From the
The
The
Although the
role of G
The
G
The structures of
PH domains suggest that the carboxyl-terminal
Fig. 2A shows the purified GST fusion proteins. The
The G
Based on the triple coiled-coil hypothesis, G
The binding of PIP
The region of the PH
domain that interacts with G
The following novel conclusions
emerge from our studies. First, we have identified the critical
residues involved in G
We thank W. Carl Stone for assisting in mutant DNA
construction and sequencing, W. Darrel Capel for purified
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunits of heterotrimeric G proteins
(G
) play a variety of roles in cellular signaling,
one of which is membrane targeting of the
-adrenergic receptor
kinase (
ARK). This is accomplished via a physical interaction of
G
and a domain within the carboxyl terminus of
ARK which overlaps with a pleckstrin homology (PH) domain. The PH
domain of
ARK not only binds G
but also
interacts with phosphatidylinositol 4,5-bisphosphate (PIP
).
Based on previous mapping of the G
binding region
of
ARK, and conserved residues within the PH domain, we have
constructed a series of mutants in the carboxyl terminus of
ARK in
order to determine important residues involved in G
and PIP
binding. To examine the effects of mutations
on G
binding, we employed three different
methodologies: direct G
binding to GST fusion
proteins; the ability of GST fusion proteins to inhibit
G
-mediated
ARK translocation to
rhodopsin-enriched rod outer segments; and the ability of mutant
peptides expressed in cells to inhibit G
-mediated
inositol phosphate accumulation. Direct PIP
binding was
also assessed on mutant GST fusion proteins. Ala residue insertion
following Trp
completely abolished the ability of
ARK to bind G
, suggesting that a proper
-helical conformation is necessary for the
G
ARK interaction. In contrast, this
insertional mutation had no effect on PIP
binding. Both
G
binding and PIP
binding were
abolished following Ala replacement of Trp
, suggesting
that this conserved residue within the last subdomain of the PH domain
is crucial for both interactions. Other mutations also produced
differential effects on the physical interactions of the
ARK
carboxyl terminus with G
and PIP
.
These results suggest that the last PH subdomain and its neighboring
sequences within the carboxyl terminus of
ARK, including
Trp
, Leu
, and residues
Lys
-Arg
, are critical for G
binding while Trp
and residues
Asp
-Glu
are important for the PH domain to
form the correct structure for binding to PIP
.
subunits of heterotrimeric G proteins
(G
)
(
)play a variety of roles in
modulating various cellular signaling cascades. These include
regulation of certain isoforms of adenylyl cyclases, activation of some
phospholipase C
isoforms and phospholipase A2, modulation of
muscarinic K
channels, mediation of the
pheromone-induced mating response in yeast, binding to retinal
phosducin, and stimulation of G protein-coupled receptor-specific
kinases such as muscarinic receptor kinase and
-adrenergic
receptor kinase (
ARK)(1, 2, 3) . Most
recently, G
has been shown to activate the MAP kinase cascade
mediated through serpentine G
protein-coupled receptors
such as that for lysophosphatidic acid, the M2-muscarinic acetylcholine
(AChR) and
-adrenergic (AR) receptors (4-6).
This activation is
p21
-dependent(5, 6) . It is now
clear that both G
and G
subunits interact with various
effectors and receptors to regulate cellular signaling.
in various signal transduction pathways has been
established, the structural and molecular basis of the interaction
between G
and its effectors has yet to be fully
understood(7) . The region of phospholipase C
interacting
with G
has been broadly mapped to the NH
-terminal
two-thirds of the protein(8) . The K
channel
has its G
-sensitive region in the cytoplasmic
carboxyl-terminal region (9). The G
-binding domain of
ARK, however, has been most intensively
studied(10, 11, 12, 13) . The
translocation and activation of the cytosolic enzyme
ARK is
mediated by the prenylated membrane-anchored G
(14) .
