From the
The pleckstrin homology (PH) domain is an approximately
100-amino-acid region of sequence homology present in numerous proteins
of diverse functions, which forms a discrete structural module. Several
ligands capable of binding to PH domain-containing proteins have been
identified including phosphatidylinositol 4,5-bisphosphate
(PIP
The pleckstrin homology (PH)
Several lines of evidence suggest that
PH domains may function to target proteins to membranes, potentially
facilitating appropriate interactions with other components of the
signal transduction pathway. The PH domain of spectrin has been
implicated as a probable site for membrane binding
(11) , and the
PH domain of PLC-
Following the observation that PH domains
and lipid binding molecules share structural similarities Harlan et
al.(16) demonstrated that fusion proteins encompassing the
PH domains from several proteins including the
Purified receptor was reconstituted into either soybean
phosphatidylcholine vesicles (20% PC) or into vesicles of defined lipid
composition. To form vesicles the required amounts of lipid in
chloro-form were dried under a stream of nitrogen, resuspended in 10
mM Tris-HCl, pH 7.2, 100 mM NaCl by vortexing, and
sonicated with a microtip sonicator. Purified
Comparison of the recently elucidated structural
features of several PH domains
(7, 8, 9, 10) reveals a degree of polarity of structure such that the
highly conserved carboxyl-terminal
We thank Darrell Capel for purified
) and the G
subunits of
heterotrimeric G proteins (G
), which bind to the
amino and carboxyl termini of the PH domain, respectively. Here we
report that the binding of G
and lipid to the PH
domain of the
-adrenergic receptor kinase (
ARK)
synergistically enhances agonist-dependent receptor phosphorylation and
that both PH domain-binding ligands are required for membrane
association of the kinase. PIP
and to a lesser extent
phosphatidylinositol 4-phosphate, phosphatidylinositol, and
phosphatidic acid were the only lipids tested capable, in the presence
of G
, of enhancing
ARK activity. In contrast,
the K
and V
for
phosphorylation of a soluble
ARK substrate (casein) was not
altered in either the presence or absence of G
and/or PIP
. A fusion protein of the
ARK
containing an intact PH domain inhibits
G
/PIP
-dependent
ARK activity. In
contrast, a mutant fusion protein in which a tryptophan residue,
invariant in all PH domain sequences, is mutated to alanine shows no
inhibitory activity. The requirement for the simultaneous presence of
two PH domain binding ligands represents a previously unappreciated
mechanism for effecting membrane localization of a protein and may have
relevance to other PH domain-containing proteins.
(
)
domain,
named for the protein in which it was first identified, is an
approximately 100-amino-acid region of sequence
homology
(1, 2) . To date more than 70 PH
domain-containing proteins have been identified including
serine/threonine and tyrosine kinases, several isoforms of
phospholipase C, GTPase-activating proteins, nucleotide exchange
factors, and cytoskeletal
elements
(1, 2, 3, 4, 5, 6) .
Whether the PH domains of these proteins share common functions and
what these functions are remain to be elucidated. Despite limited
sequence identity the structural similarities recently reported for the
PH domains of pleckstrin
(7) , spectrin
(8) , and
dynamin
(9, 10) support the classification of these
regions as discrete domains.
1 has been shown to be involved in binding this
enzyme to lipid bilayers containing phosphatidylinositol
4,5-bisphosphate (PIP
)
(12) . Furthermore, the
carboxyl terminus of the
-adrenergic receptor kinase-1 (
ARK)
has been demonstrated to bind the G
subunits of
heterotrimeric G proteins
(G
)
(13, 14) . This interaction
leads to the membrane localization of the kinase and subsequent
increased phosphorylation of agonist-occupied G protein-coupled
receptor substrates. Fusion proteins encompassing the PH domains of
several other proteins have additionally been demonstrated to bind
G
, although with varying affinities
(15) .
Importantly the G
binding site is limited to and
extends slightly beyond the carboxyl terminus of the PH
domain
(15) , raising speculation as to the function of the amino
terminus of this domain.
