Identification of a neuronal calcium sensor (NCS-1

Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
J . OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 15(1-4), 365-378 (1995)
IDENTIFICATION OF A NEURONAL CALCIUM SENSOR (NCS-1)
POSSIBLY INVOLVED IN THE REGULATION OF
RECEPTOR PHOSPHORYLATION
S. Nef, H. Fiumelli, E. de Castro, M-B. Raes', and P. Nef*
'lnstitut Pasteur de Lille, URA 1160
1, rue Calmette, 59019 Lille, France
START Unit, Department of Biochemistry, University of Geneva
30, quai Ernest-Ansermet, 1211 Geneve 4, Switzerland
ABSTRACT
Persistent stimulation of G protein-coupled receptors by agonists leads
rapidly to reduced responses, a phenomenon described as
desensitization. It involves primarily the phosphorylation of receptor sites
by specific kinases of the G protein-coupled receptor kinase (GRK) family.
The P-adrenergic receptor kinase 1 (GRK2) desensitizes agonist-activated
P2-adrenergic receptors, whereas
rhodopsin kinase
(GRK1)
phosphorylates and inactivates photon-activated rhodopsin. Little is known
about the role of calcium in desensitization. Here we report the
characterization of a novel neuronal calcium sensor (NCS) named NCS-1
possibly involved in the regulation of receptor phosphorylation. NCS-1 is a
new member of the EF-hand superfamily, which includes calmodulin,
troponin C, parvalbumin, and recoverins. By Northern analysis and in situ
hybridization, we discovered that NCS-1 is specifically expressed in the
central and peripheral nervous systems. Chick NCS-1 has 72% of amino
acid identity with Drosophila frequenin, a protein found in the nervous
system and at the motor nerve terminals of neuromuscular junctions. By
analogy with the reported function for two other members of the NCS
family, we discuss whether G protein-coupled receptors or GRKs are the
targets of neuronal calcium sensors.
365
Copyright 0 1995 by Marcel Dekker, Inc.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
366
NEF ET AL.
FIG. 1 Schematic representation of the neuronal calcium sensor NCS-1,
with 4 putative EF hand calcium binding domains. N and C represent the
N-terminus and the C-terminus of the protein, respectively. EF1 (white
box) has a cysteine residue (at position Y, see Fig. 2) instead of the
consensus aspartic acid, and therefore, represents a non-functional
calcium binding site. EF2, 3 and 4 (black or gray boxes) represents
potential calcium binding sites.
INTRODUCTION
Calcium is an important regulator of many cells, including neurons.
Recently, a novel subfamily of neuronal calcium sensors (NCS) has been
identified. The first member of this subfamily of EF-hand calcium binding
proteins to be discovered was visinin ( l ) , followed by recoverin (2),vilip
(3),and S-modulin (4). To date, nine full-length cDNAs encoding neuronspecific proteins compose this family of neuronal calcium sensors.
Recoverin is expressed in photoreceptors and in the pineal gland
(5), and is a globular protein whose three-dimensional structure has been
solved by crystallography studies (6). Recoverin crystals contained only 1
calcium although 4 putative EF-hands are predicted from the primary
structure (Fig. 1). Homologues of bovine, mouse and human recoverins
have been characterized from frog (S-modulin), and chick (visinin) retinas.
S-modulin and recoverin are the best functionally characterized neuronal
calcium sensors. They both stimulate, in a calcium-dependent manner,
the cGMP-phosphodiesterase in frog rod photoreceptors (7) via the
inhibiton of rhodopsin phosphorylation (8). Calcium-dependent effects on
receptor desensitization mediated by neuronal calcium sensors have not
yet been fully documented. Could other neuronal calcium sensors, beside
those expressed in the retina, influence G protein-coupled receptor
desensitization in the central and peripheral nervous system?
