e c o v e r i n family, b i n d s to rod o u t e r s e g m e n t m e m b r a n e s a n d p r o l o n g s t h e light activation of cyclic G M P hydrolysis w h e n the free Ca 2+ c o n c e n t r a t i o n i n c r e a s e s to m o r e t h a n 1 p,M. T h e C a Z + - d e p e n d e n t r e g u l a t i o n o f phosp h o d i e s t e r a s e by S - m o d u l i n was f o u n d to b e m e d i a t e d by r h o d o p s i n p h o s p h o r y l a t i o n ( K a w a m u r a , 1993). P h o s p h o r y l a t i o n o f r e c e p t o r m o l e c u l e s m a y b e a general m e c h a n i s m for r e g u l a t i n g signal t r a n s d u c t i o n . Thus, t h e m e m b e r s of t h e recoverin family s e e m to be CaZ+-sensitive r e g u l a t o r s of i n t r a c e l l u l a r signal transd u c t i o n systems. Recently, h i p p o c a l c i n was f o u n d to associate with a cytosolic p r o t e i n of m o l e c u l a r mass 48 k D a ( H A P - 4 8 ) in a C a Z + - d e p e n d e n t m a n n e r by blot analysis using 35S-labeled h i p p o c a l c i n ( T a k a m a t s u et al., u n p u b l i s h e d data). H A P - 4 8 was p u r i f i e d from rat brain, a n d was i d e n t i f i e d as c r e a t i n e kinase B. 35S-labeled h i p p o c a l c i n a s s o c i a t e d with p u r i f i e d c r e a t i n e kinase B subunit, b u t not with p u r i f i e d c r e a t i n e kinase M from rat muscle. R e c o v e r y o f c r e a t i n e kinase activity in t h e p o s t m i c r o s o mal s u p e r n a t a n t fraction f r o m the h i p p o c a m p u s was slightly h i g h e r in the a b s e n c e o f Ca 2+ t h a n in the p r e s e n c e of Ca 2+. T h e s e results suggest t h a t h i p p o c a l cin transfers c r e a t i n e k i n a s e B to local p l a s m a m e m b r a n e s by d e t e c t i n g the e l e v a t e d cytosolic Ca 2+ levels, w h e r e c r e a t i n e kinase p r o v i d e s a s u s t a i n e d a n d local c o n c e n t r a t i o n of h i g h - e n e r g y p h o s p h a t e as is n e e d e d for the m a i n t e n a n c e o f ion g r a d i e n t s or for the contraction of the m o l e c u l a r m o t o r s in t h e d e n d r i t e s .
A l t h o u g h the physiological functions o f h i p p o c a l c i n r e m a i n obscure, it is r e a s o n a b l e to p o s t u l a t e that hipp o c a l c i n r e g u l a t e s synaptic efficacy in h i p p o c a m p a l pyramidal neurons through cooperative interactions with Ca 2 + a n d m e m b r a n e c o m p o n e n t s .
