Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β2-adrenergic receptor (β2AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β2AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

␤-Arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and ␤-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in ␤-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in ␤-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of ␤-arrestin 2 in the presence of a phosphopeptide (V 2 R-pp) derived from the C terminus of the vasopressin type II receptor (V 2 R) or the corresponding nonphosphopeptide (V 2 R-np). V 2 R-pp binds to ␤-arrestin 2 specifically, whereas V 2 R-np does not. Activation of ␤-arrestin 2 upon V 2 R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg 394 , and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg 8 . Interestingly, binding of the polyanion heparin also leads to release of the C terminus of ␤-arrestin 2; however, heparin and V 2 R-pp have different binding site(s) and/or induce different conformational changes in ␤-arrestin 2. Release of the C terminus from the rest of ␤-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to ␤-arrestin 2 by 10-fold. Thus, the V 2 R-pp can activate ␤-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of ␤-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-␤-arrestin interactions can be modeled in vitro.Seven membrane-spanning receptors (7MSRs), 1 also referred to as G protein-coupled receptors (GPCRs), constitute the largest known family of cell surface receptors (1, 2). The human genome encodes ϳ1,000 7MSRs, which function primarily in the transmission of diverse signals (including light, odorants, chemoattractants, neurotransmitters, and hormones) from the extracellular environment to the interior of the cell (1, 2). The dynamic sensitivity of 7MSR function is in large part a function of their regulation by the G proteincoupled receptor kinase (GRK)/␤-arrestin system (1, 3). This regulation is accomplished by a two-step process involving the phosphorylation of the receptor, usually at its C terminus, by GRKs, and the subsequent binding of ␤-arrestins, which prevents further receptor activation of G proteins (desensitization) (1, 4). ␤-Arrestin binding to the receptor also f...

Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.