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.
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