Molecular Brain Research, 15 (1992) 133-140
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70469
VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain Stefan E. Lenz
a
Yvonne
Henschel
a
Dieter
Gundelfinger
Zopf
b,1 B r i t t a
Voss
a and
Eckart
D.
a
a Zentrum fiir Molekulare Neurobiologie, Universitiit Hamburg, Hamburg (FRG) and b Zentrum fiir Molekulare Biologie, Unicersitiit Heidelberg, Heidelberg (FRG)
(Accepted 21 April 1992)
Key words: Calcium binding protein; EF-hand motif; Central nervous system; Retina; Development; In situ hybridization
Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca2+-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca2+-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca2+-dependent manner, are dicussed.
INTRODUCTION T h e Ca 2+ ion plays a central role in a variety of regulatory and signalling processes in n e u r o n s 17. In many cases the function of Ca 2 ÷ d e p e n d s on the interaction with specific intracellular Ca 2 +-binding proteins which may, for example, mediate the effects of Ca 2+ on enzyme activities or control the effective concentration of the ion. T h e s e intracellular Ca2+-binding proteins can be divided into two distinct groups (for review see ref. 13). T h e first group includes proteins, like calmodulin, troponin C, parvalbumin, the calbindins and m a n y others, with E F - h a n d motifs as Ca2+-binding structures. T h e second g r o u p are the annexins, a family of proteins whose interaction with phospholipids and cell m e m b r a n e s is triggered by Ca 2÷ ions 18. T o date m o r e than 150 m e m b e r s of the family of E F - h a n d proteins have b e e n identified from m a n y different organisms. T h e y can be g r o u p e d into discrete subfamilies with varying n u m b e r s of E F - h a n d motifs 23.
Some of these E F - h a n d proteins, like calmodulin, are ubiquitously expressed and exert functions in all tissues and cell types, whereas the expression of others is restricted to one or a few tissues. Several E F - h a n d proteins, including parvalbumin 2'3'4'8, calbindin D28K 1'5'I0'15'27'30'31 and calretinin 25'26, are highly expressed in the central nervous system, but their functional significance is rather unclear. Two very similar Ca2+-binding proteins, visinin and recoverin, are specifically expressed in p h o t o r e c e p t o r cells of the retina and pinealocytes 7'11'32. Recoverin was also functionally characterized 7'22. It stimulates retinal rod guanylate cyclase at low, but not at high, Ca 2+ levels thus promoting the recovery of the p h o t o r e c e p t o r s from light-induced hyperpolarization. T h e formation of synaptic connections and the development o f excitability and chemosensitivity is characterized by a n u m b e r of structural and physiological changes in nerve cells (some of which certainly are a c c o m p a n i e d by changes in the Ca 2+ metabolism).
Correspondence: E.D. Gundelfinger, ZMNH, Universitiitskrankenhaus Eppendorf, Pavilion 22, Martinistr. 52, D-2000 Hamburg 20, FRG. Fax: (49) 40-4717-4839. 1 Present address: Dept. of Biochemistry and Biophysics, Medical School, University of California, San Francisco, CA 94143, U.S.A.
134 A
C
B E7
P1
LP1
H
M
L
B
E7
E9
Ell
E13 E15
E17 E19 P1
I kb
kb
" ~ 2 . 9 kb
"ql.6
kb
"q 2.gkb
•,,m 1 . 6 k b
28S MB
~,~ ~js
Fig. 1. Developmental expression and tissue distribution of OZ1 transcripts. Northern blots containing 5/~g poly(A) RNA (A) and 20 p,g total RNA (B,C) per lane were probed with the 421 bp OZI cDNA. A: RNAs were isolated from optic tecta at day E7 (E7) and day P1 (P1) and from day P1 liver (LP1). B: R N A was isolated at day P1 from heart (H), skeletal muscle (M), liver (L) and total brain (B). C: RNAs were isolated from total brain at indicated days of development (E7-E19, PI). A Methylene blue stain (MB) of the cross-linked Northern blot 28 before hybridization was used to estimate the amounts of applied RNA.
Using a selective cloning approach to identify genes and gene products involved in this process of functional maturation we have previously isolated a set of cDNAs of transcripts which are newly expressed in chicken optic tectum between day 7 of embryogenesis (day E7) and hatching (day P1) 33. Here we report the identification of the polypeptide encoded by one of the isolated cDNAs as a potential EF-hand Ca2+-binding protein. Its expression is not restricted to the optic tectum, but can be found in many distinct regions of the chicken brain and its synthesis is up-regulated in a differentiation-specific manner. The novel gene product shares substantial sequence similarity with visinin 32 and recoverin 7. As visinin was the first member of this subfamily of EF-hand CaZ+-binding proteins the new protein was termed VILIP (for visinin-like protein).
