(Received for publication, December 24, 1996, and in revised form, April 23, 1997)
From the ¶ Department of Microbiology and Immunology, Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107 and the Department of Pharmacology,
Medical University of South Carolina,
Charleston, South Carolina 29425
The intracellular domains of G-protein-coupled
receptors provide sites for interaction with key proteins involved in
signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of
G-protein-coupled receptors as probes to screen brain cytosol proteins.
Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR)
(His208-Arg387), M3-MR
(Gly308-Leu497), or
2A/D-adrenergic receptor (AR)
(Lys224-Phe374) were generated in bacteria as
glutathione S-transferase (GST) fusion proteins, bound to
glutathione-Sepharose and used as affinity matrices to detect
interacting proteins in fractionated bovine brain cytosol. Bound
proteins were identified by immunoblotting following SDS-polyacrylamide
gel electrophoresis. Brain arrestins bound to the GST-M3
fusion protein, but not to the control GST peptide or i3 peptides
derived from the
2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified
-arrestin and arrestin-3. The interaction of the M3-MR and
M2-MR i3 peptides with arrestins was further investigated.
The M3-MR i3 peptide bound in vitro translated
[3H]
-arrestin and [3H]arrestin-3, but
did not interact with in vitro translated or purified
visual arrestin. The properties and specificity of the interaction of
in vitro translated [3H]
-arrestin,
[3H]visual arrestin, and
[3H]
-arrestin/visual arrestin chimeras with the
M2-MR i3 peptide were similar to those observed with the
intact purified M2-MR that was phosphorylated and/or
activated by agonist. Subsequent binding site localization studies
indicated that the interaction of
-arrestin with the
M3-MR peptide required both the amino
(Gly308-Leu368) and carboxyl portions
(Lys425-Leu497) of the receptor subdomain. In
contrast, the carboxyl region of the M3-MR i3 peptide was
sufficient for its interaction with arrestin-3.
G-protein-coupled receptors possess a characteristic seven
segments of hydrophobic amino acids that likely serve as membrane spans
to form a core motif important for ligand recognition. The interaction
of agonist with the receptor initiates an ill-defined conformational
adjustment in this core motif, which is propagated to intracellular
domains of the receptor resulting in the activation of G-protein and
the initiation of intracellular signaling events. For most members of
the superfamily of G-protein-coupled receptors, the third intracellular
(i3)1 loop and the carboxyl-terminal tail
of the receptor are key sites for signal initiation and termination,
and these receptor domains also exhibit the greatest variability in
size among different subfamilies of these receptors. The largest i3
domains (100-240 amino acids) are found in receptors coupled to the
Gi, Go, and/or Gq family of
G-proteins (i.e. muscarinic, -adrenergic), whereas shorter i3 loops are found in the photoreceptor rhodopsin or
-adrenergic receptors (20-50 amino acids). During the process of
signal initiation and termination, several proteins interact with the
receptor. The interaction of arrestins with G-protein-coupled receptors is a key component of signal termination (1-5).
The arrestin family consists of visual arrestin, -arrestin,
arrestin-3, and a cone-specific arrestin termed C- or X-arrestin (6-11). In vertebrates, visual arrestin interacts with phosphorylated rhodopsin in rod cells to terminate signal propagation by interfering with receptor coupling to transducin.
-Arrestin and arrestin-3 are
widely expressed and parallel the role of visual arrestin in terms of
signal termination for G-protein-coupled receptors other than
rhodopsin. The affinity of arrestin binding to G-protein-coupled receptors is increased by receptor phosphorylation and/or activation by
agonist. Receptors of this class are phosphorylated to varying degrees
by protein kinase A and C as well as kinases specific for the activated
conformation of the receptor (G-protein-coupled receptor kinases). The
phosphorylation of the receptor by G-protein-coupled receptor kinases
and subsequent arrestin binding are intimately associated with receptor
desensitization and sequestration (12-14). Resensitization of the
receptor protein involves dissociation of bound arrestin and receptor
dephosphorylation.