The specific region of
ARK which directly interacts with and binds
G
is located within the carboxyl 125-amino-acid residue
stretch(13) , and this G
-binding domain peptide
inhibits G
-mediated phosphoinositide (PI) hydrolysis and type
II adenylyl cyclase stimulation in both a transient transfection (15) and a cell permeabilized system(16) . It has been
demonstrated that this domain can be utilized to probe and dissect a
broad range of G
-mediated signaling pathways.
-binding domain of
ARK includes a pleckstrin homology
domain (PH domain) that has been found in a variety of proteins
involved in cellular
signaling(17, 18, 19, 20, 21) .
Although the function of the PH domain is not clear, several hypotheses
have been raised and tested. The PH domains of numerous proteins appear
to bind G
to varying extents(22) . Some of these PH
domain peptides have been shown to behave as antagonists of
G
-mediated signaling in intact cells, such as
G
-mediated inositol phosphate (IP) production and
G
-mediated p21
-GTP exchange and MAP
kinase activation (23). The region responsible for binding G
,
however, is not identical to the PH domain, but rather encompasses the
carboxyl portion of the PH domain plus immediately distal sequences. A
28-mer peptide (Trp
to Ser
) containing the
carboxyl portion of the PH domain of
ARK (see Fig. 1)
inhibited G
-mediated activation of
ARK1(13) , and
desensitization of the cAMP response elicited via odorant activation of
olfactory receptors in permeabilized rat olfactory cilia(24) .
In addition, the G
binding region of retinal phosducin
contains sequences homologous with the carboxyl half of
G
-binding domain of
ARK(25) .
Figure 1:
Schematic representation of
the G-binding domain and the PH domain of
ARK and the
location of mutations constructed in this study. Both the
G
-binding domain (Pro
-Ser
) and PH
domain (Tyr
-Gln
) are located in the COOH
terminus of
ARK. The PH domain is divided into six subdomains
according to Musacchio et al. (19). Shown are subdomain 6 of
the PH domain and the adjacent sequences from
ARK1. The bold amino acids indicate the perfectly conserved amino acids among the
G
-binding active
ARK family (bovine
ARK1 and 2,
human
ARK1 and 2, and Drosophila GPRK1) (38, 39). The PH
domain consensus sequences (
, hydrophobic residue) are based on
previous publications.
Most recently,
three-dimensional structures of PH domains from pleckstrin, spectrin,
and dynamin have been
determined(26, 27, 28, 29) . The core
structures are almost superimposable, consisting of a -barrel of
seven antiparallel
-sheets and a carboxyl-terminal amphiphilic
-helix. The structural similarity to other proteins immediately
suggests that one function of PH domains is to bind small lipophilic
molecules or peptides. Indeed, some PH domains can apparently bind
phosphatidylinositol 4,5-bisphosphate (PIP
) in the cleft of
the
-barrel(30) . The PH domain of spectrin has been shown
to be the site of membrane interaction(31) . The PH domain of
the B-cell tyrosine kinase, however, binds protein kinase C (32) as well as G
subunits(33) , suggesting
that the function of PH domains is more complex.
-helix including the
most conserved Trp residue may mediate G
association. Simonds et al.(34) suggested that the G protein
,
,
and
subunits form a triple coiled-coil structure through the
amino termini and that the dissociation of G
from the G
complex allows
ARK to form a new triple coiled-coil structure
through the 28-mer peptide region encompassing the PH domains. Recent
evidence, however, demonstrates that the trypsin-digested G
subunit, in which the putative NH
-terminal coiled-coil
domain of G
is missing, still binds to
ARK(35) . In
order to determine more precisely which residues in the
G
-binding domain of
ARK are crucial for this specific
protein-protein interaction and to test the triple coiled-coil
hypothesis, this report studies the G
binding abilities of a
series of
ARK1 constructs containing mutations in the PH domain
and adjacent sequences. Effects of these mutations on the ability of
the
ARK PH domain to bind to PIP
were also examined.