To answer this question, we decided to search for neuronal calcium
sensors related to the recoverin subfamily, that are specifically expressed
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
NEURONAL CALCIUM SENSOR
367
in the nervous system. Here we report the identification of NCS-1, a novel
member of the neuronal calcium sensor family. Its expression is neuronspecific, and can be found in several structures of the central and
peripheral nervous systems. NCS-1 shares 72% of amino acid identity
with Drosophila frequenin (9). Preliminary data indicate that, at high
calcium, NCS-1 inhibits G protein-coupled receptor phosphorylation; this
suggests an important role for NCS-1 in signal transduction and perhaps
in synaptic potentiation mediated by calcium.
MATERIALS AND METHODS
Highly degenerated primers were used for PCR amplification. They
code for conserved amino acid sequences of three members of the
recoverin subfamily (vilip, visinin, and recoverin). The sense primer (5'-3
'sequence: GGGAATTC-T A T/C I G/C I A A I T T T/C T T T/C C C) and
the antisense primer (CGGGATCC-A T/C 6 A T T/C T T A/G T/A A I A T I
G C) contained a restriction site for EcoRl and BamHI, respectively. Chick
brain cDNA was prepared from 1 pg of total RNA with the reverse
transcriptase (RT) enzyme. 1/10 of the cDNA was amplified by PCR using
a slow ramp (IOU6 seconds) program for the first five cycles (steps were
1' at 92°C 1' at 35"C, slow ramp to 72"C, 3' at 72"C), followed by 35
cycles (1' at 92°C 1' at 50"C, 3' at 72°C) using a normal ramp. The RTPCR product was analyzed by agarose gel electrophoresis, and DNA with
the expected size was eluted from the gel, digested with the restriction
enzymes EcoRl and BamHI, and subcloned in the pBluescript (pBSK-)
vector (Stratagene). Both strands were sequenced according to the
dideoxy method of Sanger (10) using T3 and T7 primers.
cDNA Library Screening
lo6
phages from a chick brain cDNA library were plated on the E. Coli
strain LE392, and screened at high stringency (68°C) with a [32P]
radioactive NCS-1 probe obtained from the RT-PCR experiment. 10
368
NEF ET AL.
positive phages were characterized and their insert DNA subcloned in
pBSK- (Stratagene), and sequenced using the Sanger method (10).
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
Phvloaenetic Tree Analvsis
Progressive alignments of amino acid sequences were obtained with
Pileup (Genetics Computer Group, Madison, Wisconsin, USA). The
calculated branch lengths were obtained with the TREE program (R.F.
Doolittle, UCSD, La Jolla, USA) running on an AXPNMS system; distance
scores between each pair of aligned sequences was determined by using
the Minimum Mutation Matrix of Dayhoff (1 1).
mRNA Blot Analvsis
6 pg of total RNA obtained from diverse adult chick tissues were
electrophoresed on a 0.8% formaldehyde/agarose gel, and transferred to
Nylon membrane (Genescreen Plus). The blot was hybridized with a
[32P]-labelled chick NCS-1 probe at high stringency (68°C) for 18 hours
and washed at 50°C in 0.1 XSSC. Autoradiography was performed with an
intensifying screen for 3 days at -70°C. The autoradiogram was scanned
and a value of 1 was assigned to the signal obtained with brain.