The interaction of receptors with G-proteins, protein kinases,
arrestins, and additional entities controlling receptor trafficking apparently involves discrete motifs in cytoplasmic domains of the
receptor. The associations of these proteins with the receptor likely
occur within a signal transduction complex that may also include
various effector molecules and other proteins that influence signaling
specificity/efficiency. To define receptor subdomains important for
protein interactions and to begin to identify the components of a
signal transduction complex for G-protein-coupled receptor subtypes, we
generated peptides from the i3 loop of the M2-muscarinic
receptor (MR), M3-MR and 2A/D-adrenergic
receptor (AR) to use as probes to detect interacting proteins in bovine brain cytosol. In the present report, we determined the interaction of
the i3 loop with cytosolic proteins involved in receptor
regulation.
Radiolabeled arrestins were in vitro
translated as described previously (15). Bovine -arrestin, visual
arrestin, and arrestin-3 were also expressed in BL21 cells and purified
to homogeneity by successive chromatography on heparin- and Q-Sepharose
(13). Antibodies to protein kinase C isoforms were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and phosphatase 2A/C antibody was from Calbiochem. Anti-gelsolin monoclonal antibody (GS-2C4) was obtained from Sigma. Monoclonal antibody mAbF4C1, which
recognizes the epitope DGVVLVD present in visual arrestin,
-arrestin, and arrestin-3, was generously provided by Dr. L. Donoso
(Wills Eye Hospital, Philadelphia, PA). Glutathione-Sepharose 4B was
purchased from Pharmacia Biotech Inc. Polyvinylidene difluoride membranes were obtained from Gelman Sciences (Ann Arbor, MI).
Bovine brain
cytosolic proteins in buffer A (10 mM Tris-HCl, pH 7.5, 0.5 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose were precipitated with 40% ammonium sulfate and
pelleted by centrifugation (100,000 × g, 45 min). The
precipitated proteins were resuspended in a minimal volume of 50 mM Tris-HCl, pH 8.0, followed by extensive dialysis (4 liters of buffer A; 4 liters of buffer B (20 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 1 mM EGTA, 2 mM -mercaptoethanol). The supernatant from the 40% ammonium sulfate precipitate was brought to 90% ammonium sulfate to precipitate additional proteins and subsequently processed as described for the
40% ammonium sulfate precipitate. The dialyzed solutions were clarified by centrifugation and applied to an anion-exchange resin (DEAE-Biogel A) equilibrated with buffer B. The column was washed with
buffer B and proteins eluted sequentially with buffer B containing 100, 250, and 500 mM NaCl. Eluted proteins were desalted by
dialysis, concentrated by lyophilization, and stored at
70 °C.
The M3-MR i3 construct was obtained from Dr.
Barry Wolfe (Department of Pharmacology, Georgetown University School
of Medicine, Washington, DC) and encoded the peptide
Gly308-Leu497. The M2-MR and
2A/D-AR i3 constructs were generated from the cDNA
or genomic clones by amplification using the polymerase chain reaction.
The M2-MR subdomain was inserted into the EcoRI
restriction site of the pGEX-2T vector (Promega, Madison, WI). Using
primers containing appropriate restriction sites, the rat
2A/D-AR gene (16) segment encoding the peptide
Lys224-Phe374 was amplified by polymerase
chain reaction and subcloned into the BamHI and
EcoRI restriction sites of pGEX-2T. The M3-II
and M3-III were generated from M3-MR i3
construct (M3-I, Gly308-Leu497) by
taking advantage of a PstI restriction site (nucleotide 1918 of the rat M3 muscarinic receptor coding region). The
M3-II construct was generated by excising the
BamHI/PstI fragment from the M3-I construct with subsequent subcloning of this fragment into pGEX-3X at
the BamHI and EcoRI restriction sites via use of
an adaptor. The M3-III construct was generated using a
similar strategy with the PstI/EcoRI fragment
isolated from the M3-I construct. The M3-IV
construct was prepared by digesting the M3-I construct with HindIII removing the gene segment encoding amino acids
Lys369-Thr424. The purified plasmid containing
the amino- and carboxyl-terminal segments was then religated to yield
M3-IV. The structure of each construct used in the present
study was verified by restriction mapping and nucleotide sequence
analysis. The fusion proteins were expressed in bacteria and purified
using a glutathione affinity matrix according to the manufacturer's
instructions. Immobilized fusion proteins were either used immediately
or stored at 4 °C for no longer than 3 days. Each batch of fusion
protein used in experiments was first analyzed by SDS-PAGE and
Coomassie Blue staining.