Materials
Bovine brain G was purified
in our laboratory. The cDNA for the human
2-C10 AR was cloned in
our laboratory. The cDNA for the human M1 AChR was kindly provided by
Dr. Ernest Peralta. The cDNAs encoding G
1 and G
2 were kindly
provided by Dr. Mel Simon. Sources of other reagents were as described
previously(13, 22, 23, 25) .
Construction and Isolation of GST Fusion
Proteins
All mutants of the ARK1 carboxyl terminus (ct)
were constructed with the GST fusion vector pGEX-2T (Pharmacia) by
standard techniques utilizing the polymerase chain reaction and wild
type bovine
ARK1 as the template as
described(13, 22) . The truncated
ARK1ct fusion
protein construct (
671-689) (Fig. 1) was used as the
template for the KKKR
EEEE (K663E, K665E, K667E, R669E) mutant.
All mutations were verified by dideoxy sequencing using Sequenase
(United States Biochemical Corp.). Mutant constructs were introduced
into the Escherichia coli strain NM522 or BL21. The fusion
proteins were expressed and purified as described previously using
glutathione-agarose(22) .
Construction and Expression of Minigenes
The cDNAs
encoding various mutant proteins were amplified from the pGEX plasmid
cDNAs encoding the corresponding mutant GST fusion protein to construct EcoRI-BclI minigene cassettes and inserted into the
mammalian expression vector pRK5 as described(15) . These DNAs
were used for transfection of COS-7 cells using LipofectAMINE (Life
Technologies, Inc.) as described(23) . Expression of the mutant
peptides was determined by Western blot of whole cell lysates as
described using anti-ARKct serum(15) .
Binding of G
The
binding of bovine brain G to Fusion Proteins
to the fusion proteins and the
detection of bound G
were accomplished essentially as
described previously(22) . Briefly, 500 nM GST fusion
protein and 73 nM purified bovine brain G
were
incubated in phosphate-buffered saline, 0.01% Lubrol, and the bound
G
on the immobilized beads was detected by using antibodies
to the G
subunit (DuPont NEN). Laser densitometry was used to
quantitate the relative amounts of bound G
.
Translocation Assay of
Translocation of ARK1 to Rod Outer Segment
Membranes
ARK1 to rod outer segment
membranes and its inhibition by the fusion proteins were carried out as
described previously(12, 22) . Briefly, purified
recombinant
ARK1 and urea-stripped rod outer segment membranes
were incubated at 30 °C for 5 min in either the presence or absence
of bovine G
(185 nM). Incubations containing
G
additionally contained one of the fusion proteins (1
µM). Following incubation, samples were subjected to
centrifugation at 350,000
g for 5 min, and the
supernatant and pellet obtained were assayed for
ARK1 activity.
Quantitation of the band corresponding to phosphorylated rhodopsin was
accomplished using a Molecular Dynamics PhosphorImager.
Inositol Phosphate Assays
COS-7 cells were
cotransfected with receptor cDNA and either empty pRK5 vector DNA or
pRK-mutant ARK1 DNA. In some experiments, cells were cotransfected
with G
1 and G
2 cDNAs rather than receptor DNA. After 24 h of
incubation, cells were prelabeled with 2 µCi/ml myo-[
H]inositol for 24 h. Cells were
then stimulated for 45 min with or without agonist, and IP accumulation
was determined by Dowex anion-exchange chromatography as
described(15) .
Binding of PIP
Fusion proteins (0.5-1 µg) in
phosphate-buffered saline were incubated in polycarbonate centrifuge
tubes (7 to Fusion
Proteins
20 mm) for 10 min at room temperature.
Phosphatidylcholine (PC) vesicles or PC vesicles containing 5%
PIP
(Sigma) were added for a final lipid concentration of
0.8 mg/ml in 30 µl. After a 10-min incubation at room temperature
and 5 min on ice, the tubes were then centrifuged at 100,000
revolutions/minute (TL-100 rotor) for 15 min at 4 °C. The
supernatant was removed, and the pellet was washed once with
phosphate-buffered saline and transferred to a new tube. The percentage
of fusion protein in the supernatant and pellet was determined by using
Western blot analysis (ECL, Amersham Corp.) and densitometry.
