From the Department of Pharmacology, Medical University of South
Carolina, Charleston, South Carolina 29425 and the
Department of Microbiology and Immunology, Kimmel Cancer
Institute, Thomas Jefferson University, Philadelphia, Pennsylvania
19107
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ABSTRACT |
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We report the direct interaction of G with
the third intracellular (i3) loop of the M2- and
M3-muscarinic receptors (MR) and the importance of this
interaction relative to effective phosphorylation of the receptor
subdomain. The i3 loop of the M2- and the M3-MR were expressed in bacteria and purified as glutathione
S-transferase fusion proteins for utilization as an
affinity matrix and to generate substrate for receptor subdomain
phosphorylation. In its inactive heterotrimeric state stabilized by
GDP, brain G-protein did not associate with the i3 peptide affinity
matrix. However, stimulation of subunit dissociation by
GTP
S/Mg2+ resulted in the retention of G
, but not
the G
subunit, by the M2- and M3-MR i3
peptide resin. Purified G
bound to the M3-MR i3
peptide with an apparent affinity similar to that observed for the
G
binding domain of the receptor kinase GRK2 and Bruton tyrosine
kinase, whereas transducin
was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the
M3-MR peptide by GRK2 required both G
and lipid as is
the case for the intact receptor. Incubation of purified GRK2 with the
i3 peptide in the presence of G
resulted in the formation of a
functional ternary complex in which G
served as an adapter
protein. Such a complex provides a mechanism for specific spatial
translocation of GRK2 within the cell positioning the enzyme on its
substrate, the activated receptor. The apparent ability of G
to
act as a docking protein may also serve to provide an interface for
this class of membrane-bound receptors to an expanded array of
signaling pathways.
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INTRODUCTION |
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The precise functional roles of the intracellular domains of the family of receptors coupled to heterotrimeric G-proteins remain unclear. The seven-membrane span core motif for such receptors is fairly compact presenting a relatively small surface area at the cytoplasmic membrane face. Thus, the intracellular domains assume a significant structural presence within the receptor's microenvironment and likely play a role in receptor trafficking, signaling efficiency, receptor phosphorylation, and signal termination. The juxtamembrane segments of the third intracellular (i3)1 loops of most such receptors are critical for G-protein activation by the agonist-occupied receptor, whereas other segments of the i3 loop or other cytoplasmic domains of the receptor may interact with various accessory proteins that regulate signal propagation and contribute to the formation of a signal transduction complex on the inner face of the membrane (1-3). As part of a continuing effort to define such a signal transduction complex we used modular receptor domains to identify interacting proteins and the subdomains participating in such interactions. We initially focused on the i3 loop of muscarinic receptor (MR) subtypes.
The M2- and M4-MR primarily couple to the
pertussis toxin-sensitive G-proteins Gi/Go (4).
The M1-, M3-, and M5-MR generally couple to the Gq family of G-proteins but are also capable
of activating Go and Gi (4-6). As is the case
with most members of this receptor superfamily, there is considerable
flexibility in the type of G-protein activated by a given receptor.
Both the M2-MR and the M3-MR are phosphorylated
by the receptor kinase GRK2 in an agonist-dependent manner
(7-9). Receptor phosphorylation is a key regulatory event in the
processing of external stimuli by the cell. There are three components
required for effective receptor phosphorylation: 1) lipid, 2) G,
and 3) an activated conformation of the receptor (10-12). G
likely plays a key role in mediating receptor phosphorylation in the
intact cell. G
binds directly to the pleckstrin homology domain
of GRK2 and is postulated to mediate translocation of GRK2 from the
cytosol to the inner face of the plasma membrane. Mechanistically, how
this translocation occurs and results in receptor phosphorylation is unclear (13-16). In the present manuscript, we report the direct interaction of G
with the i3 loop of the M2-MR and
M3-MR. The interaction of G
with the i3 loop was
dependent upon G-protein activation and resulted in the formation of a
ternary complex consisting of the i3 peptide, G
, and GRK2
essentially positioning the enzyme on its substrate. In addition to its
role in receptor regulation, the interaction of G
with the i3
loop may also facilitate the interface of G-protein-coupled receptors
to diverse signaling pathways via complex formation with additional
G
-binding proteins.
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EXPERIMENTAL PROCEDURES |
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Materials--
Heterotrimer G-protein and G dimer were
purified from bovine brain as described previously (17). Transducin was
kindly provided by Dr. Heidi E. Hamm (Dept. of Molecular Pharmacology, Northwestern University). Antisera to the amino-terminal 16 amino acids
of Go
was kindly provided by Dr. Graeme Milligan (Dept. of Biochemistry and Pharmacology, University of Glasgow). Antisera to
the carboxyl-terminal 10 amino acids of G
1, which recognizes G
1-4, was generated as described (18). Monoclonal antibody 3A10
recognizes an epitope within residues 500-531 of the carboxyl terminus
of bovine GRK2 (19). GRK2 was purified from Sf9 insect cells
infected with recombinant virus as described (20). The AC2Q peptide
corresponding to amino acids Gln956 to Lys982
of adenylyl cyclase II was synthesized and purified by Biosynthesis Inc. (Lewisville, TX). All other materials were obtained as described elsewhere (1, 2).