Brain cytosol (1-200 µg)
fractions were incubated with ~5 µg of GST fusion protein bound to
the glutathione resin in 250 µl of buffer C (20 mM
Tris-HCl, pH 7.5, 70 mM NaCl) for 2.5 h at 4 °C.
The resin was washed three times with 0.5 ml of buffer C, and the
retained proteins were solubilized and applied to a denaturing 10%
polyacrylamide gel. Polyvinylidene difluoride membrane transfers were
evaluated by immunoblotting as described previously (17) using specific
antibodies. The interactions of purified arrestins with the i3 peptides
was determined in a similar manner. Arrestin binding to the
M2-MR and M3-MR peptides was also evaluated by direct binding assays using tritiated arrestins generated by in vitro translation. Aliquots (~2.5 µg) of GST fusion proteins
bound to a glutathione resin were incubated with
[3H]-arrestin (long splice variant) (1062-1600
dpm/fmol), [3H]arrestin-3 (short splice variant) (1500 dpm/fmol), [3H]visual arrestin (1300-1871 dpm/fmol),
or the [3H]
-arrestin/visual arrestin chimeras BBBA
(1990 dpm/fmol) and AABB (2010 dpm/fmol) in a total volume of 20 µl
of buffer C for 2.5 h at 4 °C with shaking. The resin was
washed three times with 100 µl of buffer C. The washed resin was
resuspended in 300 µl of buffer C, mixed with 10 ml of Ecoscint A
scintillation fluid, and the retained radioactivity quantitated by
scintillation spectrometry at ~50% efficiency. Nonspecific binding
was defined as the amount of ligand retained in parallel experiments
using a control GST resin and represented ~30% of total binding at a
concentration of 0.75 nM arrestin.
The third intracellular loop of the M2-MR,
M3-MR ,and the 2A/D-AR consists of 180 (His208-Arg387), 238 (Arg253-Gln490), and 157 (Arg218-Phe374) amino acids, respectively. The
putative first and second intracellular loops of the three receptors
are similar in size ~11-12 amino acids, 1st loop; ~18-19 amino
acids, 2nd loop) and the carboxyl-terminal tails of the
M2-MR, M3-MR, and
2A/D-AR are
23, 44, and 20 amino acids in length, respectively. As an initial
attempt to define proteins that may interact with the intracellular
domains of these G-protein-coupled receptors, we focused on the i3 loop
as it is the largest intracellular domain in this receptor group. The
juxtamembrane segments of the i3 domain are of critical importance for
receptor coupling to G-protein, whereas other segments participate in
receptor phosphorylation, receptor trafficking, and other aspects of
signal propagation.
The M2-MR (His208-Arg387),
M3-MR (Gly308-Leu497), and
2A/D-AR (Lys224-Phe374) i3
peptides were expressed in bacteria as a GST fusion protein and used to
generate an affinity matrix by saturating a glutathione-Sepharose resin
with the fusion protein (Fig. 1). The M2-MR
peptide corresponded to the entire i3 loop of the receptor. The
M3-MR i3 peptide began 45 amino acids downstream of the
amino terminus of the i3 loop and terminated seven amino acids into the
VI membrane span. The
2A/D-AR peptide began six amino
acids downstream of the amino terminus of the i3 loop and terminated at
the beginning of the VI membrane span. To determine the interaction of
these receptor-derived peptides with cytosolic proteins, we first
fractionated bovine brain cytosol to enrich for potential interacting
proteins.