ARK bound
specifically to PIP
. Furthermore, the amino terminus of the
PH domain was implicated as being the important site of lipid
interaction. The PH domain of the
ARK has thus been shown to
interact with both G
and PIP
at the
carboxyl and amino termini, respectively, of the PH domain. Here we
demonstrate that interaction of these two ligands with the intact
enzyme is required for its membrane association and activation.
Materials
Bovine ARK was overexpressed and
purified from baculovirus-infected Sf9 cells
(17) , and
G
subunits were purified from bovine brain
(18) according to previously published procedures. Purified
lipids, soybean phosphatidylcholine (
20% phosphatidylcholine
(PC)), and partially dephosphorylated casein were from Sigma. All other
reagents were of the highest grade commercially available.
Purification and Reconstitution of the
The human
-Adrenergic Receptor
-adrenergic receptor (
AR) was expressed and
purified from baculovirus-infected Sf9 cells. Briefly, Sf9 cells were
harvested 48 h postinfection with recombinant virus. The cells were
lysed and a membrane fraction prepared by centrifugation at 33,000
g. The membranes (5 mg/ml) were subsequently
solubilized with 0.25% w/v n-dodecyl
-D-maltoside, and the
AR was purified by affinity
chromatography on an alprenolol-Sepharose column as described in Ref.
19.
AR was reconstituted
into these vesicles as described previously
(20) . The
AR-containing vesicles were resuspended in 20 mM
Tris-HCl, pH 7.5, 2 mM EDTA, and the receptor concentration
was determined by radioligand binding using
I-labeled
(-)-iodocyanopindolol.
The
ARK-mediated
AR Phosphorylation
AR (40 nM) reconstituted in various lipid environments
(described in the text and figure legends) was incubated with
ARK
(10 nM) in 20 mM Tris-HCl (pH 7.5), 2.0 mM
EDTA, 10 mM MgCl
, 1 mM dithiothreitol
containing 60 µM ATP (
6000 cpm/pmol) in a total
volume of 25 µl. All assays were performed in the presence of 50
µM(-)-isoproterenol unless otherwise indicated.
Purified G
subunits (20-200 nM)
were also included in the phosphorylation reactions where indicated.
Reactions were incubated at 30 °C and stopped by addition of an
equal volume of SDS sample-loading buffer (8% SDS, 25 mM
Tris-HCl, pH 6.5, 10% glycerol, 5% mercaptoethanol, 0.003% bromphenol
blue) and electrophoresed on 10% SDS-polyacrylamide gels. The dried
gels were subjected to autoradiography and PhosphorImager analysis to
determine the picomoles of phosphate transferred to the receptor
substrate.
Construction and Purification of Glutathione
S-transferase Fusion Proteins
Glutathione S-transferase
fusion proteins derived from the ARK carboxyl terminus (ct) were
expressed in the Escherichia coli strain NM522 or BL21 and
were purified as described previously
(14) .
(
)
Binding of the
The ARK to Lipid
Vesicles
AR (3 pmol) reconstituted in vesicles composed
of either 100% PC or 95% PC, 5% PIP
was incubated in the
presence or absence of G
subunits (0.2 µg) for
1 h on ice. After incubation the receptor was added to polycarbonate
tubes (7
20 mm) (Beckman) containing purified
ARK (0.5
µg), and the reaction mixture was diluted to a final volume of 30
µl with PBS. The final lipid concentration was 1.7 mg/ml in all
assays. After incubation at room temperature for 10 min and on ice for
5 min, the tubes were centrifuged at 100,000 rpm (TL-100 rotor) for 15
min at 4 °C. The supernatant was removed and the pellet rinsed once
with PBS. The pellet was subsequently resuspended in 15 µl of PBS
and transferred to a clean tube. SDS sample-loading buffer was added to
the supernatant, and pellet fractions and the samples were
electrophoresed on 4-20% gradient polyacrylamide gels (Novex) and
subjected to Western blot analysis (ECL, Amersham Corp.) using
anti-
ARKct antibodies
(22) . The distribution of the
ARK between the pellet and the supernatant was determined by
densitometric analysis of the Western blot.
RESULTS AND DISCUSSION
Previous studies have demonstrated that the binding of
G to the PH domain of the
ARK leads to its
membrane association and enhanced receptor
phosphorylation
(13, 14, 23, 24) .
