(Received for publication, December 2, 1993; and in revised form, August 8, 1994)
From the
Invertebrate visual transduction is thought to be initiated by photoactivation of rhodopsin and its subsequent interaction with a guanyl nucleotide-binding protein (G protein). The identities of the G protein and its target effector have remained elusive, although evidence suggests the involvement of a phospholipase C (PLC).
We
have identified a phosphatidylinositol-specific PLC from the cytosol of
squid retina. The enzyme was purified to near-homogeneity by a
combination of carboxymethyl-Sepharose and heparin-Sepharose
chromatography. The purified PLC, identified as an approximately
140-kDa protein by sodium dodecyl sulfate-polyacrylamide gels,
hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP) at a
rate of 10-15 µmol/min/mg of protein with 1 µM Ca
. The partial amino acid sequence of the
protein showed homology with a PLC cloned from a Drosophila head library (PLC21) and lesser homology with Drosophila norpA protein and mammalian PLC
isozymes.
Reconstitution of
purified squid PLC with an AlF-activated 44-kDa G
protein
subunit extracted from squid photoreceptor membranes
resulted in a significant increase in PIP
hydrolysis over a
range of Ca
concentrations while reconstitution with
mammalian G
or G
1
was without effect.
These results suggest that cephalopod phototransduction is mediated by
G
-44 activation of a 140-kDa cytosolic PLC.
The molecular mechanism of photoexcitation in vertebrate vision
is well established. Photoactivated rhodopsin interacts with a
heterotrimeric G protein ()which in turn activates a
cGMP-phosphodiesterase. The resulting decrease in cGMP levels leads to
closure of cGMP-gated ion channels in the membrane and transient
hyperpolarization of the cell(1, 2, 3) .
Photoexcitation of rhodopsin in most invertebrates, on the other hand,
leads to an increase in cation permeability that results in a transient
depolarization of the cell(4, 5) . The biochemical
events initiated by the photoactivation of rhodopsin and resulting in
the opening of cation channels in the membrane of invertebrate
photoreceptors have not yet been established.
Biochemical evidence
from a number of invertebrates suggests the involvement of a G protein
in the initial events of visual transduction (6, 7, 8, 9, 10) . This
evidence includes reports of proteins cross-reacting with antibodies to
G protein subunits, toxin-mediated ADP-ribosylation, or
light-dependent GTP binding to
subunits in
squid(11, 12, 13) ,
octopus(14, 15) , and
blowfly(16, 17) . It is not clear, however, which
second messenger systems are linked to these G proteins. In Limulus, evidence of two different second messengers has
emerged from electrophysiological studies in which injection of
inositol 1,4,5-trisphosphate (12, 18) or cGMP (19) were shown to mimic the effect of light on ventral
photoreceptors, resulting in depolarization. Further evidence for
inositol 1,4,5-trisphosphate as a second messenger, at least in Drosophila, came from studies of the norpA mutant.
Phospholipase C (PLC) activity is abundant in normal Drosophila eyes and drastically reduced in norpA mutants that
exhibit defective phototransduction (20, 21, 22) . The deduced sequence of the norpA protein showed homology to PLC enzymes(23) , and
isolated norpA protein demonstrated
Ca
-sensitive phospholipid hydrolysis(24) .
The
subtype of mammalian PLC has recently been shown
to be regulated by G protein
subunits of the
G
/G
family(25, 26, 27, 28, 29) .
A protein highly homologous to this class of mammalian G proteins has
been demonstrated in Drosophila eyes(30, 31) , yet it remains to be shown whether
this G protein regulates the norpA PLC.
Recent
identification of the principle G protein from squid photoreceptors as
a 44-kDa protein closely related to mammalian G has
led to speculation that this G protein is coupled to a
PLC(32) . We present evidence in this report for the isolation
and characterization of a novel phospholipase from squid photoreceptors
that is activated by Ca
and regulated by the 44-kDa G
protein
subunit purified from the same photoreceptors. Our
results strongly suggest that G protein-activated PLC is the major
signal transduction pathway in cephalopod vision.