In the first series of experiments, we determined the interaction of
receptor subdomains with brain arrestins. Bovine brain cytosol was
fractionated by ammonium sulfate precipitation and ion exchange
chromatography and the fractions enriched for arrestins were determined
by immunoblotting (Fig. 2A, left
panel). The cytosol fraction enriched for arrestin was incubated
with the M2-MR, M3-MR or the
2A/D-AR i3 subdomains as well as the control GST
affinity matrix and arrestins retained by the matrices were determined by immunoblotting. Brain arrestins were adsorbed by the
M3-MR affinity matrix but did not interact with matrices
constructed of the GST control peptide or the i3 peptides derived from
the M2-MR or the
2A/D-AR (Fig.
2A, right panel). The amount of arrestin retained
by the M3-MR matrix was directly related to the amount of
cytosol present during incubation (Fig. 2B). Brain arrestins were not retained by GST fusion proteins containing subdomains of the
tyrosine phosphatase Syp (Src homology 2 domains, 215 amino acids), the
Na+/H+ exchanger (carboxyl terminus, 178 amino
acids), a subdomain of the M2-MR i3 loop (56 amino acids),
or the transregulatory protein c-Jun (amino terminus, 79 amino acids)
(Fig. 2C). These data indicated that the interaction of
brain arrestins with the M3-MR affinity matrix was specific
for the M3-MR peptide.
The specificity of arrestin binding to the M3-MR peptide
was further investigated by determining the interaction of the peptide with other cytosolic proteins. The distribution of protein kinase C
isoforms, phosphatase 2A, and the actin-binding protein gelsolin in the
fractionated bovine brain cytosol was determined by immunoblotting (Fig. 3, left panel). Two protein kinase C
isoforms fractionated in the 250 mM NaCl elution of the
90% ammonium sulfate precipitate. The phosphatase 2A immunoreactive
species was identified in the 250 mM NaCl elution of the
40% ammonium sulfate precipitate. Gelsolin was enriched in the 100 mM NaCl elution of the 40% ammonium sulfate precipitate.
The appropriate fraction was then incubated with the M3-MR
or the 2A/D-AR i3 affinity matrix and processed as described for arrestins. Although the M3-MR affinity matrix
adsorbed brain arrestins (Fig. 2), neither the M3-MR or the
2A/D-AR i3 peptides interacted with the protein kinase C
isoforms, phosphatase 2A, or gelsolin (Fig. 3, right
panel).
Interaction of the i3 Peptides with Arrestins
The interaction
of arrestins with the i3 peptides was investigated in more detail to
define issues of arrestin selectivity and sites of arrestin
association. In the first series of experiments, we evaluated arrestin
binding to the M3-MR using radiolabeled arrestins.
Increasing concentrations of radiolabeled -arrestin, arrestin-3, and
visual arrestin were incubated with the M3-MR or the
control GST resin. Both [3H]
-arrestin and
[3H]arrestin-3 exhibited specific binding to the
M3-MR peptide relative to the binding of the arrestins to
the control GST peptide (Fig. 4A). In
contrast, the binding of [3H]visual arrestin to the
M3-MR matrix was only slightly increased above that
observed with the GST peptide itself, consistent with the lower
affinity of visual arrestin at G-protein-coupled receptors other than
rhodopsin. Interestingly, the affinities exhibited by
[3H]
-arrestin and [3H]arrestin-3
(0.5-1.0 nM) are in the range of the Kd for arrestin binding to purified
2-AR and
M2-MR (15). The selectivity of the binding of
[3H]arrestins to the M3-MR peptide was also
observed with purified
-arrestin and visual arrestin (Fig.
4B). In the second series of experiments, the interaction of
arrestins with the i3 peptides was further evaluated using purified
-arrestin and arrestin-3. Purified
-arrestin and arrestin-3 were
adsorbed to the M3-MR matrix in a
concentration-dependent manner (Fig.