MATERIALS AND METHODS
Isolation and sequencing of cDNA clones The OZ1 cDNA clone was isolated previously from a differentiation-specific cDNA library 3-~. A cDNA library in phage Agtl0, constructed from poly(A) RNA of day PI chicken optic lobes 33, was screened with the 421 bp cDNA insert of OZ1 radioactively labeled by hexanucleotide priming 9. Nine independent clones were obtained out of 5 × 105 phages, four of which were analyzed in more detail. They were subcloned into pBluescript vectors (Stratagene) and sequenced using a T7 sequencing kit (Pharmacia). The sequences of both strands of the cDNA corresponding to the 1.6 kb transcript were determined. Northern and Southern analyses Total RNA of various tissues and developmental stages was isolated by homogenizing the tissue in an aqueous solution of 6 M urea and 3 M LiC1 (1 ml per 100 mg of tissue). The homogenate was incubated for at least 4 h on ice and pelleted for 10 min at 13,000× g. The pellet was dissolved in 3 ml buffer A (50 mM Tris-CI, pH 7.5; 0.5 M EDTA; 1% SDS), extracted several times with
Fig. 2. A: physical map of OZ1 cDNAs and corresponding mRNAs. The open reading frame encoding VILIP is shown hatched. The putative translation initiation signal (AUG start codon is underlined) and the termination codon are indicated. The cDNA sequence corresponding to the 1.6 kb m R N A will appear in the EMBL, GenBank, DDBJ Nucleotide Sequence Databases under the accession number X63530. Restriction sites are: E, EcoR] (sites introduced during cloning are given in brackets), H, HindIII, N, NdeI, P, PstI. The original 421 bp OZ1 cDNA clone contained the restriction fragment flanked by the two internal EcoRI sites. B: alignment of the predicted amino acid sequence of VILIP with the sequences of visinin 32 and recoverin 7. Amino acid residues that are identical at the same position in all three polypeptides are marked by an asterisk• Gaps included to maximize the alignment are denoted as dashes. Amino acid residues are numbered for each protein• The extent of the EF-hand motifs is indicated by a solid line above the VILIP sequence. C: alignment of the VILIP EF-hand motifs. Amino acid numbering corresponds to that in B. The structural features of the EF-hands, i.e. the amino acid side chains coordinating Ca2+-binding (X,Y,Z,-X,-Y,-Z), the consensus amino acid sequence (n, hydrophobic amino acid: - , any amino acid) and the extent of the loop and the helices 21. are indicated.
135
A cDNA map
1.6 kb mRNA
(E) P
N
EP
PE
H
(E)
II
I
II
II
I
I
CAGAAUGG
UGA
I
,
l
200 bp
I
2.9 kb mRNA
B MGKQ-NSKLAPEVMEDLVKSTEFNmHELKQW~KGFLKDCPSGRLNLEEFQ VILIP Visinin MGNSRSSALSREVLQELRASTRYTEEELSRWYEGIQRQCSDG~IRCDEFE Recoverin MGNSKSGALSKEILEELQLNTKFTEEELSSWYQSFLKECPSGRITRQEFQ **
*
*
*
*
*
**
**
*
*
**
49 50
50
**
QLYVKFFPYGDASKFAQHAFRTFDKNGDGTIDFRRFICALSITSRGSFEQ RIYGNFFPNSEPQGYARHVFRSFDTHDDGTLDFRBYIIALHLTSSGKTHL TIYSKFFPEADPKAYAQRV~RSFDANSDGTLDFKEYVIALHMTSAGKTNQ
99 100 100
KLNWAFNMYDLDGDGKITRVEMLEIIEAIYK~aVGTVIMMKMNEDGLTPEQ
149 150 150
KLE~&FSLFDVDRNGEVSKSEVLEIITAIF~IPEEERLQLPEDENSPQK KLEN~FSLYDVDGNGTISKNEVLEIVTAIF~ISPEDTKHLPEDENTPEK **
***
*
*
*
*
***
**
**
**
*
RVDKIFSKMDKNKDDQITLDEFKEAAKSDPSIVLLLQCDIQK RADKLWAYFNKGENDKIAEGEFIDGVMKNDAIMRLIQYEPKK RAEKIWGFFGKKDDDKLTEKEFIEGTLANKEILRLIQFEPQKVKEKLKEKKL *
*
*
*
**
*
*
*
191 192 202
*
C
helix--> I J
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70469
VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain Stefan E. Lenz
a
Yvonne
Henschel
a
Dieter
Gundelfinger
Zopf
b,1 B r i t t a
Voss
a and
Eckart
D.
a
a Zentrum fiir Molekulare Neurobiologie, Universitiit Hamburg, Hamburg (FRG) and b Zentrum fiir Molekulare Biologie, Unicersitiit Heidelberg, Heidelberg (FRG)
(Accepted 21 April 1992)
Key words: Calcium binding protein; EF-hand motif; Central nervous system; Retina; Development; In situ hybridization
Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca2+-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca2+-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca2+-dependent manner, are dicussed.