In Situ Hvbridization
Chick brains were dissected from E l 8 embryos, slowly freezed in dry ice,
and sectionned on a cryostat at -20°C. 10 pm sections were fixed for 15
minutes in 4% paraformaldehyde, rinced in 1XPBS, dehydrated in ethanol,
and stored at 4°C in 95% ethanol until use. Before hybridization, sections
were re-hydrated, digested for 15 minutes at 37°C with proteinase K at
1pg/ml, post-fixed for 15 minutes in 4% paraformaldehyde, acetylated for
10 minutes in O.1M triethanolamine, and dehydrated in ethanol. [ass]
radioactive antisense or sense RNA were produced by in vitro
transcription with T3 or T7 RNA polymerase from the linearized RL25
cDNA clone (930 base pairs) encoding chick NCS-1. The denatured probe
was added to the hybridation mix (NaCI 0.3M, Tris-HCI 0.02M pH 8.0,
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
NEURONAL CALCIUM SENSOR
369
EDTA 5 mM, Dextran sulfate lo%, Denhardt's lX,tRNA 0.5 mg/ml, DTT
O.lM, formamid 50%, and the radioactive probe at 50'000 cpm/ml) and
the sections were hybridized for 16 hours at 60"C, washed for 30 minutes
at 65°C in a stringent wash solution (NaCI 0.15M, Tris-HCI 0.02M pH 7.6,
EDTA 5 mM, DTT 0.1M), followed by RNAse treatment for 1 hour at 37°C
with RNAse A at 20 pg/mI. Final washes were done in 0.1X SSC for 15
minutes at 60"C, and the sections were dehydrated in ethanol, dipped in
NTB2 (Kodak) emulsion for 4-7 days, developed, mounted and
photographed using a Zeiss inverted microscope. Control sections were
prepared with a sense NCS-1 probe.
RESULTS
By PCR of chick brain cDNA with highly degenerated primers derived
from conserved amino acid sequences of three members of the recoverin
subfamily, we have amplified and sequenced several partial sequences
encoding neuronal calcium sensors. One of the PCR product encoded a
new calcium binding protein. Using this probe, we have screened a cDNA
library obtained from chick brain. We have isolated and characterized
several full-length cDNA clones. All of them, like the cDNA clone RL25,
had an open reading frame of 573 nucleotides from the start codon (AUG)
to the stop codon (TGA); it encodes a neuronal calcium binding protein
named NCS-1. Alignment of NCS-1 with nine other members of this
subfamily is shown in Fig. 2. Comparison of amino acid sequences (Table
1) revealed that Drosophila frequenin has the highest homology (72% of
identity) with chick NCS-1, suggesting that they are probably species
variants. The lowest homology (40%) is with chick visinin.
Evolutionary tree analysis (Fig. 3) indicates that all members of the
recoverin subfamily evolved from a common ancestoral gene, with
probably 4 EF-hand Ca" binding sites (Fig. 1). EF-hand site 1 is quite
divergent from the EF-hand consensus sequence but is very well
conserved in this subfamily (Fig. 2). One branch of the tree represents
members (Visinin, S-modulin, Recoverin) expressed only in the retina and
the pineal gland, another branch represents calcium sensors with broader
neuronal distribution except for hippocalcin uniquely expressed in the
hippocampus.
NEF ET AL.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
370
1
x Y
VISININ (Chick)
MGnsrSsaLs rEvlqeLras TryTEeEisr WeGFqrqCa
RgCOVERIN (Bovine)
MGnskSgaLs kEileeLqlr TkFTEeElss
VILIP (Chick/Rat)
MGKqnS.KLa pEvmedivks TeFnEhElkq
NEUROCALCIN (Rat)
MGKqnS.KLr pOjmqdLles TdFTEhE1q.s
S-MODULIN (Frog)
MGntkSgaLs kEileeLqln TkFTqeElc:
HIPPOCALCIN (Rat)
MGKqnS.KLr pEm1qdLre:- TeFaE1EiGe
MGKkaS.Kik qdtidrlttd TyYP?EZEir5
PREQWNIN (Droao.)
MGKnnS.Kia pZeledLvqr TeE‘aZqZlkG
VILIP-2 (Rat)
VILIP-3 (Rat)
MGKqnS.KLr pEv?qdLreh TeF?&?Ziqe
-53
MGKsnS.Uk pEweeLtrk TyFTEXEvqq
NCS-1 (Chick)
Consensus MGK-nS-KL- -Evl-dL--- T-XE-Ei-- W Y K G P X X S
x Y
c;1
VISININ (Chick)
RECOVERIN (Bovine)
VILIP (Chick/Rat)
NEUROCALCIN (Rat)
S-MODULIN (Frog)
AIPPOCALCIN (Rat)
PREQWNIN (Droso.)