5A). Neither arrestin was retained by the
control GST resin (Fig. 5A). As indicated above, brain
arrestins were not retained by the M2-MR or
2A/D-AR affinity matrices. However, both the
M2-MR and the
2A/D-AR i3 peptides bound
purified
-arrestin and arrestin-3 (Fig.
5B).2
The interaction of arrestins with the intact purified M2-MR
was previously characterized using in vitro translated
arrestins and -arrestin/visual arrestin chimeras (15). We thus
compared the interaction of arrestins with the M2-MR i3
peptide and the intact receptor relative to the influence of ionic
strength and the selectivity of binding for the different arrestins. As
observed for the intact purified receptor that was phosphorylated
and/or activated by agonist (15), [3H]
-arrestin
binding to the i3 peptide was first increased and then decreased with
increasing ionic strength of the incubation buffer (Fig.
6A). Previous studies using a series of
[3H]
-arrestin/visual arrestin chimeras indicated that
the selectivity of
-arrestin and visual arrestin binding to the
intact purified M2-MR and the
2-adrenergic
receptor involved specific domains of the two arrestins (15). To
determine if this selectivity of arrestin binding was maintained with
the M2-MR i3 peptide, we evaluated the binding of two
[3H]
-arrestin/visual arrestin chimeras. The BBBA
chimera consists of amino acids 1-340 of
-arrestin and amino acids
346-404 of visual arrestin. The AABB chimera consists of amino acids
1-213 of visual arrestin and amino acids 208-418 of
-arrestin. The binding of BBBA to the intact M2-MR was similar or greater
than that observed with
-arrestin, whereas the binding of AABB was intermediate between
-arrestin and visual arrestin (15). The relative binding of [3H]
-arrestin,
[3H]visual arrestin, and the
[3H]
-arrestin/visual arrestin chimeras to the
M2-MR i3 peptide, as well as the M3-MR peptide,
was similar to that observed with the intact purified M2-MR
(Fig. 6B).
Binding of Brain Arrestins,
The next series of
experiments were designed to localize subdomains of the
M3-MR i3 loop required for interaction with arrestins. The
M3-MR i3 peptide (M3-I) was divided into three
segments: an amino-terminal region (M3-II,
Gly308-Gln389), the carboxyl-terminal region
(M3-III, Val390-Leu497), and a
middle portion (Lys369-Thr424) (Fig.
7A). The M3 peptide possesses a
concentrated negative charge in the amino-terminal third and a
concentrated positive charge in the carboxyl-terminal third of the
peptide. The contribution of the middle portion was determined using
construct M3-IV in which this segment was deleted and the
peptide Gly308-Leu368 was fused to the peptide
Lys425-Leu497 (Fig. 7A). Neither
construct II or III interacted with brain arrestins or purified
-arrestin under these incubation conditions (Fig.
7B).3 However, the construct
containing both the amino and carboxyl regions of the M3-I
peptide (M3-IV) retained the ability to interact with brain
arrestins and purified
-arrestin (Fig. 7B). These data
suggest that there are at least two sites on the M3-I
peptide for
-arrestin binding and that one of these sites by itself
is insufficient. In contrast to the results with
-arrestin, the carboxyl region of the M3-MR peptide was sufficient for
interaction with arrestin-3 (Fig. 7B). Arrestin-3 bound to
the M3-I, -III, and -IV, but not to construct II,
suggesting that the binding motif for arrestin-3 is different from that
of
-arrestin.
