A ?-Arrestin Binding Determinant Common to the Second
Intracellular Loops of Rhodopsin Family G Protein-coupled
Se ´bastien Marion, Robert H. Oakley1, Kyeong-Man Kim2, Marc G. Caron3, and Larry S. Barak4
FromtheDepartmentofCellBiology,DukeUniversityMedicalCenter,Durham,North Carolina 27710
?-Arrestins have been shown to inhibit competitively G protein-
dependent signaling and to mediate endocytosis for many of the
hundreds of nonvisual rhodopsin family G protein-coupled recep-
tors (GPCR). An open question of fundamental importance con-
cerning the regulation of signal transduction of several hundred
rhodopsin-like GPCRs is how these receptors of limited sequence
homology, when considered in toto, can all recruit and activate the
two highly conserved ?-arrestin proteins as part of their signaling/
zation and internalization, the agonist-activated conformation of a
ing the GPCR and arrestin interaction. Here we identified a ?-ar-
restin binding determinant common to the rhodopsin family
GPCRs formed from the proximal 10 residues of the second intra-
cellular loop. We demonstrated by both gain and loss of function
studies for the serotonin 2C, ?2-adrenergic, ?2a-adrenergic, and
neuropeptide Y type 2 receptors that the highly conserved amino
acids, proline and alanine, naturally occurring in rhodopsin family
receptors six residues distal to the highly conserved second loop
DRY motif regulate ?-arrestin binding and ?-arrestin-mediated
internalization. In particular, as demonstrated for the ?2AR, this
occurs independently of changes in GPCR kinase phosphorylation.
These results suggest that a GPCR conformation directed by the
second intracellular loop, likely using the loop itself as a binding
inactive form to its active receptor-binding state.
transducin is competitively blocked by the binding of visual arrestin to
to terminate G protein-mediated signaling for rhodopsin family
GPCRs,5except visual arrestin is replaced by ?-arrestins. Variations in
mation of the receptor, and the ability of G protein-coupled receptor
kinases (GRK) to phosphorylate serine and threonine residues on the
C-tail and third intracellular loop of a receptor (1–5).
Receptor agonist-induced phosphorylation has long been demon-
strated to be of great importance for ?-arrestin binding, being initially
described for visual arrestin binding of the phosphorylated MII state of
light-activated rhodopsin (6–8). More recently, the formation of stable
?-arrestin complexes with agonist-activated GPCRs has been shown to
require phosphorylation of serine and threonine clusters located in the
receptor determinants that are exposed only in the active receptor con-
formation (9, 10). Supporting this alternative are observations that ago-
nist-activated GPCRs bind ?-arrestins even in the absence of GRK
phosphorylation (2). This phosphorylation-independent binding sug-
gests that determinants, resulting from conserved primary amino acid
sequences or protein secondary structural motifs, exist in all GPCRs to
regulate receptor/arrestin association. However, the receptor regions
that would comprise these arrestin-binding motifs have not been thor-
oughly defined, perhaps as a result of the sequence variability occurring
data other than for rhodopsin.
GPCRs are structurally similar in their seven transmembrane archi-
tecture and share behaviors that originate from commonly occurring
at the cytoplasmic/intracellular loop junction of transmembrane III.
The DRY motif presumably mediates interactions with both G proteins
inactive conformation in the absence of ligand (11–15). Scattered resi-
dues on the first two rhodopsin intracellular loops have been identified
as contributing to visual arrestin binding exclusive of the phosphoryl-
ated rhodopsin C-tail (4, 16, 17). In particular, a proline residue in the
rhodopsin second intracellular loop distal to the ERY motif is involved
(4, 17). In addition, computational modeling of molecular docking
residues of the second loop may directly engage transducin (18).
like GPCRs, significant differences remain. For example, regulatory
behavior in nonvisual cell systems that does not normally apply to rho-
to which a rhodopsin paradigm applies to nonvisual GPCRs is unclear
In this study we used several GPCRs to investigate the ability of nat-
* This work was supported in part by National Institutes of Health Grants NS19567 (to
M. G. C.)andHL61635(toL. S. B.).Thecostsofpublicationofthisarticleweredefrayed
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Present address: Xsira Pharmaceutical, Morrisville, NC 27560.
2Supported by Korea Ministry of Health and Welfare, KNIH Brain Research Center Pro-
gram Grant 0405-NS01-0704-0001. Present address: Dept. of Pharmacology, College
of Pharmacy, Chonnam National University, Kwang-Ju, 500-757 Korea.