However, these experiments all utilized receptor substrates presented
in heterogeneous lipid environments such as the
AR reconstituted
in crude lipid preparations or rhodopsin in rod outer segment
membranes. To investigate potential interactions between the PH domain
of
ARK and lipids,
ARs reconstituted into vesicles of defined
lipid composition were utilized. Addition of G
to
the
ARK significantly enhanced the rate and extent of receptor
phosphorylation when the substrate was presented in a heterogeneous
lipid environment (20% PC) (closed and opentriangles) but not when the receptor was reconstituted in
a homogeneous lipid environment (100% PC) (closed and opensquares) (Fig. 1A). Thus, vesicles
composed of 100% PC lack an essential cofactor required for
G
-mediated enhancement of
ARK activity. Could
this missing component be PIP
, a lipid previously
documented to bind to the amino terminus of the PH domain of
ARK
(16) ? Indeed, although
AR reconstituted into PC
vesicles containing PIP
(3 or 10%) was a poor substrate for
the
ARK (closedsymbols), addition of
G
led to a dramatic enhancement of receptor
phosphorylation (opensymbols)
(Fig. 1B). In the presence of 100 nM
G
subunits and 3 or 10% PIP
the
initial rate of receptor phosphorylation increased approximately 25-
and 65-fold, and the maximal extent of phosphorylation increased
approximately 12- and 20-fold, respectively. Thus the
ARK-mediated
phosphorylation of purified reconstituted
AR is dependent upon the
presence of two ligands, each previously shown to bind PH domains.
Figure 1:
PIP2 is required for
G-mediated activation of
ARK. A,
phosphorylation of
AR reconstituted in vesicles composed of an
impure lipid preparation (20% PC) or purified PC. Reactions were
performed in the presence of 50 µM (-)-isoproterenol
in either the presence or absence of 100 nM G
subunits.
,
AR in 20% PC;
,
AR in 20% PC
+ G
;
,
AR in 100% PC;
,
AR in 100% PC + G
. B,
phosphorylation of
AR reconstituted in vesicles composed of
purified PC containing 3 or 10% PIP
. Reactions were
performed in either the presence or absence of 100 nM
G
and in the presence of 50 µM
(-)-isoproterenol.
,
AR in 3% PIP
;
,
AR in 3% PIP
+ G
;
,
AR in 10% PIP
;
,
AR in 10% PIP
+ G
. Phosphorylation reactions were
performed as described under ``Experimental Procedures'' and
were stopped at the times indicated. The data represent the mean values
obtained from three separate determinations.
Is the simultaneous presence of G and lipid
required for the membrane association of
ARK or does PIP
interact with and directly activate a membrane-associated
G
/
ARK complex? To distinguish between these
two possibilities the kinetics of phosphorylation of a soluble
ARK
substrate (casein) were investigated. The K
and V
for
ARK-catalyzed casein
phosphorylation were unaffected when assayed in the presence of
vesicles composed of either 100% PC or 95% PC, 5% PIP
in
either the presence or absence of G
(data not
shown). These results support the hypothesis that both PH domain
ligands are required for the membrane association of the
ARK since
addition of G
and lipid did not directly activate
the kinase. Indeed, when incubated with
AR-containing vesicles,
enhanced
ARK activity (Fig. 2A) and membrane
association of the kinase (Fig. 2B) was observed only
when both PIP
and G
were present.
Under the conditions utilized in this study neither PH domain ligand
alone was sufficient to cause association of the
ARK with lipid
vesicles (Fig. 2B). Interestingly, the same pattern of
membrane association of the
ARK was observed in either the absence
or presence (data not shown) of
AR agonist. Agonist occupancy of
the receptor was, however, required for
ARK-mediated
AR
phosphorylation (data not shown).
Figure 2:
Membrane association of the ARK
requires the presence of both G
and
PIP
. A, autoradiograph showing
ARK-mediated
AR phosphorylation. The
AR was reconstituted in lipid
vesicles composed of 100% PC (PC) or 95% PC, 5% PIP
(PIP
). G
subunits were included where indicated. Phosphorylation reactions
were performed in the presence of 50 µM
(-)-isoproterenol for 10 min. The concentrations of all
components were as for B (see ``Experimental
Procedures''). B,
ARK binding to
AR-containing
vesicles composed of 100% PC (PC) or 95% PC, 5% PIP
(PIP
). G
subunits were
included where indicated, and assays were performed as described under
``Experimental Procedures.'' The distribution of the
ARK
between the supernatant (S, whitebars) and
pellet (P, blackbars) fractions is shown
±S.E. for three separate determinations. A representative
Western blot is also displayed.