INTRODUCTION T h e Ca 2+ ion plays a central role in a variety of regulatory and signalling processes in n e u r o n s 17. In many cases the function of Ca 2 ÷ d e p e n d s on the interaction with specific intracellular Ca 2 +-binding proteins which may, for example, mediate the effects of Ca 2+ on enzyme activities or control the effective concentration of the ion. T h e s e intracellular Ca2+-binding proteins can be divided into two distinct groups (for review see ref. 13). T h e first group includes proteins, like calmodulin, troponin C, parvalbumin, the calbindins and m a n y others, with E F - h a n d motifs as Ca2+-binding structures. T h e second g r o u p are the annexins, a family of proteins whose interaction with phospholipids and cell m e m b r a n e s is triggered by Ca 2÷ ions 18. T o date m o r e than 150 m e m b e r s of the family of E F - h a n d proteins have b e e n identified from m a n y different organisms. T h e y can be g r o u p e d into discrete subfamilies with varying n u m b e r s of E F - h a n d motifs 23.
Some of these E F - h a n d proteins, like calmodulin, are ubiquitously expressed and exert functions in all tissues and cell types, whereas the expression of others is restricted to one or a few tissues. Several E F - h a n d proteins, including parvalbumin 2'3'4'8, calbindin D28K 1'5'I0'15'27'30'31 and calretinin 25'26, are highly expressed in the central nervous system, but their functional significance is rather unclear. Two very similar Ca2+-binding proteins, visinin and recoverin, are specifically expressed in p h o t o r e c e p t o r cells of the retina and pinealocytes 7'11'32. Recoverin was also functionally characterized 7'22. It stimulates retinal rod guanylate cyclase at low, but not at high, Ca 2+ levels thus promoting the recovery of the p h o t o r e c e p t o r s from light-induced hyperpolarization. T h e formation of synaptic connections and the development o f excitability and chemosensitivity is characterized by a n u m b e r of structural and physiological changes in nerve cells (some of which certainly are a c c o m p a n i e d by changes in the Ca 2+ metabolism).
Correspondence: E.D. Gundelfinger, ZMNH, Universitiitskrankenhaus Eppendorf, Pavilion 22, Martinistr. 52, D-2000 Hamburg 20, FRG. Fax: (49) 40-4717-4839. 1 Present address: Dept. of Biochemistry and Biophysics, Medical School, University of California, San Francisco, CA 94143, U.S.A.
134 A
C
B E7
P1
LP1
H
M
L
B
E7
E9
Ell
E13 E15
E17 E19 P1
I kb
kb
" ~ 2 . 9 kb
"ql.6
kb
"q 2.gkb
•,,m 1 . 6 k b
28S MB
~,~ ~js
Fig. 1. Developmental expression and tissue distribution of OZ1 transcripts. Northern blots containing 5/~g poly(A) RNA (A) and 20 p,g total RNA (B,C) per lane were probed with the 421 bp OZI cDNA. A: RNAs were isolated from optic tecta at day E7 (E7) and day P1 (P1) and from day P1 liver (LP1). B: R N A was isolated at day P1 from heart (H), skeletal muscle (M), liver (L) and total brain (B). C: RNAs were isolated from total brain at indicated days of development (E7-E19, PI). A Methylene blue stain (MB) of the cross-linked Northern blot 28 before hybridization was used to estimate the amounts of applied RNA.
Using a selective cloning approach to identify genes and gene products involved in this process of functional maturation we have previously isolated a set of cDNAs of transcripts which are newly expressed in chicken optic tectum between day 7 of embryogenesis (day E7) and hatching (day P1) 33. Here we report the identification of the polypeptide encoded by one of the isolated cDNAs as a potential EF-hand Ca2+-binding protein. Its expression is not restricted to the optic tectum, but can be found in many distinct regions of the chicken brain and its synthesis is up-regulated in a differentiation-specific manner. The novel gene product shares substantial sequence similarity with visinin 32 and recoverin 7. As visinin was the first member of this subfamily of EF-hand CaZ+-binding proteins the new protein was termed VILIP (for visinin-like protein).