VILIP-2 (Rat)
VILIP-3 (Rat)
NCS-1 (Chick)
Consensus
r1YgnFFPr.s
tIYskFFPea
qlYvkFFPyG
kIYgnFFFyG
aIYskFFFda
kIYanFF?yG
kIYkqFFPqG
qlYikFFPyG
kIYanFFPyG
kIYkqFFFfG
kIY--PPP-G
.01
x
KLeWAFsifD
VISININ (Chick)
KLeWAFslYD
RECOVERIN (Bovine)
KLnWAFnmYD
VILIP (chick/Rat)
KLkWAFsmYD
NEUROCALCIN (Rat)
S-MODULIN (Frog) KLeWAFclYD
KLmWAFsmYD
HIPPOCALCIN (Rat)
KLqWAFrlYD
PREQWNIN (Droso.)
KLnWAFemYD
VILIP-2 (Rat)
KLkWAFsmYD
VILIP-3 (Rat)
KLrWAFklYD
NCS-1 (Chick)
consensus KL-WAF-1YD
151
VISININ (Chick)
RECOVERIN (Bovine)
VILIP (Chick/Rat)
NEUROCALCIN (Rat)
9-MODULIN (Frog)
HIPPOCALCIN (Rat)
PRBQWNIN (Droso.)
VILIP-2 (Rat)
VILIP-3 (Rat)
NCS-1 (Chick)
Consensus
x
RadKlwayfn
RaeKIwgffg
RvdKIFskMD
RteKIFrqMD
RtnKIwvyfg
RteKIFrqMD
RvdKIFdqMD
RvdKIFkkMD
RtdKIFrqMD
RvdrIFamMD
R-dKIF--MD
epqgyArHVF
3pkayAqHVF
DaskFAqHaF
DaskFAeHVF
DpkayAqHVF
DaskFAeHVF
DpskFAslVF
DaskFAqHaF
DaskFAeHVF
DptkFAtfVF
DpskFA-HVP
EF2
2-Y
-x
-2
EF1
2-Y-x - 2 5 5
dGrircdeFe
sGqLdaagE;
SG-L---eP-
. ..
- L r
hlTSaGkthl
hmTSaGKtn;
SiTSrGsfes:
SvTSrGkleq
mm?SsGkanc
IDFrEFIikL SvTSrGriiC
RfeDeNnDGs IeFeEFIrh: SvTSkr;nlde
RiF;):kVgDGT IDFrEFicAL S-JTSrGsfe,::
KtFDtNsDGT I3FrEF::AL
SvTSrt2kles
nvFDeNkDGr IeFaEFIqAL Sv3rSi:de
F.-?D-N-DDT IDPrEFIiAL SVTSZG-le;
RsFCtNdDGT
RsF3aPjsDGT
Rt?C!d’g3GT
RtF3aNgDGT
RsFDaNn3GT
RcF3:NaDGT
EF3
Y 2-Y-x - 2
vDrnGevsks E v L E T i t A i f
vDgnGtIskn EvLEIvtAIf
lDgdGkItrv EMLEZleAiY
iDgnGyIska EMLEIvqAIY
vDgnGtInkk EvLEIitAIf
1DgnGyIsre EMLEIvqAZY
vDndGyItre EMynivdAIY
lDgdGrItr1 EMLEIieAIY
lDgnGyIsrs EMLEIvqAiY
lDndGyItrn EMLdIv6AZY
1Dg-Q-I-r- BMLEIV-AIY
EF4
Y 2-Y-x - 2
kgendKiaeg E ? : d m n d
kkdDdKLtek ZF;ejtiank
knkDdqitLd EFktaaiCsDP
tnrDgKLaLe EFirqaKsDP
kkdDdKLteg EFiggivknk
tnnDgKLsLe EFirgaKs3P
knhDgKLtLe EFregsKaD?
qdkDdqitLe EFkeaaKsD?