Plasmid Constructions and Protein Expression-- The M2-MR i3 peptide (His208-Arg387; MW = 19,603 plus GST) and the GRK2-ct (Tyr466-Leu689; MW = 26,073 plus GST) constructs were generated as described previously (1, 13). The construct encoding the PH-domain (Met1-Gln196; MW = 22,965 plus GST) of Bruton tyrosine kinase (Btk-PH) was kindly provided by Dr. Owen N. Witte (Dept. of Microbiology and Molecular Genetics, Howard Hughes Medical Institute, UCLA). The full-length rat M3-MR cDNA was kindly provided by Dr. Tom Bonner (Laboratory of Cell Biology, NIMH). The M3-MR i3 peptide (Arg252-Gln490; MW = 26,471 plus GST) and the M3-MR carboxyl terminus (Asn547-Leu589; MW = 5,351 plus GST) constructs were generated by DNA amplification using the polymerase chain reaction and inserted into the BamHI and EcoRI restriction sites of the pGEX-4T-1 vector. To generate the M3-MR i1 peptide (Lys93-Tyr104; MW = 1,449 plus GST) and the M3-MR i2 peptide (Asp164-Lys182; MW = 2,374 plus GST) constructs, complementary oligonucleotides from these regions were synthesized and annealed prior to ligation into the BamHI and EcoRI restriction sites of the pGEX-4T-1 vector. The structure of each construct used in the present study was verified by restriction mapping and nucleotide sequence analysis. GST fusion proteins were expressed in bacteria and purified on a glutathione-Sepharose matrix as described previously (1). GST and GST fusion proteins were eluted from the resin with 10 mM glutathione and subsequently concentrated/desalted by ultrafiltration (Centricon 3).
Protein Interaction Assays-- Purified receptor subdomains (~5 µg) immobilized on a glutathione resin (2-10 µl of packed resin) or eluted from the resin were incubated with G-protein or GRK2 in a total volume of 250 µl of buffer A (20 mM Tris-HCl, pH 7.5, 0.6 mM EDTA, 1 mM dithiothreitol, 70 mM NaCl, 0.01% Thesit) at 4 °C for 90 min with gentle rotation. In experiments using receptor subdomains not tethered to resin, samples were subsequently incubated with 10 µl of packed resin at 4 °C for 20 min with gentle rotation. The resin was washed three times with 0.5 ml of buffer A at 4 °C, and the retained proteins were solubilized in Laemmli sample buffer and applied to a denaturing 10% polyacrylamide gel. Polyvinylidene difluoride membrane transfers were evaluated by immunoblotting (1).
Phosphorylation of the M3-MR i3 Peptide by
GRK2--
The incubation conditions for phosphorylation reactions were
essentially as described previously for the intact M2-MR,
M3-MR, and -adrenergic receptor (AR) (10, 21, 22).
Briefly, the reaction was carried out in a total volume of 50 µl of
buffer (20 mM Tris-HCl, pH 7.2, 2 mM EDTA, 7 mM MgCl2) containing 2-4 pmol of the
GST-M3-MR i3 peptide fusion protein and 50 nM
GRK2 with or without various additions as described in the figure
legends. Unless indicated otherwise, all phosphorylation reactions
contained 300 µM PI. The reactions were initiated by
addition of 0.1 mM [
-32P]ATP (500-1000
cpm/pmol) and incubated at 30 °C for 40 min. The reactions were
stopped by addition of 50 µl of 2 × Laemmli sample buffer and
electrophoresed on 10% SDS-polyacrylamide gels. The gels were dried
and subsequently exposed to Kodak XAR-5 film for 1 to 12 h. The
amount of peptide phosphorylation was determined by cutting the
phosphorylated bands from the gel and quantitation by liquid
scintillation spectrometry.
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RESULTS AND DISCUSSION |
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The interaction of G-protein subunits with the i3 peptides
(M2-MR i3 = His208-Arg387,
M3-MR i3 = Arg252-Gln490) was
first addressed using heterotrimeric G-protein or dissociated G-protein
subunits. The i3 peptides were bound to a glutathione-Sepharose matrix
and incubated with bovine brain G-protein in the presence of GDP/EDTA
or GTPS/Mg2+, which would either stabilize the
heterotrimer or promote subunit dissociation, respectively. The resin
was washed, and retained proteins were determined by SDS/PAGE and
subsequent immunoblotting with either a Go
or G
common antisera. In its heterotrimeric form, immunoblotting with either
antisera indicated that G-protein did not effectively interact with the
i3 peptide resin (Fig. 1A). However, following activation and subunit dissociation, G
, but not Go
, bound to the i3 peptides (Fig. 1A).