MATERIALS AND METHODS
Isolation and sequencing of cDNA clones The OZ1 cDNA clone was isolated previously from a differentiation-specific cDNA library 3-~. A cDNA library in phage Agtl0, constructed from poly(A) RNA of day PI chicken optic lobes 33, was screened with the 421 bp cDNA insert of OZ1 radioactively labeled by hexanucleotide priming 9. Nine independent clones were obtained out of 5 × 105 phages, four of which were analyzed in more detail. They were subcloned into pBluescript vectors (Stratagene) and sequenced using a T7 sequencing kit (Pharmacia). The sequences of both strands of the cDNA corresponding to the 1.6 kb transcript were determined. Northern and Southern analyses Total RNA of various tissues and developmental stages was isolated by homogenizing the tissue in an aqueous solution of 6 M urea and 3 M LiC1 (1 ml per 100 mg of tissue). The homogenate was incubated for at least 4 h on ice and pelleted for 10 min at 13,000× g. The pellet was dissolved in 3 ml buffer A (50 mM Tris-CI, pH 7.5; 0.5 M EDTA; 1% SDS), extracted several times with
Fig. 2. A: physical map of OZ1 cDNAs and corresponding mRNAs. The open reading frame encoding VILIP is shown hatched. The putative translation initiation signal (AUG start codon is underlined) and the termination codon are indicated. The cDNA sequence corresponding to the 1.6 kb m R N A will appear in the EMBL, GenBank, DDBJ Nucleotide Sequence Databases under the accession number X63530. Restriction sites are: E, EcoR] (sites introduced during cloning are given in brackets), H, HindIII, N, NdeI, P, PstI. The original 421 bp OZ1 cDNA clone contained the restriction fragment flanked by the two internal EcoRI sites. B: alignment of the predicted amino acid sequence of VILIP with the sequences of visinin 32 and recoverin 7. Amino acid residues that are identical at the same position in all three polypeptides are marked by an asterisk• Gaps included to maximize the alignment are denoted as dashes. Amino acid residues are numbered for each protein• The extent of the EF-hand motifs is indicated by a solid line above the VILIP sequence. C: alignment of the VILIP EF-hand motifs. Amino acid numbering corresponds to that in B. The structural features of the EF-hands, i.e. the amino acid side chains coordinating Ca2+-binding (X,Y,Z,-X,-Y,-Z), the consensus amino acid sequence (n, hydrophobic amino acid: - , any amino acid) and the extent of the loop and the helices 21. are indicated.
135
A cDNA map
1.6 kb mRNA
(E) P
N
EP
PE
H
(E)
II
I
II
II
I
I
CAGAAUGG
UGA
I
,
l
200 bp
I
2.9 kb mRNA
B MGKQ-NSKLAPEVMEDLVKSTEFNmHELKQW~KGFLKDCPSGRLNLEEFQ VILIP Visinin MGNSRSSALSREVLQELRASTRYTEEELSRWYEGIQRQCSDG~IRCDEFE Recoverin MGNSKSGALSKEILEELQLNTKFTEEELSSWYQSFLKECPSGRITRQEFQ **
*
*
*
*
*
**
**
*
*
**
49 50
50
**
QLYVKFFPYGDASKFAQHAFRTFDKNGDGTIDFRRFICALSITSRGSFEQ RIYGNFFPNSEPQGYARHVFRSFDTHDDGTLDFRBYIIALHLTSSGKTHL TIYSKFFPEADPKAYAQRV~RSFDANSDGTLDFKEYVIALHMTSAGKTNQ
99 100 100
KLNWAFNMYDLDGDGKITRVEMLEIIEAIYK~aVGTVIMMKMNEDGLTPEQ
149 150 150
KLE~&FSLFDVDRNGEVSKSEVLEIITAIF~IPEEERLQLPEDENSPQK KLEN~FSLYDVDGNGTISKNEVLEIVTAIF~ISPEDTKHLPEDENTPEK **
***
*
*
*
*
***
**
**
**
*
RVDKIFSKMDKNKDDQITLDEFKEAAKSDPSIVLLLQCDIQK RADKLWAYFNKGENDKIAEGEFIDGVMKNDAIMRLIQYEPKK RAEKIWGFFGKKDDDKLTEKEFIEGTLANKEILRLIQFEPQKVKEKLKEKKL *
*
*
*
**
*
*
*
191 192 202
*
C
helix--> I J
