(Received for publication, January 21, 1997, and in revised form, March 18, 1997)
From the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California at San Francisco, San Francisco, California 94110
Phosducin-like protein (PhLP), a widely expressed
ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and
Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of
G signaling in retina and pineal gland. However, although
phosducin has a well characterized role in retinal phototransduction,
function of the PhLP remains unclear. In this study we examine the
ability of PhLP to bind G
dimer in vitro and in
vivo. Using PhLP glutathione S-transferase fusion
proteins, we show that PhLP directly binds G
in
vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of G
to both the amino- and
C-terminal halves of PhLP. Protein-protein interactions between G
and PhLP are inhibited by the
subunit of Go and
Gi3, suggesting that PhLP can bind only free G
.
Finally, we show that PhLP complexes, at least partially, with G
in vivo. Following overexpression of epitope-tagged PhLP
together with G
1
2 proteins in COS-7
cells, a PhLP-G
complex is co-immunoprecipitated by monoclonal
antibody directed against the epitope tag. Similarly, polyclonal
anti-PhLP antibody co-precipitates endogenous PhLP and G
proteins
from NG108-15 cell lysates. These data are consistent with the
hypothesis that PhLP is a widely expressed modulator of G
function. Furthermore, because alternate forms of the PhLP transcript
are expressed, there may be functional implications for the existence
of two G
-binding domains on PhLP.
Heterotrimeric guanine nucleotide-binding proteins (G proteins)
play a major role in transmembrane signaling processes by transducing
extracellular signals from the superfamily of heptahelical cell surface
receptors to their appropriate intracellular effectors (1, 2). In its
trimeric form, G is inactive, and the G
subunit binds a
molecule of GDP. Upon ligand binding, the receptor catalyzes the
exchange of GDP for GTP on G
that causes its activation and
dissociation from the tightly bound G
complex.1 Inactivation and reassociation of
the heterotrimer is initiated by the hydrolysis of bound GTP into GDP
by an intrinsic GTPase activity of the G
subunit. It is now known
that both the free GTP-bound G
and the G
dimer can bind and
regulate downstream effectors including adenylyl cyclases,
phospholipases, and ion channels, and thereby modulate second messenger
levels and ion flux (3).
The discovery of several specific G binding proteins has recently
shed light on new roles for G
in the propagation and termination
of cellular signaling. The dimer has been shown to recruit
-adrenergic receptor kinase (
ARK)2 to
its membrane-associated receptor substrate and thus initiate receptor
desensitization (4, 5). This process occurs via direct binding of
G
to the C terminus of a putative pleckstrin homology domain on
ARK (6). Furthermore, the responsiveness of G protein-regulated
signaling systems may be directly modulated through the interaction of
G
subunits with intracellular regulatory proteins. For instance,
phosducin, a phosphoprotein mainly expressed in the retina and pineal
gland, inhibits the phototransduction cascade by scavenging
subunits of the G protein transducin (Gt), thus preventing
their reassociation with the Gt
subunit (7, 8). Because
phosducin has a higher affinity for Gt
than does
Gt
, it has been suggested that the formation of the phosducin/Gt
complex is a major factor regulating
photoreceptor responsiveness (9). From in vitro binding and
co-transfection assays, it was proposed that phosducin may also compete
with other targets for G
binding, such as
ARK and
phospholipase C type
2 (10, 11).
We recently isolated a rat brain cDNA encoding a phosducin-like
protein (PhLP), which has 65% amino acid homology to phosducin (12).
We also described several 5-end splice variants that generate two
predicted isoforms of the protein: PhLP long (PhLP) of 301 amino acids
containing the entire coding sequence and PhLP short
(PhLPS) of 218 amino acids missing the first 83 N-terminal residues of PhLP (12, 13). Based on sequence homology with phosducin,
we have suggested that PhLP proteins regulate G
signaling in
nonretinal tissues. In favor of this hypothesis, a recent report showed
that recombinant PhLPS inhibits several G
functions
in vitro (14). Interestingly, these authors suggested that
unlike phosducin (11, 15), the N terminus of PhLP was unlikely to contain a G
-binding domain.
To more directly characterize the interaction of PhLP with G, we
studied PhLP binding to G
both in vivo and in
vitro. Our results here, using in vitro binding studies
with a series of truncated PhLP/glutathione S-transferase
(GST) fusion proteins, show that PhLP binds
through a bipartite
binding domain. The G
-PhLP interaction was confirmed by
co-immunoprecipitation of the complex from cell lysates. Our findings
support the hypothesis that PhLP can modulate G
function (14) in
many tissues through direct protein-protein interactions. Regulation of
PhLP/G
interactions could be an important factor in controlling G
protein signaling.