The membrane association of
ARK in the presence of
AR requires both 5% PIP
and G
and would appear to account for the
enhanced rate and extent of receptor phosphorylation observed under
these conditions. Increasing the concentration of PIP
and
G
enhances the initial rates of
ARK
phosphorylation of
AR in a dose-dependent fashion (Fig. 3).
As the concentration of G
increases from 20
nM to an apparently saturating concentration of 120
nM a dramatic increase in the V
of the
enzyme and a modest increase in the apparent affinity for PIP
is observed (Fig. 3). Analysis of this data using the
ALLFIT program
(25) reveals slope factors greater than 1, an
observation that may suggest positive cooperativity.
Figure 3:
Dose-dependent activation of the ARK
by PIP
and G
. Purified
AR was
reconstituted in vesicles composed of PC containing various
concentrations of PIP
. The initial rate of phosphorylation
of these receptor substrates by the
ARK was subsequently
determined in the absence of any additions (
) or in the presence
of 20 nM (
), 40 nM (
), 120 nM
(
), or 200 nM (
) G
subunits.
The results shown represent the mean values from two separate
determinations. The EC
values for PIP
binding
are 5.7% PIP
(20 nM G
),
4.35%PIP
(40 nM G
), 4.0%
PIP
(120 nM G
), and 3.0%
PIP
(200 nM G
). These values
were determined using the ALLFIT program. Constraining parameters such
that all curves shared the same EC
value had a significant
deleterious effect on the quality of the fit (p <
0.01).
Which lipids in
the presence of G are capable of mediating
membrane association of the
ARK? To address this question
AR
was reconstituted in a lipid background of PC in the presence of an
EC
concentration of PIP
(3%) or 3% of various
other lipids. At low G
subunit concentrations (20
nM) the only lipids capable of promoting
ARK
phosphorylation of
AR were PIP
and to a much lesser
extent phosphatidylinositol 4-phosphate (PIP) (Fig. 4, left
panel). This pattern of lipid specificity is similar to that
observed by Harlan et al. (16), who looked directly at the
binding of PH domain-containing fusion proteins to lipid vesicles.
PIP
and (with a lower affinity) PIP were the only lipids
capable of promoting membrane association of these proteins. Raising
the concentration of G
(200 nM) increases
the extent of
AR phosphorylation and appears to increase the
apparent affinity of the
ARK for lipid. Under these conditions
other precursors (phosphatidylinositol and PIP) or metabolites
(phosphatidic acid) of PIP
become effective promoters of
ARK-mediated
AR phosphorylation (Fig. 4, right
panel). Thus, although at 20 nM G
PIP
is approximately 4-fold more effective at
promoting
ARK-mediated
AR phosphorylation than PIP
(Fig. 4, left panel), at a higher concentration of
G
(200 nM), PIP
and PIP were
equally effective (Fig. 4, right panel). Of the lipids
tested only PIP
, PIP, phosphatidylinositol, and
phosphatidic acid were capable of promoting G
activation of the
ARK (Fig. 4, right panel).
Figure 4:
PIP most effectively mediates
lipid/G
enhancement of
ARK activity. Purified
AR reconstituted in 97% PC, 3% of the indicated lipids was
phosphorylated for 10 min in the absence of G
(whitebars) or alternatively in the presence
of either 20 nM G
(leftpanel, black bars) or 200 nM
G
(right panel, black bars). PA,
phosphatidic acid; PI, phosphatidylinositol, PE,
phosphatidylethanolamine; MAG, monoacylglycerol
(1-monopalmitoyl-rac-glycerol (C16:0)); DAG,
diacylglycerol (1,2-dioleoyl-rac-glycerol (C18:1,
[cis]-9)); ceramides (type III); type III);
GC, galactocerebrosides (type II). The results shown represent
the mean values obtained from at least three separate
determinations.
