Abstract
Homologous desensitization of beta2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta2-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta2-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.
Highlights
Least for some receptors) the continuous presence of agonist [4]
It has been reported that the C terminus of the dopamine D1 receptor imposed an inhibitory effect on arrestin binding to the receptor, which could be relieved by its GRK-mediated phosphorylation [21]
We analyzed the role of the 2AR C terminus for -arrestin binding and receptor internalization
Summary
Arrestin-independent Role of 2AR C-tail in Internalization tagged wild-type or mutant 2AR was determined in membranes prepared from transiently transfected HEK293 cells as described [23]. Cells were transiently cotransfected with YFP-tagged Ligand Binding of the Receptor Mutants—It has previously wild-type or mutant 2AR, Arr2-CFP, and GRK2. -Arrestin translocation was analyzed in HEK293 cells, (Fig. 1) bound 125I-cyanopindolol with fairly similar affinities which were cotransfected with FLAG-tagged wild-type or Ligand binding parameters in membranes from HEK293 cells transiently expressing FLAG- or YFP-tagged wild-type or mutant human  2AR Binding constants for 125I-CYP and isoproterenol were determined from saturation experiments and competition experiments, respectively.
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