tnnDgKLaLe EFikgaKsDP
knaDgKLtLq EFqegsKaDP
kn-D-KLtLe EPiegaiC-DP
1DFrEYI;AL
1DFkEXviAL
:DFrEFIcAL
IDFrEFIiAL
1DFkEYm:AL
~
: r
_ _ <
KMipeeerlq IPEDEnaPqK
IcMiSpe”,t<r.;ZEDEnT?ES
KMVgtv;?;lk nnE3glT?Eq
KMVssv..:.k aPEDEaT?HX
KMinaedr;<:. 1PEDEnTFEK
sZEDE8XZR
KMVSSV..T;,X
qMVgqq..gc, .sZ3,Er2TPqK
KMVgCviT>,r PaqDglTPqq
KMVSSV..T,< .:?ZDEa?PSR
qMVgnt . .:-e :?EeEn??ER
KMV------- -PEDE:.TPZR
...
L L
aImrLi>!e;
-
~f...........
eIlrLicfey; , ; s v X i k l s t L < l
sIViLL3c61 5 ,. .~. . . . . . . .
siVrliCcc~saaJq: . . . . . .
eIlrLiQyep znvkdk;kt.<kr
SIVriLCicJs ssasqf . . . . . .
rIVqaLaigg J...........
sIVlLL2cdm y i x . . . . . . . . . .
s1VrLLC;cdp ssasqf . . . . . .
sIVqaLalyd glv . . . . . . . . .
sIVrLLQ--p - - - - - - - - - - - -
FIG. 2 Alignment of ten neuronal calcium sensor amino acid sequences.
Sequences were obtained from EMBL and Swissprot data bases; NCS-1
accession number is L27420. X, Y, Z and -X, -Y, -Z represent the
positions of amino acids around a calcium ion in an EF-hand.
NEURONAL CALCIUM SENSOR
37 1
Table 1. Percentage of amino acid identity between ten members of the
neuronal calcium sensor subfamily.
NCSl VILIB VILIPZ FREQUENIN
SMOOULIN
NEUROCALCIN VlLlPl
40
46
49
51
40
46
30
43
47
51
62
RECOVERIN
83
49
51
VILIP-1
57
69
89
53
67
46
69
NEUROCALC IN
58
90
66
57
87
49
SMODULIN
43
48
45
41
47
HIPPOSALC IN
93
50
67
68
53
57
VILIP-2
57
72
55
VILIP-3
59
VlSlNlN
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
HIPPEALGIN
FREQUENIN
41
47
RECOVERIN
63
I
I
I
I
I
Common
Ancestor
FIG. 3 Phylogenetic tree for the neuronal calcium sensor (NCS) subfamily.
The relationship of a set of genetic sequences was deduced from a tree
analysis which gives more information than analysis based on percent
identity or homology. The general topology of the tree gives information
on how the sequences should be grouped, while evolutionary distances
separating different sequences are shown by the branch lengths.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
372
NEF ET AL.
Using RNA blot analysis with the NCS-1 probe (Fig. 4) and PCR (data
not shown) we observed that neuronal tissues exhibit strong NCS-1
mRNA expression, whereas liver, heart, muscle, and lung do not. We
observed also a faint signal with intestines, probably due to the peripheral
nervous ganglia present in this tissue. In brain and eye extracts, a single
protein of 22kDa was detected by protein blot analysis with
immunopurified polyclonal antibodies against recombinant NCS-1 (data
not shown). Only one copy of the NCS-1 gene was found in the chick
genome after extensive PCR amplification, cDNA screening, and genomic
DNA blot analysis.
Using in situ hybridization, we observed that antisense RNA NCS-1
probes labelled intensively several structures in the central (Fig. 5-7) and
peripheral nervous system such as spinal ganglia, and motoneurons (data
not shown).
A sagittal section of the chick brain (Fig. 5a) clearly indicated that NCS1 mRNA is present in the cerebellum, the forebrain, the brain stem, and
the midbrain. Control experiment with a NCS-1 sense probe (Fig. 5b, 6b,
7b) indicated, as expected, no signal in these structures.