References Dizhoor, A.M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K.A., Philipov, P.P., Hurley, J.B. and Stryer, L. (1991) Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science, 251: 915-918. Dizhoor, A.M., Ericsson, L.H., Johnson, R.S., Kumar. S., Olshevskaya, E., Zozulya, S., Neubert, T.A., Stryer, L., Hurley J.B. and Walsh, K.A. (1992) The NH 2 terminus of retinal recoverin is acylated by a small family of fatty acids. J. Biol. Chem., 267: 16033-16036. Gray-Keller, M.P., Polans, A.S., Placzewski, K. and Detwiler, P.B. (1993) The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods. Neuron, 10: 523-531. Heizmann, C.W. and Hunziker, W. (1991) Intracellular calcium-binding proteins: more sites than insights. Trends Biochem. Sci., 16: 98-103. Hidaka, H. and Okazaki, K. (1993) Neurocalcin family: a novel calcium-binding protein abundant in bovine central nervous system. Neurosci. Res., 16: 73-77. Kajimoto, Y., Shirai, Y., Mukai, H., Kuno, T. and Tanaka, C. (1993) Molecular cloning of two additional members of the neural visinin-like Ca2+-biuding protein gene family. J. Neurochem., in press. Kawamura, S. and Murakami, M. (1991) Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods. Nature, 349: 420-423. Kawamura, S. (1993) Rhodopsin phosphorylation as a mechanism of cyclic GMP phosphodiesterase regulation by S-modulin. Nature, 362: 855-857. Kobayashi, M., Takamatsu, K., Saitoh, S., Miura, M. and Noguchi, T. (1992) Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus. Biochem~ Biophys. Res. Commun., 189: 511-517. Kobayashi, M., Takamatsu, K., Saitoh, S. and Noguchi, T. (1993) Myristoylation of hippocalcin is linked to its calcium-dependent membrane-association properties. J. Biol. Chem., in press. Linder, M.E., Pang, I.-H., Duronio, R.J., Gordon, J.F., Sternweis, P.C. and Gilman, A.G. (1991) Lipid modifications of G protein subunits: myristoylation of G0a increases its affinity for bg. J. Biol. Chem., 266: 4654-4659. McGinnis, J.F., Stepanik, P.L., Baehr, W., Subbaraya, I. and Lerious, V. (1992) Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein. FEBS Lett., 302: 172-176. Resh, M.D., and Ling, H.-P. (1990) Identification of a 32K plasma membrane protein that binds to the myristoylated amino-terminal sequence of p60 v-sr~.Nature, 346: 84-86. Saitoh, S., Takamatsu, K., Kobayashi, M. and Noguchi, T. (1993) Distribution of hippocalcin mRNA and immunoreactivity in rat brain. Neurosci. Lett., 157: 107-110. Takamatsu, K., Kitamura, K. and Noguchi, T. (1992a) Isolation and characterization of recoverin-like Ca2+-binding protein from rat brain. Biochem. Biophys. Res. Commun., 183: 245-251.
295 Takamatsu, K., Kobayashi, M. and Noguchi, T. (1992b) Immunohistochemical localization of recoverin-like Ca2+-binding protein (P23k) in mouse brain. Acta Histochem. Cytochem., 25: 533-536. Towler, D.A., Gordon, J.I., Adams, S.P. and Glaser, L. (1988) The biology and enzymology of eukaryotic protein acylation. Ann. Rev. Biochem., 57: 69-99.
Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H. and Miki, N.(1990) Visinin: a novel calcium binding protein expressed in retinal cone cells. Neuron, 2: 469-476. Zozulya, S. and Stryer, L (1992) Calcium-myristoyl protein switch. Proc. Natl. Acad. Sci. USA, 89: 11569-11573.
Neuroscience Research, 17 (1993) 291-295 © 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0168-0102/93/$06.00 NSR 00670
Update article
Hippocalcin: a calcium-binding protein of the EF-hand superfamily dominantly expressed in the hippocampus Ken Takamatsu and Tetsuya Noguchi Department of Physiology, Toho University School of Medicine, Ohta-ku, Tokyo 143, Japan (Received 11 June 1993; accepted 1 July 1993)
Key words: Hippocalcin; Ca2+-binding protein; EF-hand; Brain; Hippocampus; Myristoylation; Membrane association Summary Hippocalcin is a recently identified Ca2+-binding protein with three EF-hand structures, dominantly expressed in the hippocampal pyramidal layer. The complete amino acid sequence of hippocalcin deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22 574 daltons, and has a striking sequence homology to those of visinin, recoverin, S-modulin, neurocalcins and neural visinin-like proteins. Hippocalcin binds 3 mol of Ca 2÷ per mol of protein at submicromolar Ca 2+ levels, and associates the plasma membrane in a Ca2+-dependent manner. Hippocalcin is myristoylated at its NH2-terminal glycine residue, and this modification is a key event in terms of its membrane-association property.