Figure 1:
SDS-polyacrylamide gel and
immunoblot analysis of squid retinal fractions. Samples of squid
retinal membrane preparations and extracted proteins were prepared as
described under ``Experimental Procedures'' and subjected to
electrophoresis through 10% polyacrylamide. a, the gel was
stained with Coomassie Blue; b, the proteins were transferred
to nitrocellulose and immunoblotted with antisera specific for
mammalian G/G
. Lane 1, 5 µg of
squid eye homogenate; lane 2, 5 µg of squid retinal
membranes; lane 3, 2 µg of 2 M KCl membrane
extract.
Figure 2: a, chromatography of soluble fraction from squid retina through CM-Sepharose. See ``Experimental Procedures'' for explanation. b, SDS-polyacrylamide gel electrophoresis of fractions obtained from CM-Sepharose. Aliquots (10 µl) of the indicated fractions were subjected to electrophoresis through 7.5% polyacrylamide gel. Proteins were visualized with Coomassie Blue. Numbers at the side of the gel indicate the migration of protein standards.
Figure 3: a, chromatography of squid retinal PLC through heparin-Sepharose. See ``Experimental Procedures'' for explanation. b, SDS-polyacrylamide gel electrophoresis of fractions obtained from heparin-Sepharose. Aliquots (10 µl) of the indicated fractions were subjected to electrophoresis as described in Fig. 2.
Figure 4: Comparison of the amino acid sequences of peptide fragments obtained from squid eye PLC with those deduced for PLC21, norpA, and Rat1. The preparation and sequencing of the peptide fragments from purified squid eye PLC are described under ``Experimental Procedures.'' The sequences are given in one-letter code. The sequences showing identity with those in the squid PLC are shown in bold letters. Amino acid residues are numbered at the beginning of each line.
Figure 6:
Effect of G-44 on PLC activity at
varying calcium concentrations. Purified PLC (2 ng) was incubated with
phospholipid vesicles containing PIP
and indicated
concentrations of free Ca
in the presence (
) or
absence (
) of 8 ng of squid retinal G
-44. Assays were
performed as described under ``Experimental
Procedures.''
Figure 5:
Effect of G proteins on PLC activity.
Purified PLC was incubated with phospholipid vesicles containing
PIP and indicated concentrations of purified squid eye
G
-44 (
), bovine retinal G
(
), or rat
brain G
1
(
) in the presence of AlF
and 1 µM free Ca
. Results shown
are representative of independent experiments using three separate PLC
preparations.
The results of our experiments presented here clearly
demonstrate the presence of a PLC enzyme in squid photoreceptors. The
140-kDa enzyme was readily purified from the cytosolic fraction of
squid photoreceptors and shared similar properties with several
mammalian phospholipase C subtypes. The catalytic properties of the
enzyme toward PI were very different from those toward PIP.
Over all concentrations of calcium up to 1 mM, the purified
enzyme had a higher specific activity for PIP
than PI and
was 10 times more active with PIP
as substrate at 1
µM free calcium than with PI at 1 mM free
calcium. The squid protein was active over a broad pH range from 5.5 to
8 but was almost inactive at pH 9. With the exception that squid PLC
was more active at neutral pH, this substrate preference most resembles
that reported for the mammalian PLC
subtype(42) , but is
quite distinct from that reported for the only other invertebrate PLC
purified to date, namely Drosophila norpA gene product, that
is maximally active at pH 9 and inactive at pH 6. Squid PLC appears to
be a uniquely basic protein that is not recognized by antisera to
either the
,
, or
classes of vertebrate PLC isozymes. (
)Sequence data obtained from CNBr fragments of the purified
protein showed best homology to the Drosophila neuronal PLC
clone, PLC21, with lesser homology to the norpA gene product.
This latter finding was unexpected in that norpA clearly
functions importantly in fly vision, and it may signify an evolutionary
divergence among invertebrate photoreceptors. Additional neuronal
signaling function(s) for norpA based upon cellular
localization has recently been suggested, and our findings may further
signify that additional PLC isozymes participate in visual signaling.
Full analysis of the relationship of this protein to other members of
the PLC family of proteins, however, must await elucidation of the
complete amino acid sequence of the squid PLC and studies of its
cellular distribution.
