Interaction of Arrestins with Intracellular Domains of Muscarinic and α2-Adrenergic Receptors

Abstract

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein- G-protein-coupled receptors possess a characteristic seven segments of hydrophobic amino acids that likely serve as membrane spans to form a core motif important for ligand recognition. The interaction of agonist with the receptor initiates an ill-defined conformational adjustment in this core motif, which is propagated to intracellular domains of the receptor resulting in the activation of G-protein and the initiation of intracellular signaling events. For most members of the superfamily of Gprotein-coupled receptors, the third intracellular (i3) 1 loop and the carboxyl-terminal tail of the receptor are key sites for signal initiation and termination, and these receptor domains also exhibit the greatest variability in size among different subfamilies of these receptors. The largest i3 domains (100 -240 amino acids) are found in receptors coupled to the G i , G o , and/or G q family of G-proteins (i.e. muscarinic, ␣-adrenergic), whereas shorter i3 loops are found in the photoreceptor rhodopsin or ␤-adrenergic receptors (20 -50 amino acids). During the process of signal initiation and termination, several proteins interact with the receptor. The interaction of arrestins with G-protein-coupled receptors is a key component of signal termination (1-5).The arrestin family consists of visual arrestin, ␤-arrestin, arrestin-3, and a cone-specific arrestin termed C-or X-arrestin (6 -11). In vertebrates, visual arrestin interacts with phosphorylated rhodopsin in rod cells to terminate signal propagation by interfering with receptor coupling to transducin. ␤-Arrestin and arrestin-3 are widely expressed and parallel the role of visual arrestin in terms of signal termination for G-proteincoupled receptors other than rhodopsin. The affinity of arrestin binding to G-protein-coupled receptors is increased by receptor phosphorylation and/or activation by agonist. Receptors of this class are phosphorylated to varying degrees by protein kinase A and C as well as kinases specific for the activated conformation of the receptor (G-protein-coupled receptor kinases). The phosphorylation of the receptor by G-protein-coupled receptor kinases and subsequent arrestin binding are intimately associated with receptor desensitization and sequestration (12)(13)(14). Resensitization of the receptor protein involves dissociation of bound arrestin and receptor dephosphorylation.The interaction of receptors with G-proteins, protein kinases, arrestins, and additional entities controlling receptor trafficking apparently involves discrete motifs in cytoplasmic domains of the receptor. The associations of these proteins with the receptor likely occur within a signal transduction complex that may also include various effector m...

Structure and Function of the Third Intracellular Loop of the 5‐Hydroxytryptamine2A Receptor: The Third Intracellular Loop Is α‐Helical and Binds Purified Arrestins

“…E. coli JM109 cells and Pfu polymerase were obtained from Stratagene (La Jolla, CA, U.S.A.). Visual arrestin (v-arrestin), p-arrestin (arrestin-1), arrestin-3 (also known as arrestin-2), and a nonsubtype-selective anti-arrestin rabbit polyclonal antibody (F4C1) were obtained as described previously (Wu et al, 1997). P-Arrestin and arrestin-3 murine monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY, U.S.A.).…”

“…G,,-coupled receptors are characterized by rather long third intracellular loops (i3), which, in some cases, are important for G, , coupling (Oksenberg et al, 1995;Hill-Eubanks et al, 1996) and arrestin binding (Wu et al, 1997). The structures of these loops are currently unknown as is their ultimate function.…”

Regulation of α2AR trafficking and signaling by interacting proteins

“…All three α 2 AR subtypes interact with arrestin 3 [44][45][46]. The α 2B AR also interacts with arrestin 2 with a relatively lower affinity than that with arrestin 3 [45].…”

“…In addition to 14-3-3ζ and spinophilin, arrestin also binds to the 3i loop of α 2 AR [44][45][46]. As discussed below, interaction of the 3i loop with these scaffolding proteins regulates both α 2 AR trafficking and signaling.…”

Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases

“…Fully mature receptors then move from the TGN to the plasma membrane. At the plasma membrane GPCRs may undergo internalization upon stimulation by their ligands [4][5][6]. Internalized receptors are either recycled back to the plasma membrane or targeted to the lysosome for degradation.…”