Design and Isolation of Mutant Fusion
Proteins
We focused on subdomain 6 of the PH domain and its
adjacent sequences to construct various mutants of the ARK
carboxyl-terminal peptide as shown in Fig. 1. The Trp
residue in subdomain 6 is perfectly conserved in all PH domains
as are several hydrophobic residues. In addition, clusters of acidic
(Asp or Glu) and basic (Lys or Arg) residues are found in the
G
binding region of PH domains. Thus, we made a series of
point mutations around the most conserved Trp
in
ARK1 and also reversed the ionic charge of the acidic and basic
regions. In addition, since this region is predicted, by computer
analysis, to form an
-helix we inserted 1 or 2 Ala residues to
disrupt the proper orientation of the helical structure.
ARK1ct fusion protein mutant KKKR
EEEE co-migrates with the
truncated wild type fusion protein (
671-689) since the
mutant is derived from the
671-689. Other mutant fusion
proteins derived from the wild type fusion protein (GST-
ARKct;
Pro
-Leu
) migrate as expected except the
E646K mutant. This mutant migrates faster than the wild type due to
either a change in the ionic strength or some degradation. Because the
mutant peptide migrates close to the
671-689, it is likely
that sequences up to Ser
are still intact.
Figure 2:
Isolation of mutant GST-PH domain fusion
proteins and binding to bovine brain G. A, Coomassie
Blue-stained SDS-polyacrylamide gel of various GST fusion proteins used
in this study. B, Western blot assessing G
binding
ability of the various GST fusion proteins. The position of the
subunit of G
is indicated by the arrowhead. GST and
wild type GST-
ARK-ct were used as negative and positive controls,
respectively. C, the relative amounts of bound G
as
the mean percentage ± S.D. or range of the band intensity of
wild type GST-
ARK-ct.
G
The
G Binding to Mutant Fusion Proteins
binding abilities of these mutant GST fusion proteins (Fig. 2A) were assessed by the direct binding assay
shown in Fig. 2B. The truncated wild type
671-689, still significantly binds G
(although
weaker than the wild type), consistent with our previous data (13).
When the basic residues in the truncated protein were changed to acidic
residues (KKKR
EEEE), the G
binding activity was
completely lost, suggesting that these basic residues are crucial for
the G
binding ability of
ARK. All other mutations
altered G
binding to varying extents, with the most
deleterious mutations being W643A and L647G. K645E, E646K, and
DSD
KKK still display some binding, although significantly weaker
than the wild type (Fig. 2, B and C). Since Arg
and Ser are most frequently substituted with Trp according to the
Dayhoff's table(36) , W643S and W643R were also tested.
The binding of these mutants is similar to that of W643A. Finally, the
effect of 1 or 2 Ala residue insertions after Trp
(WA and
WAA, respectively) was assessed. These insertions completely block
binding activity, suggesting that the proper orientation of residues in
the
-helix is disrupted.
Inhibition of G
In order to
confirm the relative binding affinity of the mutant fusion proteins, a
fixed concentration (1 µM) of each was tested for the
ability to inhibit G-mediated Translocation of
ARK1 to Membranes by Mutant Fusion Proteins
-mediated translocation of
ARK1 to
rod outer segment membranes. As shown in Fig. 3, the wild type
ARK1ct fusion protein significantly inhibits G
-mediated
translocation of
ARK1. The truncated mutant,
671-689,
also inhibits the translocation, but less effectively than the wild
type, consistent with the direct G
binding data. Other
mutants are less effective inhibitors, but the relative efficacy among
the mutants is consistent with the direct binding data. For example,
the mutations with two Ala insertions (WAA) had no inhibitory effect,
but K645E and E646K, which bind more G
than WAA in the direct
binding assay (Fig. 2B), slightly inhibited
G
-mediated
ARK translocation. Thus, the relative
inhibitory effects of these fusion proteins correlate well with the
relative binding activities observed in the direct G
-binding
assay. The DSD
KKK mutant, which shows moderate G
binding in the direct binding assay, did not inhibit translocation of
ARK1. However, the inability of this mutant to bind
PIP
, as described later, probably is the reason for this
discrepancy.
