Abstract
beta-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 receptors (GPCR). An open question of fundamental importance concerning 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 beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)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 beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, 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 patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.
Highlights
-arrestin affinity for individual receptors and between different G protein-coupled receptors (GPCR) primarily rest on the following two factors: the agonist-induced conformation 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 [1,2,3,4,5]
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
Occurrence of Conserved Residues in the Second Intracellular Loop of Class I GPCRs—Examination of the human GRCR data base demonstrated 360 class I rhodopsin-like entries of which 244 contained glutamic/aspartic residues followed by arginine residues (E/D)R at the DRY motif. 175 entries were randomly selected from a list of predominantly deorphanized class I GPCRs and were evaluated for the frequency of residue occurrence at positions 1–23 of the second loop, with the lead DRY motif residue defined as position 1
Summary
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 Biosciences. Plasmids—FLAG- and GFP-tagged 5-HT2c nonedited receptors were described previously [21]. 2AR, NPY2R, and ␣2aAR refer 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). Receptor Phosphorylation and Sequestration Assay—Receptor phosphorylation and sequestration assay in HEK-293 cells have been done as described previously [24]. 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
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