3To whom correspondence may be addressed. E-mail: email@example.com.
4To whom correspondence may be addressed. Tel.: 919-684-5433: Fax: 919-681-8641;
5The abbreviations used are: GPCR, G protein-coupled receptor; 5HT, 5-hy-
green fluorescent protein; GRK, G protein-coupled receptor kinase; NPY, neuropep-
tide Y; GTP?S, guanosine 5?-3-O-(thio)triphosphate; ELISA, enzyme-linked immu-
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 5, pp. 2932–2938, February 3, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
2932 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 5•FEBRUARY 3, 2006
acids downstream of the DRY motif, to modulate ?-arrestin/receptor
interactions. ?-Arrestin translocation and receptor endocytosis were
adrenergic, and neuropeptide Y2 receptors. Our data combined with
sequence analysis of over 175 human rhodopsin family GPCRs suggest
that in these receptors a contiguous 10-amino acid region beginning
with the DRY motif forms a phosphorylation-independent structural
determinant for binding ?-arrestin.
Materials—[3H]Adenine for measurement of cAMP generation and
125I-cyanopindolol for receptor binding were purchased from
PerkinElmer Life Sciences, and [3H]CGP-12177 was from Amersham
were from Sigma. Norepinephrine was from Bioanalytical Systems
(West Lafayette, IN). The anti-phospho-?2AR (Ser-355/Ser-356) was
from Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids—FLAG- and GFP-tagged 5-HT2c nonedited receptors
were described previously (21). NPY2R receptor was cloned by total
to receptors with a hemagglutinin tag at the N terminus. Receptor
cDNA containing mutations for the ?2AR-P138A, 5HT2cR-P159A,
?2aAR-A138P, and NPY2R-H159P were generated by standard PCR
methods using a proofreading polymerase (Pfu; Stratagene). ?-Arres-
tin2-GFP was made as described (23).
Antagonist Binding, Agonist Binding, cAMP Assays, and GTP?S
Binding—These procedures in which receptor expression levels were
viously (5, 22).
Receptor Phosphorylation and Sequestration Assay—Receptor phos-
described previously (24). Measurement of receptor surface expression
by ELISA was performed under nonpermeabilized conditions (25).
Microscopy and ?-Arrestin Translocation—Confocal microscopy of
HEK-293 cells containing either ?-arrestin2-GFP and one of the ?2AR,
?2AR, or NPY2R receptor variants or the 5HT2cR-GFP wild type or
mutant was performed as described (23) using a Zeiss LSM-510.
Occurrence of Conserved Residues in the Second Intracellular Loop of
Class I GPCRs—Examination of the human GRCR data base (www.g-
pcr.org) demonstrated 360 class I rhodopsin-like entries of which 244
contained glutamic/aspartic residues followed by arginine residues
of predominantly deorphanized class I GPCRs and were evaluated for
the frequency of residue occurrence at positions 1–23 of the second
Fig. 1A shows these relative frequencies determined for receptors
where the lead second loop residue is an aspartic acid. (Note, where the
lead residue was glutamic acid, a similar frequency result was obtained
in this loop region, particularly at positions 2 and 8.
Fig. 1B presents a plot of the next most common group, the hydro-
phobic residues Iso/Leu/Val/Phe in red plotted among the basic group
(blue). In Fig. 1B, the red and blue horizontal dashed lines show the
frequency at which a member of the hydrophobic or basic group,
hydrophobic group members Iso/Leu/Val/Phe occur at position 6 98%
of the time, a level comparable with the 99% occurrence of arginine at
position 2. It is also apparent from Fig. 1A that at position 9 a proline
(Pro-9) or alanine (Ala-9) occurs 90% of the time.
Fig. 1C summarizes the position frequency analysis in terms of a
composite of the most probable receptor sequence. Sub-sequences
formed from the first 12 and 11 positions of the 23 positions match the
mouse olfactory receptor Olfr843 and human prokineticin receptor 1
exactly, and the most probable match for the entire sequence occurs
with the second loop of the human galanin receptor. In the composite
second loop, the first 10 residues are highly conserved with positions
1–3 the DRY motif, positions 4–7 and 10 hydrophobic amino acids,
ency toward basic residues.