PhLP and PhLPS cDNAs were cloned
in our laboratory (12). The GST-phosducin construct was obtained from
Dr. Cheryl Craft (University of Southern California). G and
Go
proteins purified from bovine brain were kindly
provided by Dr. Eva Neer (Brighman and Woman's Hospital, Boston, MA).
G
1 and G
2 expression vectors were kind
gifts from Dr. H. Bourne (University of California at San Francisco).
Purified recombinant G
1
2 was generously
provided by Dr. Rene Onrust in the Bourne laboratory. Recombinant
Gi
3 protein was from Calbiochem.
The full-length PhLP (amino acids 1-301) as
well as different regions of the protein corresponding to amino acid
residues 84-301 (referred to as PhLPS), 1-167, 1-115,
1-70, 50-167, 84-167, 161-301, and 200-301 were expressed as GST
fusion proteins. DNA fragments encoding PhLP and its derivatives were
amplified by polymerase chain reaction using rat PhLP cDNA as
template and 5- and 3
-primers containing BamHI and
EcoRI sites, respectively. The amplified fragments were
ligated in frame with the 3
-end of the coding region of GST into
BamHI and EcoRI sites of the pGEX-2T vector
(Pharmacia Biotech Inc.). The resultant constructs were verified by DNA
sequencing using the chain termination method (Sequenase version 2.0, U. S. Biochemical Corp.) and used to transform Escherichia
coli strain BL21.
An epitope-tagged PhLP expression vector was generated by fusing an 8 amino acid peptide from the hemagglutinin (HA) of influenza virus to
the C terminus of PhLP. Sense and antisense oligonucleotides corresponding to the HA epitope (YDVPDYAS), flanked by a 5
EcoRI site and a 3
NotI site, were synthesized,
annealed, and inserted into pcDNA3 (Invitrogen) between
EcoRI and NotI sites. The full-length PhLP coding
sequence, amplified as described above, was then ligated between the
BamHI and EcoRI sites of the modified pcDNA3
vector, so as to fuse the PhLP C terminus in frame with the 5
-end of the HA tag.
Fusion protein expression was induced with 0.1 mM isopropyl-1-thiol--D-galactopyranoside
for 90 min, and the proteins were solubilized and purified on
glutathione-Sepharose 4B resin (Pharmacia) by the Sarkosyl method (16).
In a typical binding assay, following immobilization on
glutathione-agarose beads, fusion proteins at a final concentration of
0.5-1.0 µM were incubated with 50-100 nM
G
purified from bovine brain in 50 µl of phosphate-buffered saline (PBS) containing 0.01% Lubrol for 2 h at 4 °C.
Following six washes in 200 µl of PBS containing 0.01% Lubrol, the
beads were resuspended in SDS sample buffer and boiled for 10 min. The eluted proteins were separated with 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto nitrocellulose membranes using standard methods. The blots were probed with a polyclonal anti-
antiserum (1:1000; DuPont NEN) and processed using
the enhanced chemiluminescense detection system (Amersham Corp.).
Occasionally, blots were stripped and reprobed with a polyclonal
anti-GST antiserum (1:4000; Santa Cruz).
The entire coding region of PhLPS cDNA was amplified by polymerase chain reaction and fused in frame with the maltose binding protein coding region in the vector pMAL-c2 (New England Biolabs). The maltose binding protein-PhLPS fusion protein migrated at approximately 72 kDa on SDS-polyacrylamide gels as expected. The fusion protein was purified to apparent homogeneity by amylose resin chromatography exactly as described by the manufacturer (New England Biolabs) and was injected into rabbits by a commercial source (CalTag) for generation of a polyclonal antiserum. This antiserum was affinity-purified over a column of GST-PhLPS coupled to CnBr-activated Sepharose 4B (Pharmacia).
Cell Culture and Transient DNA TransfectionNG108-15 cells were grown as described (12) in Dulbecco's modified Eagle's medium (DMEM) containing 10% serum+ (JRH Biosciences). COS-7 cells (3 × 105 cells/well) were seeded 48 h before transfection in 6-well plates in DMEM supplemented with 10% fetal bovine serum. Cells were incubated for 5 h in serum-free DMEM with DNA plasmids premixed with lipofectAmine (Life Technologies, Inc.) and were then incubated overnight at 37 °C in DMEM containing 10% fetal bovine serum. The total amount of DNA in all transfections was 2 µg/well. When required, the empty pcDNA3 vector was used to maintain a constant amount of DNA.