In the retina at E l 8 (Fig. 6a), a strong signal for NCS-1 was detected in
three different layers: the ganglion cell layer (GCL), the inner nuclear layer
(INL), and the outer nuclear layer (ONL). The control section hybridized
with a sense NCS-1 probe was negative (Fig. 6b).
In the optic tectum (Fig. 7a) a strong patchy signal was detected in the
stratum griseum centrale (SGC), and a faint signal in the laminae c, g, and
i of the stratum griseum et fibrosum superficiale (SGFS). The lowest
structure of the optic tectum, the neuroepithelium gave also a patchy
signal.
In the cerebellum, when observed at higher magnification (data not
shown), NCS-1 mRNA was mostly detected between the granular layer
(GL) and the molecular layer (ML) which correspond either to Purkinje
cells or to basket cells.
DISCUSSION
In this work, we have reported the RT-PCR cloning and the neuronal
distribution of chick NCS-1, a novel calcium binding protein of the
373
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
NEURONAL CALCIUM SENSOR
FIG. 4 Tissue distribution of NCS-1. An abundant signal corresponding to
mRNA of 3000 bases is present in chick brain, cerebellum, and at a lower
level in eye. A faint signal (-1-3% of the brain signal) was reproducibly
detected either by Northern analysis or by PCR with total RNA from
intestines. A scan of the autoradiogramm is shown.
NEF ET AL.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
374
Fia. 5 NCS-1 in situ hybridization to the avian brain. Sagittal sections
through the telencephal, mesencephal and cerebellum of E l 8 chick brain
were hybridized with antisense (A) and sense (B) NCS-1 probes.
375
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
NEURONAL CALCIUM SENSOR
FIG. 6 NCS-1 in situ hybridization to the avian retina. Sections of the chick
retina at E l 8 were hybridized with antisense (A) and sense (B) NCS-1
probes. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner
nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer;
photoreceptor layer; PE, pigmented epithelium.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
376
NEF ET AL.
FIG. 7 NCS-1 in situ hybridization to the avian optic tectum. Antisense (A)
and sense (B) NCS-1 probes were hybridized with sections from the chick
optic tectum at E18. NE, neuroepithelium; SAC, stratum album centrale;
SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum
superficiale (laminae c, g, i); SO, stratum opticum.
recoverin subfamily. To date, ten members of the neuronal calcium sensor
family have been identified. Each member appears to be expressed by a
unique combination of neurons. A good illustration is hippocalcin which is
present only in the hippocampus. In the cerebellum, vilip is abundant in
the granular layer but absent from basket or Purkinje neurons (3),
whereas it is the opposite for NCS-1 mRNA which is abundant in basket
or Purkinje cells and less abundant granular cells (Fig. 5). In the retina,
visinin is restricted to the outer nuclear layer (ONL) ( l ) ,vilip is present in
the gangion cell layer (GCL) and in the inner nuclear layer (INL) (3),
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
NEURONAL CALCIUM SENSOR
377
whereas NCS-1 is present in all three layers (ONL, INL, GCL) (Fig. 6). It
remains to be determined by electronic microscopy studies where these
neuronal calcium sensors are located within a neuron (presynaptic?, cell
body?, post-synaptic densities?).
NCS-1 is probably the avian homologue of Drosophila Frequenin (72%
of amino acid identity) whose central and peripheral neuronal location has
been reported (9). The overexpression of frequenin in transgenic flies
produces a shaker-like phenotype; it has been proposed that it is due to
an enhanced facilitation of neurotransmitter release at the neuromuscular
junction. However, it is not yet clear how a neuronal calcium sensor such
as frequenin could function at the synapse between a motor neuron and a
muscle; does frequenin regulate the phosphorylation of a voltage-gated
calcium channels or play a role in the calcium-dependent fusion of
synaptic vesicles with the pre-synaptic membranes?
Among the neuronal calcium sensor subfamily, frog S-modulin and
bovine recoverin (43 to 47% of amino acid identity with chick NCS-1) are
the best characterized neuronal calcium sensors with respect to function.