As is apparently the case with most complex signal processing
systems, G-protein-coupled receptors likely operate within a signal
transduction complex that is either preexisting or is generated by the
biological stimuli. The components of such a signaling complex are
unclear but might include proteins that influence events at the
receptor-G-protein or G-protein-effector interface or contribute to the
formation of the signal transduction complex itself (Refs. 17 and 18,
and references therein). As part of an effort to identify components of
this signal transduction complex, we initiated two experimental
approaches. The first approach was based on a biological activity and
was designed to detect factors that might influence the transfer of
signal from receptor to G-protein (17, 18). The second approach,
described in the present report, involved the identification of
proteins capable of associating with receptors of this class. In the
latter approach, we used intracellular domains of G-protein-coupled
receptors as "bait" to search for interacting proteins in brain
cytosol. Receptor subdomains were generated from the i3 loop of the
M2-MR, M3-MR, and 2-AR. Both the
muscarinic receptor subtypes and the
2-AR are capable of
coupling to multiple G-proteins and effectors in a cell type-specific
manner. Activation of the M2-MR results in inhibition of
adenylyl cyclase, whereas the M3-MR couples to the inositol
phosphate/protein kinase C signaling pathway. The
2A/D-AR is generally associated with inhibition of
adenylyl cyclase, but activation of this receptor also influences
signal transduction pathways involving phospholipases, p21ras,
the mitogen-activated protein kinase signaling pathway, and various ion
channels (see Ref. 17, and references therein).
We first evaluated the interaction of the i3 loops with the arrestin
family of proteins, which are implicated in receptor uncoupling and
internalization. The arrestins exhibited a specific interaction with
the i3 receptor subdomains. The receptor subdomain probes did not
interact with selected protein kinase C isoforms, cytosolic phosphatase
2A, or the actin-binding protein gelsolin. However, both -arrestin
and arrestin-3 bound to the i3 loops of the muscarinic receptor
subtypes and the
2A/D-AR. The major observations
concerning this interaction are as follows. First, the interaction of
-arrestin and arrestin-3 with the M3-MR involved different regions of the i3 loop. Second, the i3 domains from the
muscarinic receptors and the
2A/D-AR differed in their
ability to interact with endogenous bovine brain arrestins in a crude cytosol fraction versus purified recombinant
-arrestin
and arrestin-3. Third, the interaction of arrestins with the i3 loop
peptides occurred in the absence of peptide phosphorylation.
Both -arrestin and arrestin-3 are widely expressed, but exhibit a
heterogeneous intra-tissue distribution, and both arrestins are
alternatively spliced to generate a short and long form of the protein.
Although arrestins clearly interact with the receptor protein (15,
19-22), the sites of interaction and the selectivity among the
different arrestins and receptor families outside the visual system are
undefined. However, the present study indicates that there are receptor
motifs that are indeed capable of distinguishing the two types of
arrestin. Within the M3-MR i3 loop, amino acids Gly308-Leu368 and
Lys425-Leu497 are required for binding of
-arrestin and the partially purified brain arrestin, whereas amino
acids Gly308-Leu368 are not required for
binding of arrestin-3. The differential interaction of the two
arrestins with the M3-MR subdomain may relate to the charge
distribution within the receptor peptide segments and the structural
properties of the two arrestins.
Although the i3 peptides from the M2-MR, M3-MR
and 2A/D-AR all bound purified arrestins, only the
M3-MR was capable of interacting with brain cytosol
arrestins. The inability of brain arrestins to interact with the
M2-MR and
2-AR may simply reflect
differences in the relative affinities of the different receptor
subdomains for the arrestins. However, there were no apparent
differences in the efficiency of the interaction of the three receptor
subdomains with the purified arrestins under these experimental
conditions, suggesting that other factors may be regulating this
interaction. It is possible that the brain arrestins are slightly
different from the recombinant arrestins, and that this difference
contributes to selective interactions with receptor families.
Alternatively, perhaps there are additional proteins in the
fractionated brain cytosol that impede arrestin binding to the
M2-MR and
2A/D-AR, but not the
M3-MR i3 peptides. Such proteins may interact with motifs
in the i3 peptide or perhaps with arrestin itself.
Interaction of visual arrestin with rhodopsin or -arrestin and
arrestin-3 with the
2-AR or the M2-MR
involves multiple contact sites that impart apparent positive
cooperativity to the reaction (15, 19-21). Arrestin binding to the
receptor is proposed to first involve an ionic interaction that senses
the phosphorylation and activation state of the receptor. If the
receptor is phosphorylated or agonist-occupied, the apparent affinity
of arrestin binding to the receptor is increased. The phosphorylated
receptor subdomain likely forms a component of the arrestin binding
site, although phosphorylation-dependent conformational
shifts in the intracellular regions of the receptor may also reveal
sites that participate in arrestin binding. Whereas the binding of
arrestin to rhodopsin is highly dependent upon receptor phosphorylation
and activation, a truncated splice variant of visual arrestin binds to
nonphosphorylated rhodopsin. The truncated and full-length visual
arrestins differ in their subcellular distribution, with the former
constitutively localized to disc membranes, while the latter associates
with the disc membranes in a light-dependent manner (23).