Immunoprecipitation2 × 106 transfected
COS-7 cells or 4 × 106 NG108-15 cells were washed
twice with ice-cold PBS and lysed in Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40, 2 µg/ml aprotinin and leupeptin, 20 µg/ml soybean trypsin
inhibitor). After 15 min of incubation on ice, insoluble material was
removed by centrifugation at 10,000 × g at 4 °C for
10 min, and the lysate was precleared in the presence of protein
A-agarose (Santa Cruz) for 30 min. COS-7 cell lysate was then incubated
with 5 µg of monoclonal antibody 12CA5 (Boehringer Mannheim) to the
HA tag, whereas NG108-15 cell lysate was incubated with 5 µg of
affinity-purified polyclonal anti-PhLP or an equivalent amount of
preimmune serum. The incubations were conducted overnight at 4 °C
before precipitation in the presence of protein A-agarose. The
immunoprecipitates were washed four times with PBS containing 0.01%
Lubrol, and protein complexes were eluted in SDS sample buffer and
analyzed by Western blot using the 12CA5 antibody and/or a monoclonal
antibody to the G1 subunit (Transduction Laboratories).
To determine whether PhLP directly interacts with G, we
generated PhLP and PhLPS GST fusion proteins and examined
their ability to bind G
purified from bovine brain. Following
immobilization of the fusion proteins on glutathione-agarose beads,
G
binding was detected by Western blot analysis using an anti-
antiserum. As controls, we tested G
binding by the GST protein
itself and GST fused to phosducin (GST-phosducin), a known
G
-binding protein. The fusion proteins migrate at their expected
molecular weight as visualized by Coomassie Blue-staining (Fig.
1A, lower panel) and can be
detected with an anti-GST antibody on Western blot analysis (Fig.
1B, lower panel). In addition, GST-PhLP proteins are recognized by an affinity-purified polyclonal antiserum directed against PhLPS (data not shown).
As previously reported by other investigators (11), GST-phosducin bound
detectable amounts of G at a molar ratio of
:GST-phosducin of approximately 1:7 (Fig. 1A, upper panel).
Under similar conditions, GST-PhLPS and GST-PhLP also
retained G
(Fig. 1A, upper panel). By
contrast, GST protein did not bind G
subunits even when present at a 3-fold higher concentration than the GST-PhLP proteins (Fig. 1A, upper panel), indicating that G
binding
by the fusion proteins is specified by the PhLP protein sequence. The
affinity of phosducin for G
was previously reported to be in the
nanomolar range (11, 14). Under our experimental conditions, GST-PhLP
appeared to have a slightly lower affinity for G
than
GST-phosducin but of the same order of magnitude because titration with
varying amounts of G
protein showed that GST-PhLP retained only
slightly lower amounts of G
than did GST-phosducin (Fig.
1B, upper panel).
To map the G binding domain of PhLP, we examined recombinant
G
1
2 interaction in vitro with
GST fusion proteins containing various regions of PhLP. Fig.
2 shows an alignment of PhLP deletion constructs with
the full-length PhLP. Each construct produced a protein that migrated
at the expected molecular weight on SDS-PAGE (data not shown).
Surprisingly, we found G
binding activity of PhLP at two areas in
the N- and C-terminal regions. In the N-terminal half of PhLP
(PhLP1-167), amino acids 50-115 appeared sufficient for
G
binding, because PhLP1-115 and
PhLP50-167 retained G
, whereas PhLP1-70
and PhLP84-167 did not (Fig. 2). The 50-115 region
contains an 11-amino acid stretch (57-67: TGPKGVINDWR) that is
perfectly conserved between PhLP and phosducin and is known to be
essential for G
binding by phosducin (11). Furthermore, the
crystal structure of the Gt
-phosducin complex,
reported while this manuscript was in preparation, showed that this
highly conserved sequence has extensive and tight interactions with the
center of the G
propellar (17). This conserved sequence region of
PhLP (amino acids 57-67) may be important for binding of G
because PhLP84-167 totally lacked
binding, whereas
PhLP50-167 had essentially full binding activity. However,
although the 57-67 region may be important for G
binding by
PhLP, additional elements seem required because PhLP1-70
did not bind G
.
Additional deletions revealed that the C-terminal half of PhLP
(PhLP161-301) also binds G. This binding activity
was further localized to the C-terminal 101 residues of PhLP
(PhLP200-301) (Fig. 2). This C-terminal binding domain may
explain why PhLPS (PhLP84-301), which does not
contain the 57-67 conserved sequence, still retained significant
G
binding activity.
The crystal structure of Gt -phosducin also showed
two spatially and possibly functionally distinct domains in phosducin (17). These two domains roughly correspond to the N-terminal and
C-terminal halves of the molecule and do not interact with each other
but both contact G
. The N-terminal domain may compete with G
,
whereas the C-terminal, thioredoxin-like domain was suggested to be
responsible for G
translocation away from the membrane (17).