Similar experiments with transducin resulted in undetectable G
binding to the M2- or M3-MR i3 peptide (Fig.
1A) consistent with the distinct functional properties of
transducin and brain G-protein (~80% of which is Go)
(23). These data suggest that the i3 loop presents a motif for brain
G
binding and that the G
domain recognized by this motif is
masked by the G
subunit.
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The interaction of the i3 peptides with G was also observed using
purified bovine brain G
that was physically separated from brain
G
(Fig. 1, B and C). Purified G
did not
bind to GST control, protein-tyrosine phosphatase 1C (PTP1C),
Na+/H+ exchanger or c-Jun fusion proteins (Fig.
1B) and also did not interact with the M3-MR i1,
M3-MR i2, or M3-MR carboxyl terminus peptides
(Fig. 1C). Although the M3-MR i3 peptide
interacted with brain G
, it did not recognize transducin
(Fig. 1C). As brain G
and transducin
differ in
their
subunits (23), these data indicate that the
subunit plays
an important role in the recognition of G
by the receptor
subdomain.
To provide additional insight as to the relative properties of G
binding to the i3 peptides, the M3-MR i3 was compared with G
binding domains of the proteins, GRK2 and Btk. GRK2
phosphorylates selected G-protein-coupled receptors following their
activation by agonist, whereas Btk is a tyrosine kinase involved in
human X chromosome-linked immunodeficiency. The binding of G
to
the M3-MR i3, GRK2-ct, and Btk-PH was dependent upon
G
concentration and exhibited similar affinities although maximum
binding varied (Fig. 2). The direct
association of G
with the receptor subdomain may relate to
earlier reports indicating that G
plays diverse roles in
signaling by G-protein-coupled receptors (23-28).
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G is postulated to mediate translocation of GRK2 from the cytosol
to the membrane leading to phosphorylation of activated adrenergic,
muscarinic, neurokinin, and other receptors of this structural class.
Mechanistic aspects of this translocation are unclear. G
may
increase the amount of membrane-associated GRK2 by using the acylated
subunit of G
to anchor to hydrophobic sites in the membrane
bilayer. A second possibility is that the binding of G
to GRK2
initiates a conformational change in the enzyme that reveals a
"membrane binding domain" of GRK2. Another possibility is that
G
may provide an anchor to the membrane by interacting with the
i3 loop of the activated receptor. In such a situation, the kinase
would be positioned adjacent to its substrate suggesting that one role
of G
is to "position" enzyme and substrate facilitating the
phosphorylation reaction. If such a hypothesis is true, then G
should promote the formation of a ternary complex consisting of the
activated receptor, G
, and GRK2. We addressed these issues
experimentally using the M3-MR i3 peptide.
We first determined if the M3-MR i3 peptide was a suitable
substrate for phosphorylation by GRK2 and if so, how this
phosphorylation compared with that observed with the intact
agonist-activated receptor itself. As is the case for the intact
M2-MR, M3-MR, and 2-AR,
phosphorylation of the M3-MR i3 peptide required both
G
and the acidic lipid PI when assayed under ionic conditions
similar to cytosol (Fig. 3A).
G
and PI stimulated phosphorylation of the i3 peptide in a
synergistic manner (Fig. 3A). The M3-MR i3 peptide was phosphorylated by GRK2 to a stoichiometry of ~1-3 mol of
phosphate/mol of peptide, which is comparable to the results obtained
with the intact, agonist-activated M2-MR,
M3-MR, and
2-AR. The ability of G
to
stimulate GRK2-mediated phosphorylation of the M3-MR i3
peptide was concentration-dependent, and it was blocked by
the AC2Q peptide, a 27-residue peptide derived from adenylyl cyclase
type II that blocks G
regulation of various effector proteins
(Fig. 3, B and C) (29). The interaction of G
with the M3-MR i3 peptide described earlier was
also blocked by the AC2Q peptide (data not shown).
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As observed for the intact agonist-activated 2-AR,
M2-MR, and M3-MR (9, 21, 22), the
phosphorylation of the M3-MR i3 peptide was sensitive to
increased ionic strength of the incubation buffer (Fig.