That the PH domain of the ARK is the region of the enzyme that
interacts with both G
and PIP
is
suggested by previous studies demonstrating direct interactions between
these ligands and fusion proteins encompassing the PH domain of this
enzyme
(15, 16) . Furthermore, here we show that a
carboxyl-terminal fusion protein of the
ARK, which contains the PH
domain, inhibits G
/PIP
-dependent
ARK activity (Fig. 5,
ARKct). However, a
fusion protein in which a tryptophan residue invariant in all PH domain
sequences is mutated to alanine (Trp
in the
ARK) and
which shows no binding to either G
or
PIP
has no inhibitory activity (Fig. 5,
Trp
mutant).
Figure 5:
Inhibition of
G/PIP
-mediated
ARK activation by
the PH domain of
ARK. Purified
AR reconstituted in 97% PC, 3%
PIP
was phosphorylated in the presence of 20 nM
G
for 10 min. The glutathione
S-transferase fusion proteins
Pro
-Leu
of the
ARK
(
ARKct) and a mutant fusion protein in which Trp
of the
ARK is mutated to alanine (Trp
mutant) were included in the phosphorylation reactions
at the concentrations indicated. The results represent the means
±S.E. for three separate
determinations.
The data presented
demonstrate that effective membrane localization of the ARK, which
enhances both the rate and extent of phosphorylation of receptor
substrates, requires the simultaneous presence of two PH domain ligands
(PIP
and G
). Neither PH domain binding
ligand alone is sufficient to affect this functional activation of the
enzyme. The requirement for a specific lipid to effect
G
-mediated activation of the
ARK was
previously unappreciated since purified receptor preparations
reconstituted in heterogeneous lipid environments have been exclusively
utilized as substrates for this kinase. Increasing the concentration of
G
appears to increase the affinity of the
ARK
for lipid. Thus, binding of a ligand to the carboxyl terminus may
affect binding of a ligand to the amino terminus of the PH domain. The
precise mechanism and potentially cooperative nature of multiple ligand
binding to the PH domain of the
ARK are currently under
investigation.
-helix and three loops
connecting the
-strands,
/
,
/
, and
/
, fall on opposite faces of the
domain. These three loops represent the most variable region of PH
domains and provide the potential for differences in ligand
specificity. Thus, although in this and previous studies the PH domain
of the
ARK has been shown to interact with PIP
and
G
(15, 16) not all PH domains have
been demonstrated to bind to these specific ligands, and various other
molecules have been implicated as ligands for these domains. The PH
domain of spectrin has been implicated as binding to specific sites in
bovine brain membranes
(11) , and the PH domain of Bruton
tyrosine kinase in addition to binding G
(26) has been shown to bind protein kinase C
(21) . The
number and variety of ligands for PH domains remain to be elucidated;
however, the synergistic activation of the
ARK by G
and lipid suggests that reexamination of the binding of these
particular ligands to the PH domains of other proteins may be fruitful.
In particular, proteins that have been shown to bind weakly to
G
or PIP
might display a higher
affinity for these ligands and potentially altered functional
characteristics when assayed in the presence of both. The potentially
cooperative binding of multiple ligands to PH domains may also in part
explain the failure of glutathione S-transferase-PH domain
fusion proteins to interact with protein ligands in either the two
hybrid yeast system or when utilized to screen T7 promoter-based
bacterial expression libraries
(9) . It remains to be determined
if the membrane localization of proteins via multiple ligand binding is
a function common to all PH domains.
,
-subunits of heterotrimeric
G proteins;
AR,
-adrenergic receptor;
ARK,
-adrenergic receptor kinase; PC, phosphatidylcholine;
PIP
, phosphatidylinositol 4,5-bisphosphate; PIP,
phosphatidylinositol 4-phosphate; PBS, phosphate-buffered saline; ct,
carboxyl terminus.
ARK and
G
subunits, W. Carl Stone for DNA sequencing and
purification of the
ARKct fusion protein, W. Carl Stone and Dr.
Jeffrey L. Arizza for generation of the anti-
ARK polyclonal sera,
Grace P. Irons for virus and cell culture, and Dr. Mario Tiberi for
analysis of data using the ALLFIT program.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.