A number of guanyl nucleotide-binding
proteins have been identified in invertebrate photoreceptors, but the
role of these proteins in visual transduction has not been clearly
elucidated. The G-44 that we have isolated from squid
photoreceptors appears to be the same as a protein previously reported
as the major G
subunit in squid photoreceptors that is closely
related in amino acid sequence to mammalian
G
(32) . Indeed, our G
-44 was shown here
to cross-react with antibodies raised against mammalian
G
. The homology of G protein
subunits in both
squid and Drosophila eyes to the G
/G
family of vertebrate G proteins that modulate PLC enzymes of the
subtype led us to investigate the ability of G
-44 to
regulate the squid PLC. The results of our experiments reported here
demonstrate that the effects of G
-44 on squid PLC are very similar
to those reported for G
on PLC
(25, 26, 27, 28, 29) .
The presence of G
-44 effectively increased PIP
hydrolysis by the enzyme over a range of Ca
concentrations from 30 nM to 10 µM. This
activity appeared to be specific to this
subunit, and we have not
been able to modify the enzyme's activity with other G protein
subunits or the purified
subunits. Our experiments
were performed using G
-44 extracted from photoreceptors treated
with either GTP
S or AlF
in order to maximally
activate the G protein. We have also observed substantial activity
using G
-44 extracted in the absence of added nucleotide or
activators. A possible explanation of this basal activity is the
presence of some other PLC-activating factor in the G
-44
preparation that affects PLC activity independent of guanyl
nucleotides. Our data showing complete inhibition of PLC activation
following immunoprecipitation with G
antisera,
however, indicate that the activity in the samples was attributed to
G
-44 rather than a contaminant. We consider it more probable that
this basal activity is the result of intrinsic GTP binding to G
-44
in our membranes which were prepared under full illumination, and we
are currently addressing this issue using dark-adapted animals. Several
groups have reported proteins modified by both cholera and pertussis
toxins in squid, and one report has shown a protein identified by
antisera raised against G
(13) . In our
preparations we did not detect proteins using polyclonal antisera
raised against bovine G
. However, this may have been
the result of different extraction conditions used in our experiments,
and further work would be needed to examine the role of other G
proteins in invertebrate photoreceptors.
Data from several
laboratories have reported that PLC enzymes are modified during
purification and storage possibly by contaminating proteases. Park et al.(43) have recently reported that the
Ca-dependent enzyme calpain can cleave PLC
from 150 kDa to 100- and 45-kDa fragments that retain
full PIP
hydrolyzing activity and sensitivity to
Ca
but are devoid of G
activation. In
squid retina we saw evidence of only a single species of PLC activity
associated with the 140-kDa protein characterized here. Indirect
evidence of proteolysis of the 140-kDa protein by a
Ca
-dependent enzyme was found during our early
preparations when impure PLC samples were applied to hydroxyapatite; we
observed rapid and complete loss of the 140-kDa protein and PLC
activity associated with fractions containing 94- and 40-kDa fragments.
We are currently examining the regulation of these PLC hydrolysis
fragments by G
-44.
The solubility of squid PLC, in contrast to
G protein-regulated mammalian PLC which is mostly
membrane-associated in brain, may reflect the association of the two
enzymes with their respective G protein activators. PLC
interacts with a membrane-bound activator G
,
while our data indicate that squid G
-44 may be soluble under some
conditions. Solubility has thus far been uniquely associated with
retinal transducins in vertebrates, and this may indicate greater
similarities between mammalian G
and invertebrate
G
-44 than is readily apparent from their amino acid sequences.
This raises the possibility that G
-44 dissociates from the disc
membrane after interaction with photoactivated rhodopsin and associates
with the cytosolic PLC. Our data presented here clearly demonstrate
that such an interaction can occur. The detailed examination of the
molecular interaction of G
-44 with rhodopsin and PLC will be the
subject of further reports.
A growing body of evidence suggests that
light-dependent activation of PLC does take place in invertebrate
photoreceptors and our data are the first demonstration of G protein
regulation of PLC in such a system. As we have shown, under the
regulation of G-44, PLC is activated, and the concentration of
inositol 1,4,5-trisphosphate and presumably diacylglycerol in
invertebrate photoreceptors is increased. It remains to be demonstrated
how these second messengers regulate the opening of cation channels
that leads to the depolarization of the photoreceptor membrane.