At high Ca2' concentrations, they increase the light sensitivity and lifetime
of cyclic GMP-phosphodiesterase in frog rod photoreceptors (7). These
effects are mediated by the inhibition of rhodopsin phosphorylation in a
calcium-dependent manner (8). If NCS-1 exhibits the same function as Smodulin and recoverin, it is very likely that these neuronal calcium sensors
act on G protein-coupled receptor kinases (GRKs). Indeed, it has recently
been demonstrated that bovine p26 kDa recoverin can form a calciumsensitive complex of 94 kDa with a protein the size of rhodopsin kinase
(67 kDa) (12) and that up to the last stage of rhodopsin kinase purification,
recoverin is present as the main contaminant (13).
ACKNOWLEDGMENTS
This work was supported by a grant from the Swiss National
Foundation (31.32623.91). P.N. is a START fellow of the Swiss National
Foundation. We would like to thanks Bernard Vandenbunder to help us
with the in situ experiments, and Marie-Claire Velluz for the subcloning
and DNA sequencing.
NEF ET AL.
378
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by Hug Belle Idee
For personal use only.
REFERENCES
1. Yamagata, K.; Goto, K.; Kuo, C. H.; Kondo, H. and Miki, N. Visinin: a
novel calcium binding protein expressed in retinal cone cells. Neuron
4, 469-76, 1990
2. 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. Recoverin: a
calcium sensitive activator of retinal rod guanylate cyclase. Science
251, 915-8, 1991
3. Lenz, S. E.; Henschel, Y.; Zopf, D.; Voss, B. and Gundelfinger, E. D.
VILIP, a cognate protein of the retinal calcium binding proteins visinin
and recoverin, is expressed in the developing chicken brain. Brain Res
Mol Brain Res 15, 133-40, 1992
4. Kawamura, S.; Takamatsu, K. and Kitamura, K. Purification and
characterization of S-modulin, a calcium-dependent regulator on
cGMP phosphodiesterase in frog rod photoreceptors. Biochem
Biophys Res Commun 186, 41 1-7, 1992
5. Korf, H. W.; White, B. H.; Schaad, N. C. and Klein, D. C. Recoverin in
pineal organs and retinae of various vertebrate species including man.
Brain Research 595, 57-66, 1992
6. Flaherty, K. M.; Zozulya, S.; Stryer, L. and McKay, D. B. Threedimensional structure of recoverin, a calcium sensor in vision. Cell 75,
709-716, 1993
7. Kawamura, S.; Hisatomi, 0.;Kayada, S.; Tokunaga, F. and Kuo, C. H.
Recoverin has S-modulin activity in frog rods. J Biol Chem 268, 1457982, 1993
8. Kawamura, S. Rhodopsin phosphorylation as a mechanism of
cyclic GMP phosphodiesterase regulation by S-modulin. Nature 362,
855-7, 1993
9. Pongs, 0.;Lindemeier, J.; Zhu, X. R.; Theil, T.; Engelkamp, D.; KrahJentgens, I.; Lambrecht, H. G.; Koch, K. W.; Schwemer, J.;
Rivosecchi, R.; Mallart, A.; Galceran, J.; Canal, I.; Barbas, J. A. and
Ferrus, A. Frequenin, a novel calcium-binding protein that modulates
synaptic efficacy in the drosophila nervous system. Neuron 11, 15-28,
1993
lO.Sanger, F.; Nicklen, S. and Coulson, A. R. Dideoxy sequencing
method for DNA. Procedings of the National Academy of Science
U.S.A. 74, 5463-5466, 1977
11.Feng, D. F. and Doolittle, R. F. Phylogenetic tree analysis. Methods in
Enzymology 183, 375-387, 1990
12.Gorodovikova, E. N. and Philippov, P. P. The presence of a calciumsensitive p26-containing complex in bovine retina rod cells. FEBS letter
335, 277-279, 1993
13.Palczewski, K., McDowell, J. H. and Hargraves, P. A. Purification and
characterization of rhodopsin kinase J Biol Chem 263, 14067-14073
(1988)

Download Report