-Arrestin and arrestin-3 also bind to nonphosphorylated
M2-MR or
2-AR and high affinity binding is
less dependent upon the activation state of the receptor than is the
interaction between visual arrestin and rhodopsin (15, 23). The
possible interaction of arrestins with nonphosphorylated receptors is
also suggested by the ability of phosphorylation-defective mutants to
undergo receptor desensitization and sequestration. An important role
for arrestin interaction with a nonphosphorylated receptor is also
apparent for visual signaling in invertebrates (24). In the dipteran
Calliphora, arrestin interaction with the receptor protein
clearly precedes receptor phosphorylation, and indeed the
arrestin-receptor complex is the preferred kinase substrate (24). As
the interaction of arrestins with the i3 peptides in the present study
occurred without peptide phosphorylation, the i3 loop or perhaps other
domains of G-protein-coupled receptors appear capable of serving as a docking site for arrestin independent of receptor phosphorylation. Indeed, the properties of arrestin binding to the M2-MR i3
peptide appeared similar to those exhibited by the purified receptor
protein that was phosphorylated and/or activated by agonist. The
binding of
-arrestin and arrestin-3 to the i3 peptides may also
relate to the agonist-induced conformational changes responsible for initiating the signaling cascade. In the absence of agonist, the regions of the receptor involved in G-protein activation are stabilized in a conformation that acts as a "brake" on signal initiation (25).
Such a conformational "brake" may be released by discrete mutations
in the i3 loop of some receptors, such that the receptor becomes
constitutively active (26). An analogous situation may occur when the
i3 loop is separated from the conformational restrictions imposed by
membrane spans of the receptor and assumes an "activated and/or
accessible conformation" that is recognized by arrestin.
The demonstration that the experimental approach using receptor subdomains as probes for receptor-associated proteins resulted in the detection of protein-protein interactions of clear biological relevance underscores the potential utility of the system to identify additional interacting proteins that may contribute to the formation of a signal transduction complex. Such interacting proteins may play an important role in directing the receptor-initiated signal to a specific effector pathway. In contrast to signaling events in the visual system where the components are localized and the interactions between the individual molecules are relatively specific, other G-protein-coupled receptors operate in diverse cell types and couple to multiple G-proteins and effectors. Perhaps, such receptors have evolved larger i3 loops to maintain the fidelity of the signaling system by providing sites for interaction with additional accessory proteins that influence receptor trafficking and/or signaling specificity and efficiency.
We thank Dr. Barry Wolfe, Dr. Tatsuya Haga
(Department of Biochemistry, Institute for Brain Research, Faculty of
Medicine, University of Tokyo, Japan), Dr. Larry Fliegel (Department of Biochemistry and Pediatrics, University of Alberta, Alberta,
Canada), Dr. Gen-Shen Feng (Department of Biochemistry and
Molecular Biology, Indiana University School of Medicine), and Dr.
Steven Rosenzweig (Department of Pharmacology, Medical University
of South Carolina, Charleston, SC) for providing the M3-MR,
M2-MR (56-amino acid segment for the third intracellular
loop), Na+/H+ exchanger, Syp, and c-Jun fusion
protein constructs, respectively. We also thank Dr. Larry Donoso for
mAbF4C1 and Dr. Vsevolod Gurevich (Sun Health Research Institute, Sun
City, AZ) for purified arrestins. The expression vector containing the
peptide derived from the third intracellular loop of the
2A/D-adrenergic receptor was generated by Dr. Sally
Kadkhodayan in the laboratory of Dr. Lanier.