Mutagenesis in the Second Loop—Mutation of Arg-2 in the DRY
motif has been shown to enhance ?-arrestin binding, resulting in con-
Percent Frequency of Occurrence
D R Y L A I V H P L R Y R R L R T P R R A
1412 208 16
10 12 14 16 18 20 22 24
DRY - Hydrophobic Residues
DRY - Basic Residues
FIGURE 1. Homology of the N-terminal portion of the second intracellular loop
among various rhodopsin-like GPCRs. A, frequency of occurrence by position for 136
the DRY motif in 136 GPCRs. C, composite sequence representing the most probable
substitutions and second most probable ones (above) generated from analysis of the
second loop composition of 175 GPCRs.
FEBRUARY 3, 2006•VOLUME 281•NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2933
stitutively activated/desensitized receptors (24, 27). However, the func-
tional dependence on ?-arrestin binding from point substitutions
beyond the DRY motif in residues 4–10 has received scant attention,
to decrease visual arrestin binding (4, 17). Because the crystal structure
modeled through the ExPASy Proteomics Server software DeepView
(28) if Ala-9 substitution could preserve a Pro-9 like conformation for
(Protein Data Bank access code 1U19) (29), which has a completely
resolved polypeptide chain and is also in agreement with the model of
the rhodopsin oligomer (30–32).
changes in the second loop resulting from an alanine for proline substi-
tution (Fig. 2A), the differences in the planar (?) and rotational (? and
?) bond angles (in degrees) about the C-? carbon were calculated (Fig.
practically identical to the native one, perturbations in the bond angles
quickly dampen within 1–2 residues of the proline (Fig. 2B). Moreover,
Pro-9 (Fig. 2C) occupies the center of an accessible region at the lower
intracellular face of rhodopsin that has been demonstrated as a docking
determinant for the G protein transducin (18).
tution in the Second Intracellular Loop of a Constitutively Active Sero-
tonin Receptor—Serotonin 5HT2c receptors naturally exist in several
distinct protein isoforms secondary to RNA editing (33, 34). Editing
with different degrees of constitutive activity (34, 35). RNA editing
changes amino acids 6, 8, and 10 (Ile, Asn, and Ile) that surround the
second loop Pro-9 and impairs the ability of 5-HT2cR isoforms to con-
fore investigated whether Pro-9 in the 5HT2cR played an active role in
this ?-arrestin regulation by assessing the functional consequences of a
Pro-9 to Ala-9 substitution. Additionally, introducing an Ala-9 was
residue to occur at this position in GPCRs (Fig. 1A).
We first investigated whether substitution of Pro-9 in the most con-
stitutively active 5HT2cR isoform (i.e. nonedited INI isoform) could
intracellular vesicular pattern of the constitutively internalized GFP-
much more pronounced plasma membrane presence. To quantify the
extent to which the proline/alanine substitution modifies 5HT2cR dis-
tribution, we measured by ELISA the cell surface expression of the
receptors in the presence of the inverse agonist SB206553 (Fig. 3B),
which has been proven to disrupt ?-arrestin/5HT2cR constitutive
interaction (21). 15 min of SB206553 treatment resulted in an absolute
26 ? 3% increase of Pro-9 5HT2cR expression at the cell surface. No
significant variation of the Ala-9 5HT2cR was detected. After 30 min of
inverse agonist treatment, 30 ? 3 and 8 ? 3% increases in cell surface
expression were obtained for the Pro-9 and Ala-9 5HT2cRs, respec-
tively, suggesting that in contrast to the 5HT2cR-INI a majority of the
Ala-9 5HT2cRs already resides at the plasma membrane in the absence
of agonist and therefore interacts much less well with ?-arrestins.
position 9. C, putative arrestin-interacting face formed from residues 3, 4, and 7–10 and
centered on proline 9 of the second intracellular loop.
stitutively active 5HT2cR. A, representative confocal images of HEK-293 cells tran-
siently transfected with 4 ?g of either 5HT2cR-GFP (top panel) or 5HT2cR-P159A-GFP
sion of FLAG-5HT2cR (f) or FLAG-5HT2c-P159A (?) was measured by ELISA before and
sent the mean of three independent experiments done in triplicate. C and E, co-immu-
?-arrestin2-GFP or G?qthat was co-immunoprecipitated in three independent experi-
ments each normalized by the amount of receptor expressed is presented. Bar, 10 ?m.