Based on sequence homology, Gaudet et al. proposed a similar structure for PhLP and predicted the G
interacting residues in
this protein (17). The regions occupied by these amino acids, depicted
in Fig. 2, correspond to residues 54-69 and 114-152 in the N-terminal
domain and to residues 240-247 and 270-277 in the C-terminal domain.
Our results are in perfect agreement with these predictions and also
suggest that the N-terminal and C-terminal domains can interact with
G
independently. Because these domains may affect different
functions of G
, they might be useful tools to study different
aspects of G
regulation as suggested by Gaudet et al.
(17).
Previous studies have demonstrated that the binding of G to G
subunit inhibits its interaction with phosducin or
ARK (15, 18). In
contrast, the N-terminal domain of the G protein-gated K+
channel as well as the small GTPase, ADP-ribosylation factor were shown
to interact with either G
alone or trimeric G
(19, 20).
We found that recombinant Gi
3 inhibited
G
binding to GST-PhLP (Fig. 3). Similarly,
Go
-GDP
S but not Go
-GTP
S partially abolished the interaction of G
to GST-PhLP (data not shown). Together, these results suggest that only free G
can interact with PhLP. Western blot analysis with a common anti-
antiserum (DuPont NEN) indicated that neither Gi
3 or
Go
was retained on GST-PhLP along with the G
dimer
(Fig. 3 and data not shown). In addition,
Gi
3 by itself did not bind to GST-PhLP (Fig.
3). It should be noted that a relatively high concentration of
recombinant Gi
3 was required to totally
eliminate G
binding to GST-PhLP (Fig. 3). This may reflect the
fact that G
interacts more tightly with PhLP than with G
subunit, as was previously found for phosducin and Gt
interaction with G
(17).
To demonstrate G-PhLP interaction in vivo, the complex
was immunoprecipitated following overexpression of the proteins in COS-7 cells. For these experiments, the C terminus of PhLP was tagged
with a HA epitope. COS-7 cells were transiently transfected with
plasmids encoding PhLP-HA, G
1 and G
2 subunits, or a combination of these proteins. Expression of the proteins in COS-7 cells was monitored by Western blot analysis using monoclonal anti-HA and anti-
1 antibodies (Fig. 4A, lower
panels). PhLP-HA was specifically precipitated by anti-HA antibody
(Fig. 4A, upper panel). In addition, this
antibody co-precipitated overexpressed
G
1
2 subunits, only in cells co-expressing
PhLP-HA (Fig. 4A, upper panel). We also detected
G
in immunoprecipitates from cells transfected only with PhLP-HA
plasmid (Fig. 4A, upper panel), suggesting that
PhLP-HA interacts with both overexpressed and endogenous G
subunits.
Interaction of endogenous PhLP and G was examined in NG108-15
neuroblastoma × glioma cells because this cell line expresses high basal levels of PhLP.3 Endogenous PhLP
protein from NG108-15 cell lysates was immunoprecipitated by an
affinity-purified polyclonal antiserum directed against PhLPS. On Western blot analysis of NG108-15 cell lysates,
this antiserum recognized a single band migrating at 46 kDa, the
expected molecular mass for full-length PhLP protein (data not shown)
(12). The antiserum also immunoprecipitated a 46-kDa protein from
[35S]methionine-labeled NG108-15 cells (data not shown).
Anti-PhLP immunoprecipitates contained G
as detected by Western blot
analysis (Fig. 4B). Preimmune serum did not precipitate G
(Fig. 4B). These results confirm the overexpression studies
(Fig. 4A) and suggest that PhLP might exist, at least
partially, as a complex with G
subunits in vivo.
In conclusion, these studies have documented the direct interaction of
PhLP with G through both in vivo and in
vitro analyses. Our deletion analysis, together with the recent
crystal structure of the phosducin-Gt
complex,
suggests that two potentially independent domains on PhLP interact with
G
. This complements prior studies on PhLP that suggested that
regions beyond the N terminus were involved in inhibition of G
function in vitro (14). Because the two domains of PhLP are
predicted to interact with functionally different regions of G
(17)
and we have previously shown the existence of multiple forms of the
PhLP transcript, it is tempting to speculate that alternate forms of
PhLP might produce distinct changes in G
signaling. For example,
PhLPS contains predominantly the C-terminal G
-binding
domain and thus might produce different kinetics or extent of changes
in G
function than the full-length PhLP protein. It remains to be
determined which of the diverse G
cellular effects are
functionally modified by PhLP-G
interactions.
We thank Steven Barhite, Ivan Diamond, Ulrike Heberlein, and Robert Messing at the Ernest Gallo Clinic and Research Center for many helpful discussions during the course of these studies. We also thank Henry Bourne, Cheryl Craft, Eva Neer, and Rene Onrust for gifts of proteins and plasmids as noted under "Experimental Procedures."