Figure 3:
Inhibition of G-dependent
translocation of
ARK1 to rod outer segment membranes by various
mutant fusion proteins. Each fusion protein was included in the
G
-mediated
ARK-translocation assays described under
``Experimental Procedures'' at 1 µM. In the
absence of G
, 5.1 ± 3.0% (n = 5) of
the total
ARK1 activity was associated with the membrane fraction.
In contrast, when assays were performed in the presence of G
,
59 ± 5.0% (n = 5) of the total
ARK1
activity was membrane-associated. *, less than the G
control, p < 0.05.
Effects of Mutant
Since bacterially expressed
proteins are utilized in the above two assays, we constructed plasmid
minigenes encoding the same mutant ARKct Peptides on
G
-mediated IP Production
ARKct domains and expressed
them in mammalian cells. In COS-7 cells, agonist stimulation of
transiently expressed
2-ARs produces pertussis toxin-sensitive,
G
-mediated PI hydrolysis. In contrast, agonist stimulation of
M1-AChRs produces pertussis toxin-insensitive, G
-independent
PI hydrolysis mediated via Gq
subunits. We utilized these two
pathways to examine the ability of co-expressed mutant
ARKct
peptides to selectively antagonize Gi
-mediated PI hydrolysis.
Expression of each mutant peptide was confirmed by Western blot as
described previously(15) , demonstrating that the level of
cellular expression of all mutant peptides was similar (data not
shown). Co-expression of wild type
ARKct peptide resulted in
70% attenuation of
2-AR-mediated IP production, while the
mutant
ARKct peptides WAA, L647G, and W643A had no significant
effect (Fig. 4A). These mutants were shown to have the
lowest G
binding activities according to the direct
G
binding assay and the translocation assay ( Fig. 2and
3). Other mutant peptides, K645E, Q642G, E646K, and K644E, inhibited
2-AR-stimulated IP production. When assayed for the ability to
antagonize M1-AChR-mediated IP production, none of the peptides
exhibited significant activity (Fig. 4B). It is of note
that the wild type peptide, which binds PIP
, did not
attenuate the M1-AChR-mediated PI hydrolysis by phospholipase C,
suggesting that the PH domain peptide does not sequester the
phospholipase C substrate, PIP
, under these conditions. The
effect of the mutant peptides on IP production provoked by
co-expression of G
and G
was also determined. The results are
consistent with the
2-AR data (Fig. 4C),
demonstrating that the inhibitory effects are dependent on the ability
to sequester G
subunits specifically in intact cells.
Figure 4:
Comparison of the effects of mutant PH
domain peptides on IP production mediated by 2-C10 AR, M1 AChR,
and tranfected G
. COS-7 cells were transiently transfected
with plasmid DNA encoding the a2-C10 AR (A), M1 AChR (B) (0.2 µg/well), or G
1 and G
2 (C) (1
µg each/well) plus empty pRK5 vector or the indicated mutant PH
domain peptide minigene (2 µg/well). Cells were stimulated with the
appropriate agonists (
2 AR, 1 µM UK14304; M1-AChR, 1
mM carbachol) IP production is represented as a percentage of
the maximal agonist-induced stimulation (A and C), or
fold stimulation (B) observed in vector cotransfected cells.
Each column represents mean ± S.E. values for three to five
separate experiments. * signifies value less than control, p < 0.01.