2934 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 281•NUMBER 5•FEBRUARY 3, 2006
tin2 (25). Therefore, by using a co-immunoprecipitation strategy, we
tested whether the proline substitution affects this constitutive associ-
ation (Fig. 3C). Under basal conditions an alanine substitution of Pro-9
led to a 4-fold decrease in the amount of ?-arrestin2-GFP co-immuno-
precipitated by the 5HT2cR (Fig. 3, D and C).
It is well established that 5HT2c receptors interact with Gqto stim-
ulate phospholipase C (36, 37) and that receptor RNA editing silences
constitutive activity by affecting G protein coupling efficiency (37, 38).
Consequently, we investigated by co-immunoprecipitation of the
receptor with Gq(Fig. 3E) whether Pro-9 substitution would also affect
Gqcoupling to the constitutively active 5HT2cR. Under basal condi-
tions and compared with the wild type, the Ala-9 5HT2cR produced a
4-fold decrease of co-immunoprecipitated Gq(Fig. 3F). Altogether
Pro-9 as well as naturally occurring editing processes in positions 6, 8,
and 10 regulate receptor coupling to the cognate G protein as well as
Substitution of Proline 9 in ?2-Adrenergic Receptor—We next evalu-
stitutively active receptor. For this purpose we used the well character-
ized ?2AR. We assessed ligand binding characteristics and G protein
coupling for both wild type and mutant receptors (Table 1). The data
presented in Table 1 are consistent with a relatively mild loss of G
substitution of Pro-9 affects coupling between the receptor and its cog-
was measured by receptor-stimulated binding of nonhydrolyzable GTP
analogues (Fig. 4A). A 1 log decrease in affinity for the Ala-9 ?2AR was
observed (Table 1 and Fig. 4A).
Taken together, the agonist and GTP?S binding data would suggest
that second messenger signaling should decrease at least mildly in the
Ala-9 ?2AR. Most surprisingly, an opposite trend in signaling was
regulates GPCR desensitization through facilitation of GRK/?-arrestin
signaling/G protein coupling (coupling measured as GTP?S binding)
for the Ala-9 ?2AR relative to the Pro-9 ?2AR (ratios of 1.00 wild type
to desensitize. Therefore, we tested the extent of agonist-mediated
?-arrestin2-GFP translocation to these receptors.
plasma membrane (Fig. 4C, top panels). Although ?-arrestin2-GFP
recruitment to the Ala-9 ?2AR approaches a qualitatively similar distri-
bution to that of wild type receptor after 15 min with saturating con-
centrations of agonist (data not shown), at early time points (1–4 min)
the wild type receptor demonstrates greater ?-arrestin association (Fig.
4, C and D). This significant delay in redistribution of ?-arrestin to the
mutant ?2ARs. ?2AR (f) and ?2AR-AF (E) containing membranes prepared from HEK-293
?2AR and Pro-9 mutant ?2ARs. HEK-293 cells were transfected with 2.5 ?g of cDNA for the
tates of membranes from HEK-293 cells transfected with hemagglutinin-tagged receptor
terenol (iso) for 15 min at 37°C and evaluated for receptor internalization using [3H]CGP-
GraphPad Prism. Means were shown to be significantly different at p ? 0.05. Results are
representative of four independent experiments with data presented as mean ? S.E. F,
HEK-293 cells transiently transfected with 1.5 ?g of either ?2AR or ?2AR-AF were
exposed or not to 10 ?M of isoproterenol for 15 min at 37 °C. Cells were then homoge-
nized and receptors were immunoprecipitated in the presence of phosphatase inhibi-
tors. Representative immunoblots depict the total amount of receptor immunoprecipi-
tated (bottom blot) and the amount of receptor phosphorylated on serines 355 and 356
Binding characteristics of the Ala-9 and Pro-9 ?2ARs
61 ? 13
26 ? 3
3.4 ? 1.5
34 ? 10
Maximum amount of cAMP
R in high affinity state
39 ? 2
16 ? 1
EC50for cAMP production
Maximum amount of GTP?S binding GTP?S EC50
7 ? 3
81 ? 19
1.00 ? 0.05
0.83 ? 0.04
1.00 ? 0.05
1.34 ? 0.07
4.9 ? 1
4.4 ? 2
FEBRUARY 3, 2006•VOLUME 281•NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2935
the observed enhancement in signaling.
