Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors

Abstract

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central “crest” of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M2 muscarinic receptor, so that agonist activation of the M2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M2, whereas its interactions with other receptors, including the β2-adrenergic receptor and the D1 and D2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β2-adrenergic and D2 dopamine receptors, while reducing its interaction with the D1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes.

Genome Editing Provides New Insights into Receptor-Controlled Signalling Pathways

“…The HEK293 cells genome-edited to lack expression of both arrestin-2 and arrestin-3 have also been used to complement other approaches. In studies of mutated variants of arrestin-3 that appeared to interact spontaneously with the M2 muscarinic acetylcholine receptor it was concluded that Lys 139 Ilearrestin-3 was indeed able to interact with this receptor in an agonist-independent fashion [48]. In a similar vein, [49] used HEK293 cells lacking both arrestin-2 and arrestin-3, but also engineered additionally to lack expression of the endogenously expressed 2-adrenoceptor, to study functional outcomes of two conformations (tail and core) of GPCR-arrestin complexes.…”

Arrestins: Introducing Signaling Bias Into Multifunctional Proteins

“…38 Mutants were tested for their binding to β2AR, M2 muscarinic receptor, D1 and D2 dopamine receptors, and Y1 and Y2 neuropeptide Y receptors. 50–52 The results were encouraging. Replacement of just two residues enhanced arrestin-3 preference for some receptors over others ~50–60-fold.…”

“…51 Certain mutations selectively affected arrestin interactions with inactive (predocking) and activated GPCRs. 50,52 The good news is that these data suggest that the construction of nonvisual arrestins with relatively narrow receptor specificity is feasible. The bad news is that testing even a few dozen arrestin mutants with ~400 nonodorant GPCRs expressed in every mammalian species is hardly realistic.…”

“…Chimera construction, 38,44,89 site-directed mutagenesis, 45–47,50–52,90 peptide interaction studies, 91 EPR, 21,92 NMR, 93 and X-ray crystallography 48,49,56,94 all suggest that the receptors engage the concave sides of both arrestin domains (Figs. 1 and 2).…”

Arrestin mutations: Some cause diseases, others promise cure

“…The results of the targeted manipulation of these putative receptor-discriminator residues suggest that the construction of nonvisual arrestins with narrow receptor specificity is feasible: certain double mutants demonstrate 50–60-fold preference for some GPCRs over others, 19 and a number of mutations differentially affect arrestin interactions with distinct functional forms of several GPCRs. 101–103 Generally speaking, preactivating mutations often reduce receptor specificity of arrestins. 94,96 However, this is so in case of active phosphorylated receptors, whereas the same mutations enhance binding only to cognate unphosphorylated GPCRs.…”