Binding of Mutant Fusion Proteins to
PIP
Some PH domains including that of ARK have
been shown to bind PIP
in the cleft of the
NH
-terminal
-barrel(30) . The ability of the
mutants to bind PIP
was assessed and compared to the
G
binding ability. Binding of mutant GST fusion proteins to
PC vesicles containing 5% PIP
was examined using a
centrifugation assay. The nonspecific binding of GST-
ARKct wild
type fusion protein to PC vesicles is relatively high (45-60% in
pellet after centrifugation) (Fig. 5) in comparison to native
ARK. Nonetheless, the binding of the wild type fusion protein to
PIP
-containing PC vesicles is clearly significantly higher,
since no fusion protein is detected in the supernatant. Interestingly,
much less nonspecific binding to PC vesicles was observed for some
mutant fusion proteins such as G642G,
671-689, and
KKKR
EEEE (Fig. 5). Thus, the carboxyl end of the fusion
protein appears to be lipophilic. The W643A mutant and the DSD
KKK
mutant bind significantly less to PIP
-containing PC
vesicles (Fig. 5), suggesting that the
-barrel structure
necessary for the PIP
binding was impaired by these
mutations. The DSD
KKK mutant did not inhibit the translocation of
ARK (Fig. 3) perhaps due to its weak PIP
binding. Other mutants exhibited significant translocation to
PIP
-containing vesicles (Fig. 5). Interestingly, the
Ala insertion, WA, and the KKKR
EEEE mutants, which did not bind
to G
, still contained PIP
binding activity. On
the other hand, a mutant which still had some G
-binding
activity (DSD
KKK) showed less PIP
binding. These data
suggest that crucial residues for the G
binding activity are
quite different from those for PIP
binding.
Figure 5:
PIP-mediated translocation of
various GST-PH domain fusion proteins. The percentage of the fusion
protein in PC vesicles (open bars) or 5% PIP
/95%
PC vesicles (shaded bars) are presented. The results shown
represent the mean values obtained from three separate determinations.
* signifies a fusion protein that does not show significant binding to
the PIP
-containing vesicles in comparison to PC
vesicles.
binding region of the PH domain of
ARK
is in a 125-amino-acid residue stretch located within the carboxyl
terminus (13). This region includes the 28-mer peptide
(Trp
-Ser
) that shows inhibitory activity on
the G
-mediated activation of
ARK(13) . The region
of this 28-mer peptide is the putative triple coiled-coil domain and
encompasses the most conserved last subdomain of the PH
domain(34) . The homology between two G
-binding
proteins,
ARK and phosducin, starts at subdomain 4 of the PH
domain of
ARK and continues to the adjacent sequences of the
ARK PH domain(25) . Based on these observations, we made a
series of mutants from the region encompassing the last subdomain of
the PH domain. Mutations at Trp
, Leu
, or
within the cluster of basic residues located more distally most
effectively reduced the ability of
ARK to bind to G
and
were less capable of inhibiting G
-mediated IP production.
These residues are fairly conserved among the PH domain-containing
proteins (Fig. 1). Notably, the Trp residue in subdomain 6 is
100% conserved in the PH domain sequences. Moreover, the crucial
residues for G
binding such as Trp
,
Leu
, Lys
, and Lys
are 100%
conserved among members of the
ARK family (from human to bovine)
and the Drosophila GPRK1 that bind G
(Fig. 1).
, G
, and
G
subunits form a coiled-coil structure through their amino
termini, and the dissociation of G
from G
subunits
allows
ARK to form a new triple coiled-coil(34) . The
hydrophobic and ionic interactions of the residues within the 28-mer
peptide region were thought to explain the mechanism of binding of
ARK and G
subunits. Based on this model,
Lys
, Lys
, and Glu
provide
crucial hydrogen bonds, and Trp
and Leu
participate in the hydrophobic interactions. However, the
three-dimensional structures of the PH domains of pleckstrin, spectrin,
and dynamin suggest that the Trp residue faces the inside of the
protein rather than localizing to the surface of the
helix(26, 27, 28, 29) . Moreover, the
trypsin-digested G
subunit, which lacks the coiled-coil sequence
of G
, still binds to the PH domain of
ARK and
spectrin(35) . Together with our results that the
Lys
, Lys
, and Glu
mutants
still bind to G
and inhibit G
-mediated IP
production, these observations suggest that the interaction between
G
and
ARK cannot simply be explained by the tripled
coiled-coil model. Nonetheless, the fact that the alanine insertions
after Trp
impair G
binding activity suggests
that this region could form an
-helical structure like other PH
domains, thereby playing a crucial role in interacting with
G
.