Confidence for a putative arrestin-binding role of the ?2AR second
intracellular loop depends to a large degree on a lack of change in the
ity to undergo downstream ?-arrestin-directed behaviors. Fig. 4E
depicts an autoradiograph of the basal and agonist-mediated phospho-
rylation of purified ?2AR wild type and mutant receptors. Of the two
major kinase families that regulate ?2AR phosphorylation in HEK cells,
ager analysis showed no significant differences in the agonist-mediated
enhancement of phosphorylation among the wild type and mutant for
the same amount of receptor. In addition we assessed that agonist-
induced GRK-dependent phosphorylation of serines 355 and 356 in
Pro-9 ?2AR and in the Ala-9 mutant ?2AR was preserved (Fig. 4F).
These two serines account for 48% of the GRK-dependent phosphoryl-
ation (49). The phosphorylation signal was identical between wild type
and mutant receptors when normalized for total immunoprecipitated
receptor (data not shown). Together these results suggest that GRK-
mediated phosphorylation remains intact in the Ala-9 ?2AR.
The magnitude of GPCR internalization also provides a quantitative
reflection of receptor/?-arrestin association and was assessed in HEK-
293 cells (Fig. 4E). Consistent with the previous ?-arrestin2-GFP trans-
location results, a 22 ? 3% decrease in sequestration of the Ala-9 ?2AR
was observed in comparison to Pro-9 ?2AR. Moreover, the additional
which may be part of a common G protein binding determinant among
decreases the endocytosis as well as substantially reducing ?-arrestin
translocation (data not shown).
Effects of Proline 9 Insertion in the Second Intracellular Loop of ?2a-
Adrenergic and NPY2 Receptors—With agonist exposure the ?2aAR has
a much weaker endocytic response than either the ?2bAR or ?2cAR (41,
42). Similarly, the NPY2R internalizes poorly compared with the NPY1
and -4 receptors (43, 44). Both the ?2aAR and NPY2R lack proline at
tigated these two receptors to determine whether substitution of an
alanine by a proline at position 9 would result in greater agonist-medi-
ated recruitment of ?-arrestin2 and a more robust internalization of
these receptors. Fig. 5A shows the cellular distribution of ?-arrestin2-
significantly greater amount of ?-arrestin2 to the plasma membrane.
Following 15 and 30 min of norepinephrine stimulation, quantification
nalizes 3.6-fold more than the wild type Ala-9 ?2aAR (Fig. 5B).
We also assessed after 15 min of NPY stimulation the ?-arrestin2-
GFP distribution in cells expressing equivalent amounts of either the
wild type His-9 or Pro-9 NPY2R (Fig. 5C). His-9 NPY2R by contrast to
Pro-9 NPY2R only weakly recruits ?-arrestin2 from the cytosol. Quan-
tification by ELISA measurement of surface receptor expression (Fig.
5D) verifies a greater magnitude in endocytosis for the Pro-9 NPY2R
ization is measurable, but by contrast 17 ? 3% of the Pro-9 NPY2R
NPY2R internalize, a 3.3-fold augmentation of endocytosis.
Taken together, the loss and gain of function data for the four recep-
tors indicate that a proline at second intracellular loop position 9 favors
ligand-activated receptor recruitment of ?-arrestin. Moreover, this
property, as illustrated by the ?2AR, occurs without changes in the
extent of GRK phosphorylation of the receptor.
An important unrealized goal for understanding GPCR signal trans-
for ligand-mediated interaction between the ?-arrestin and each of the
hundreds of GPCRs outside the visual system. A similar statement also
applies to G proteins and GPCRs, and indeed a role for second loop
determinant for receptor, and recognize, dock, and activate arrestin for
every agonist-bound GPCR. Although candidate motifs from sequence
and structural considerations alone have not been forthcoming, a clue
may lie in the common behaviors receptor-?-arrestin complexes
undergo upon ligand activation. The translocation of the receptor-?-
arrestin complexes to coated pits is one behavior that relies on recogni-
tion and docking. If function recapitulates structure, then observing
how conserved substitutions in analogous receptor segments affect
portion of the common arrestin-binding determinant.
The DRY motif is a highly invariant segment in several hundred rho-
nalized ?2aAR and NPY2 receptors. A and C, representative confocal images of nore-
pinephrine- (A) or NPY (C)-induced ?-arrestin2 (?arr2)-GFP recruitment in HEK-293 cells
transiently expressing ?2aAR (A) or NPY2R (C) (left panels) or their Pro-9 counterparts
ent experiments done in triplicate. Bar, 10 ?m.
2936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 5•FEBRUARY 3, 2006