Introduction The calcium ion is a major second messenger whose intracellular receptors include a number of structurally related Ca2+-binding proteins (Heizmann and Hunziker, 1991). These Ca2+-binding proteins transduce the Ca 2÷ messages into a variety of physiological responses. A new family of intracellular Ca2+-binding proteins with three EF-hand structures, referred to as the recoverin family, has been found in the retina (Yamagata et al., 1990; Dizhoor et al., 1991; Kawamura and Murakami, 1991; McGinnis et al., 1992) and in the brain (Takamatsu et al., 1992a; Hidaka and Okazaki, 1993; Kajimoto et al., 1993). Retina-derived members of the recoverin family are distributed in the photoreceptor cells, and regulate photo-signal transduction
Correspondence to: Dr. K. Takamatsu, Department of Physiology, Toho University School of Medicine, 5-21-16, Ohmori-nishi, Ohta-ku, Tokyo 143, Japan. Tel.: 81-3-3762-4151, ext. 2342; Fax: 81-3-37628225.
systems via prolonging the light activation of cyclic GMP phosphodiesterase in a Ca2+-sensitive manner (Kawamura, 1993; Gray-Keller et al., 1993). Although there is no evidence concerning the physiological function of brain-derived members of the recoverin family, their structural similarity to retina-derived members leads us to postulate that they might have similar functions and act as Ca2+-sensitive regulators in signal transduction systems in the brain. We recently identified a novel member of this family, and found dominant expression of it in the hippocampus by Northern blot and immunoblot analyses, designating it hippocalcin (Kobayashi et al., 1992).
Distribution of hippocalcin In situ hybridization analysis revealed that hippocalcin mRNA was expressed abundantly in the pyramidal cells of the hippocampus; however, moderate-to-weak signals could be detected in the Purkinje cells of the
292 cerebellum, the d e n t a t e g r a n u l e cells a n d pyramidal cells of cerebral cortex layers II to VI a n d weak signals in the large n e u r o n a l cells of the c a u d a t e - p u t a m e n (Saitoh et al., 1993). T h e distribution of hippocalcin i m m u n o r e a c t i v i t y closely m a t c h e d that of m R N A . In most cell types, hippocalcin i m m u n o r e a c t i v i t y was localized in the cytoplasm a n d plasma m e m b r a n e of cell bodies a n d dendrites, suggesting the involvement of hippocalcin in postsynaptic n e u r a l functions. T h e dist r i b u t i o n of hippocalcin is very different from those of other m e m b e r s of the b r a i n - d e r i v e d recoverin family, such as P23k ( T a k a m a t s u et al., 1992b), n e u r o c a l c i n ( H i d a k a and Okazaki, 1993) a n d n e u r a l visinin-like proteins (Kajimoto et al., 1993). Since each of these proteins seems to exist a n d work in a different p o r t i o n in the brain, hippocalcin probably has an i m p o r t a n t role in the n e u r a l functions in the h i p p o c a m p u s .
Primary structure of hippocalcin T h e complete a m i n o acid s e q u e n c e of hippocalcin d e d u c e d from the c D N A is c o m p o s e d of 195 residues, has a calculated m o l e c u l a r mass of 22574 daltons, a n d has a striking s e q u e n c e homology to those of r e t i n a - d e rived m e m b e r s (such as visinin, recoverin a n d S-rnodulin; 4 0 - 5 0 % ) a n d b r a i n - d e r i v e d m e m b e r s (such as n e u r o c a l c i n s a n d n e u r a l visinin-like proteins; 8 0 - 9 0 % ) of the recoverin family (Kobayashi et al., 1992). In t w o - d i m e n s i o n a l gel electrophoresis, hippocalcin migrated as a single spot, m o l e c u l a r mass 23 kDa a n d isoelectric point 5.2 u n d e r CaZ+-free conditions, a n d as a double spot, m o l e c u l a r mass 21 kDa a n d 5.3 u n d e r CaZ+-loaded conditions, indicating that hippocalcin shows C a 2 + - d e p e n d e n t c o n f o r m a t i o n a l changes.