to the PH domain of
ARK was impaired by the W643A mutation. According to the
three-dimensional structure from other PH domains, the Trp residue
interacts with the core of the
-barrel, most likely contributing
to domain stability. We have made several Trp mutants from other PH
domain-containing proteins, and bacterial expression of these mutants
was lower, confirming that the Trp residue and perhaps the carboxyl
-helix are important to stabilize the domain structure.
(
)Therefore, the most conserved Trp residue is critical
for both G
binding and stablization of the
PIP
-binding cleft in the NH
terminus of the
ARK PH domain. Mutations in the cluster of anionic residues
located in the beginning of the carboxyl
-helix (DSD
KKK
mutation) attenuated binding to PIP
. Although the
DSD
KKK mutant binds G
as well as K645E or E646K, the
ability of the DSD
KKK mutant to inhibit G
-mediated
ARK translocation to rod outer segment membranes was less than
that of K645E or E646K, consistent with the evidence that both
PIP
and G
are required for PH domain-mediated
membrane translocation of
ARK(37) . Other mutants with
markedly impaired G
binding still possess PIP
binding activity, suggesting that these mutations do not result
in global misfolding of the domain peptides.
has been mapped using guanine
nucleotide releasing factor and phospholipase C
, demonstrating
that the NH
-terminal half of PH domain is not necessary for
G
binding activity(22) . Later, this was confirmed by
using the PH domain of Bruton tyrosine kinase(33) . Apparently,
the carboxyl
-helix of PH domain mediates the interaction with
G
. One function of the NH
-terminal region of the
PH domain, in contrast, is binding to PIP
(30). Moreover,
the PH domain of Bruton tyrosine kinase has been shown to bind protein
kinase C at the NH
-terminal region of the PH domain, based
on the evidence that the mutation of Arg
in the PH domain
resulted in lower protein kinase C binding capacity (32). Considering
the heterogeneity in sequences of PH domains, various other molecules
have been implicated as ligands for the PH domain. Since the molecules
which bind PH domains seem to be quite diverse ranging from lipids to
macromolecules, the function of the PH domain and the mechanisms of PH
domain action may be quite complex and delicately regulated. In at
least one instance, binding of G
and PIP
to the
PH domain of
ARK appears to be cooperative, since binding of the
COOH terminus of the PH domain to G
potentially affects
PIP
binding(37) .
binding to the
ARK PH domain and
localized these within subdomain 6 of the PH domain and the adjacent
region. Second, although the region can form an
-helical structure
which is crucial for the G
interaction, the
G
-
ARK interaction cannot simply be explained by a triple
coiled-coil model. Third, the effects of mutations in the
ARK PH
domain on the ability to bind G
are distinct from those on
PIP
binding. Finally, the most highly conserved Trp in
subdomain 6 of the PH domain is absolutely required for both
G
and PIP
binding.
,
subunits of heterotrimeric G proteins; PH, pleckstrin
homology; AR, adrenergic receptor; AChR, muscarinic cholinergic
receptor;
ARK,
-adrenergic receptor kinase; PIP
,
phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; GST,
glutathione S-transferase; IP, inositol phosphate; PI,
phosphoinositide;
ARKct, carboxyl terminus of
ARK.
ARK, rod
outer segment membranes, and G
, Sabrina T. Exum for assisting
tissue culture, and Drs. J. A. Pitcher and J. Inglese for helpful
discussions throughout the course of this work. We also thank Donna
Addison and Mary Holben for excellent secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.