4A), and this apparently
reflects the loss of a direct interaction between GRK2 and the i3
peptide (Fig. 4B, upper panel). Addition of
G
to the incubation buffer reversed the inhibition of
M3-MR i3 peptide phosphorylation by increased salt
concentrations, suggesting that G
facilitated the interaction of
GRK2 with its substrate. The interaction of G
with the
M3-MR i3 peptide was actually augmented by increasing the
concentration of KCl or NaCl (Fig. 4B, lower
panel), an effect that was directly opposite to the influence of
increased ionic strength on i3 peptide/receptor phosphorylation and the
association of GRK2 with the M3-MR i3 peptide (Fig. 4,
A and B, upper panel). Thus, under
ionic conditions similar to those found in cytosol, GRK2 did not
effectively phosphorylate the intact agonist-activated receptor or the
i3 peptide unless G
was present. Indeed, addition of G
resulted in the association of GRK2 with the M3-MR i3
peptide affinity matrix in the presence of cytosolic concentrations of
potassium (Fig. 4C). This association is likely mediated by
a ternary complex consisting of the receptor subdomain, G
, and
GRK2. These data indicate that G
served as a docking protein to
allow formation of this ternary complex, which is apparently required
for effective phosphorylation by GRK2.
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There are three important points concerning the interaction of G
with the M3-MR i3 peptide. First, the interaction of the M3-MR i3 peptide with G
requires dissociation of
heterotrimeric G-protein. Second, the inability of the
M3-MR i3 peptide to recognize transducin
indicates
that the interaction with G
is isoform-selective. Third, it is
hypothesized that when the M3-MR i3 peptide is expressed free of the conformational constraints imposed by the receptor's membrane spans, it assumes an activated conformation (see
"Discussion" in Ref. 1). This hypothesis is supported by structural
analysis of the cytoplasmic domains of rhodopsin where key atomic
distances were identical with those observed by chemical cross-linking
of the intact activated receptor (30, 31). Such an interpretation is
also supported by the interaction of arrestin with the i3 loop of the
M2-MR, M3-MR, and
2-AR (1) and
the ability of GRK2 to phosphorylate the M3-MR i3 peptide
in a manner similar to that observed with intact, agonist-activated
G-protein-coupled receptors.
The dependence of G binding to the i3 peptide upon G-protein
dissociation is of particular note relative to both potential downstream signaling events and various aspects of receptor regulation. Upon receptor activation, G
may be "trapped" by the
M3-MR i3 loop and present a motif that is recognized by
GRK2 positioning the kinase in close proximity to its substrate, the
activated receptor. Although such a scenario may not be operative for
all GRKs or G-protein-coupled receptors, it is of particular interest for several reasons. G
is suggested to interact with several molecules involved in signal propagation. The regulation of
structurally diverse molecules by G
suggests that its structure
has unique properties that perhaps facilitate simultaneous interaction
with more than one protein. These structural properties might reside in
the repetitive elements of the WD-40 motifs and would be conducive to
the function of G
as an adaptor protein. The apparent ability of
G
to act as a docking protein suggests that G
may anchor the formation of a signal transduction complex. Such an anchor may also
allow the interface of selected G-protein-coupled receptors to
signaling pathways involving soluble tyrosine kinases and/or low
molecular weight G-proteins leading to activation of mitogenic signaling pathways and/or changes in cellular architecture.
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ACKNOWLEDGEMENTS |
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We thank Dr. Owen N. Witte, Dr. Gen-Shen Feng
(Dept. of Biochemistry and Molecular Biology, Indiana University School
of Medicine), Dr. Larry Fliegel (Dept. of Biochemistry and
Pediatrics, University of Alberta, Alberta, Canada), and Dr.
Steven Rosenzweig (Dept. of Pharmacology, Medical University of South
Carolina) for providing the Btk-PH, PTP1C,
Na+/H+ exchanger, and c-Jun fusion protein
constructs, respectively. We thank Dr. Tom Bonner for the rat
M3-muscarinic receptor cDNA and Dr. Heidi E. Hamm for
transducin and transducin . The expression vector containing the
peptide derived from the third intracellular loop of the
M3-muscarinic receptor was generated by Dr. Pablo Escriba
in the laboratory of Dr. Lanier. We appreciate the technical assistance
of Lisa Kless in the laboratory of S. M. L. and the contributions of
Dr. Jane Dingus and Bronwyn Tatum for G-protein purification in the
laboratory of J. D. H.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants NS24821 (to S. M. L.), DK37219 (to J. D. H.), and GM47417 (to J. L. B.) and Council for Tobacco Research Grant 2235 (to S. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Established Investigator of the American Heart Association.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2574; Fax: 803-792-2475; E-mail: laniersm{at}musc.edu.
1
The abbreviations used are: i3, third
intracellular loop; MR, muscarinic receptor; AR, adrenergic receptor;
MW, calculated molecular weight; GST, glutathione
S-transferase; PI, phosphatidylinositol; PAGE,
polyacrylamide gel electrophoresis; GTPS, guanosine
5'-O-(3-thiotriphosphate).
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REFERENCES |
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