TABLE 1 DISTRIBUTION OF HIPPOCALCIN MRNA AND IMMUNOREACTIVITY IN RAT BRAIN (MODIFIED FROM SAITOH ET AL., 1993) Area
mRNA
Immunoreactivity
Olfactory bulb Cerebral cortex Layer I Layer 11 Layers III and IV Layers V and VI Hippocampus CA1 and2 CA3 CA4 Dentate gyrus Subiculum Entorhinal cortex Septal nuclei Caudate-putamen Fornix Mamillary nuclei Thalamus Hypothalamus Anterior part Posterior part Ventral part Amygdaloid complex Midbrain Cerebellum Molecular layer Purkinje cell layer Granule cell layer Deep cerebellar nuclei Internal capsulecerebral peduncle Pons-medulla Spinal cord
-
-
-++ + +
++ + +
+++ +++ + + + + -
++ +++ ++ + + + ± :k -
-
+ -
++ -
++ -
-
+ -
Intensities of the signals are expressed as follows: + + +, most intense: + +, intense; +, moderate; +, weak; - , not detected.
Calcium-binding activity T h e predicted a m i n o acid s e q u e n c e of hippocalcin contains three putative CaZ+-binding d o m a i n s of the E F - h a n d structure. This structural f e a t u r e is c o m m o n to all m e m b e r s of the recoverin family. Blots with 45Ca d e m o n s t r a t e d that m a n y m e m b e r s of the recoverin family, including hippocalcin, b i n d Ca 2+, indicating that these p r o t e i n s specifically b i n d Ca 2 + at submicromolar or lower c o n c e n t r a t i o n s in solution. I n d e e d , P23k, a b r a i n - d e r i v e d m e m b e r of this family, has b e e n r e p o r t e d to have two CaZ+-binding sites with dissociation constants of 0 . 2 / ~ M a n d 1 3 / z M ( T a k a m a t s u et al., 1992a). T r y p t o p h a n fluorescence spectroscopy of recoverin indicated that recoverin c o n t a i n s at least two
Ca2+-binding sites f u n c t i o n i n g at s u b m i c r o m o l a r Ca 2+ levels (Dizhoor et al., 1991). T h e third of the three E F - h a n d d o m a i n s does n o t seem to satisfy the structural criteria of a high-affinity CaZ+-binding site. However, all three fusion p r o t e i n s of each E F - h a n d d o m a i n with m a l t o s e - b i n d i n g p r o t e i n showed CaZ+-binding activity on 4SCa-blot analysis, indicating that hippocalcin may have the ability to b i n d 3 mol of Ca 2+ per mol of p r o t e i n at s u b m i c r o m o l a r Ca 2+ levels (Kobayashi et al., 1993). Since s e q u e n t i a l b i n d i n g of Ca 2+ to hippocalcin causes c o n f o r m a t i o n a l changes a n d the t h r e e - d i m e n sional c o n f o r m a t i o n of a p r o t e i n m o l e c u l e is crucial to its Ca2+-binding affinity, the Ca2+-binding p r o p e r t y of
293 native hippocalcin might differ from that of the fusion proteins.
not known whether NH2-terminal acyl heterogeneity is limited to photoreceptor proteins or is common to the members of recoverin family.
N-Myristoylation of hippocalcin Membrane-association property The NH2-terminal sequences of all members of the recoverin family meet the criteria for NHz-terminal myristoylation: a glycine residue at the NH 2 terminus, and a serine or threonine residue at position 5 (Towler et al., 1988). Since rabbit reticulocyte lysate was known to contain N-myristoyl transferase activity, [3H]myristic acid incorporation into hippocalcin was examined in vitro (Kobayashi et al., 1993). 3H-label was associated with newly synthesized hippocalcin and was resistant to 1 M hydroxylamine treatment, suggesting an amide linkage involved in the myristoylation of protein. In the case of mutagenic (Glya-Ala 1) hippocalcin, no 3H-label incorporation could be detected. Since native hippocalcin comigrated precisely with myristoylated recombinant hippocalcin on two-dimensional gels, but not with nonmyristoylated recombinant hippocalcin, it seems likely that native hippocalcin is myristoylated. Recent mass spectrometry findings indicate that NH2-terminal peptides derived from recoverin contain a mixture of saturated and unsaturated aac fatty acids; however, the heterogeneous acylation was not detected in previous analyses of myristoylated proteins, except one other photoreceptor protein, transducin (Dizhoor et al., 1992). Since structural analyses of the NHz-terminal peptides of other members of the recoverin family, including hippocalcin, have not been established, it is
While proteins which undergo myristoylation have diverse functions and are found at various locations within cells, in several cases this modification has been shown to be related to their association with cell membranes (Towler et al., 1988). Although myristoylated hippocalcin is soluble in the Ca 2+-free form, it shows a fairly tight membrane association in the Ca2+-bound form. The myristoylation of hippocalcin is a key event in terms of its Ca2+-dependent membrane-association properties (Kobayashi et al., 1993). Hippocalcin increases hydrophobicity by accepting Ca2+; however, the hydrophobic interaction with phospholipids cannot fully explain the Ca2+-dependent membrane association of hippocalcin. These findings suggest that the NH2-terminal myristic acid on hippocalcin interacts with the lipid bilayer and assists in interactions with other membrane proteins. Recently, it has been reported that myristoylated recoverin, but not the nonmyristoylated form, bound to phosphatidylcholine vesicles as well as rod outer segment membranes in a Ca2+-dependent manner, suggesting that a specific protein receptor for myristoylated recoverin is not required for its Ca2+-dependent membrane association (Zozulya and Stryer, 1992). However, the finding that recoverin binds to a concanavalin A-Sepharose column
Hippocalcin 6-Neurocalcin NVP-1 Recoverin visinin
MGKQNSK-LRPEMLQDLRENTEFSELELQEWYKGFLKDCPTGILNVDEFKKIYANFFPYAMPPKFAEHVF
69
*******_*********************************************************** *******_****--**************************************************** ******************************************************************* **NSR*SA*SR*V**E**AS*RYT*E**SR**E**QR**SD*RIRC***ER**G****NSE*QGY*RQ**
69 69
Hippocalcin 6-Neurocalcin NVP-1 Recoverin Visinin
RTFDTNSDGTIDFREFIIALSVTSGGRLEQKLMWAFSMYDLDGNGYISREEMLEIVQAIYKMVSSV--MK
137 137
*****************************************************************--** ********************************************************************** ****************************************************************** *********************************************************************
70 70
139 140 140
195 MPEDESTPEKRTEKIFRQMDTNNDGKLSLEEFTRGAKSDPSIVRLLQCDPSSAFPVLS Hippocalcin ******************************************************** 193 6-Neurocalcin ************************************************** 191 NVP-1 202 L****N*****A***WGFFGKKD*D**TEK**IE*TLANKE*L**I*FE*QKVKEKLKEKKL Recoverin 192 L****NS*Q**AD*LWAYFNKGEND*IAEG**ID*VMKNDA*M**I*YE*KK visinin Fig. 1. Alignmentof the deduced amino acid sequence of hippocalcin and homologousproteins. Amino acids identicalto hippocalcinare marked by " *2' Hippocalcin (Kobayashiet al., 1992), d-Neurocalcin (Hidaka and Okazaki, 1993), NVP-1; neural visinin-likeprotein-1 (Kajimotoet al., 1993), Recoverin(Dizhoor et al., 1991), Visinin(Yamagataet al., 1990).
294 c o n t a i n i n g i m m o b i l i z e d b l e a c h e d r h o d o p s i n indicates an i n t e r a c t i o n b e t w e e n r e c o v e r i n a n d r h o d o p s i n (Dizh o o r et al., 1991). T h e possibility that t h e N H z - t e r m i nal myristic acid on p r o t e i n s is p r i m a r i l y involved in p r o t e i n - p r o t e i n i n t e r a c t i o n s is s u g g e s t e d by r e c e n t studies on pp60 Src a n d an a s u b u n i t of g u a n i n e nuc l e o t i d e - b i n d i n g p r o t e i n s ( R e s h a n d Ling, 1990; L i n d e r et al., 1991).
Functional aspects R e c o v e r i n was d i s c o v e r e d as t h e activating factor of g u a n y l a t e cyclase to m e d i a t e t h e C a 2 + - d e p e n d e n t stimulation o f cyclic G M P resynthesis d u r i n g t h e recovery of the electrical light r e s p o n s e ( D i z h o o r et al., 1991). R e c e n t l y , r e c o v e r i n has b e e n r e p o r t e d to p r o l o n g the rising p h a s e of t h e light r e s p o n s e , while having little to no effect on the kinetics o f r e s p o n s e recovery, suggesting t h a t it delays t e r m i n a t i o n o f the t r a n s d u c t i o n casc a d e r a t h e r t h a n affect resynthesis o f cyclic G M P and r e c o v e r t h e d a r k c u r r e n t ( G r a y - K e l l e r et al., 1993). S - m o d u l i n , a n o t h e r r e t i n a l m e m b e r of the r e c o v e r i n family, b i n d s to rod o u t e r s e g m e n t m e m b r a n e s a n d p r o l o n g s t h e light activation of cyclic G M P hydrolysis w h e n the free Ca 2+ c o n c e n t r a t i o n i n c r e a s e s to m o r e t h a n 1 p,M. T h e C a Z + - d e p e n d e n t r e g u l a t i o n o f phosp h o d i e s t e r a s e by S - m o d u l i n was f o u n d to b e m e d i a t e d by r h o d o p s i n p h o s p h o r y l a t i o n ( K a w a m u r a , 1993). P h o s p h o r y l a t i o n o f r e c e p t o r m o l e c u l e s m a y b e a general m e c h a n i s m for r e g u l a t i n g signal t r a n s d u c t i o n . Thus, t h e m e m b e r s of t h e recoverin family s e e m to be CaZ+-sensitive r e g u l a t o r s of i n t r a c e l l u l a r signal transd u c t i o n systems. Recently, h i p p o c a l c i n was f o u n d to associate with a cytosolic p r o t e i n of m o l e c u l a r mass 48 k D a ( H A P - 4 8 ) in a C a Z + - d e p e n d e n t m a n n e r by blot analysis using 35S-labeled h i p p o c a l c i n ( T a k a m a t s u et al., u n p u b l i s h e d data). H A P - 4 8 was p u r i f i e d from rat brain, a n d was i d e n t i f i e d as c r e a t i n e kinase B. 35S-labeled h i p p o c a l c i n a s s o c i a t e d with p u r i f i e d c r e a t i n e kinase B subunit, b u t not with p u r i f i e d c r e a t i n e kinase M from rat muscle. R e c o v e r y o f c r e a t i n e kinase activity in t h e p o s t m i c r o s o mal s u p e r n a t a n t fraction f r o m the h i p p o c a m p u s was slightly h i g h e r in the a b s e n c e o f Ca 2+ t h a n in the p r e s e n c e of Ca 2+. T h e s e results suggest t h a t h i p p o c a l cin transfers c r e a t i n e k i n a s e B to local p l a s m a m e m b r a n e s by d e t e c t i n g the e l e v a t e d cytosolic Ca 2+ levels, w h e r e c r e a t i n e kinase p r o v i d e s a s u s t a i n e d a n d local c o n c e n t r a t i o n of h i g h - e n e r g y p h o s p h a t e as is n e e d e d for the m a i n t e n a n c e o f ion g r a d i e n t s or for the contraction of the m o l e c u l a r m o t o r s in t h e d e n d r i t e s .
A l t h o u g h the physiological functions o f h i p p o c a l c i n r e m a i n obscure, it is r e a s o n a b l e to p o s t u l a t e that hipp o c a l c i n r e g u l a t e s synaptic efficacy in h i p p o c a m p a l pyramidal neurons through cooperative interactions with Ca 2 + a n d m e m b r a n e c o m p o n e n t s .
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Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H. and Miki, N.(1990) Visinin: a novel calcium binding protein expressed in retinal cone cells. Neuron, 2: 469-476. Zozulya, S. and Stryer, L (1992) Calcium-myristoyl protein switch. Proc. Natl. Acad. Sci. USA, 89: 11569-11573.