Agonist-regulated Interaction between α2-Adrenergic Receptors and Spinophilin

Abstract

The three ␣ 2 -adrenergic receptor (␣ 2 AR) 1 subtypes are members of the type II, biogenic amine-binding, G protein-coupled receptor family. These receptor subtypes all couple via the G i /G o family of GTP-binding proteins to the inhibition of adenylyl cyclase, inhibition of voltage-dependent calcium channels, potentiation of potassium currents via G protein-coupled, inwardly rectifying potassium channels, activation of phospholipase D, and activation of MAP kinase in native cells (1-4). In heterologous cell systems, these receptors also couple to the activation of a variety of signaling molecules, including Ras (5-7), p70 S6 kinase (8), MAP kinase (9, 10), and phospholipase D (11).Although all three ␣ 2 ARs appear to activate similar signaling pathways, differences in the cellular trafficking of these subtypes have been reported, both in naive cells and following agonist activation. Subtype-selective differences in agonistelicited ␣ 2 AR redistribution have been noted in several experimental systems (12-18). The ␣ 2B AR subtype is readily internalized following agonist activation, whereas the ␣ 2A AR subtype typically is not (14, 18). The ␣ 2C AR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14). The ␣ 2 AR subtypes also manifest different trafficking itineraries in polarized Madin-Darby canine kidney II (MDCKII) cells, even in the absence of agonist treatment. The ␣ 2A AR subtype is targeted directly to the basolateral surface (19), whereas the ␣ 2B AR subtype is delivered randomly to both the apical and basolateral surfaces but is selectively retained on the basolateral surface (t1 ⁄2 ϭ 10 -12 h) in contrast to its rapid loss from the apical surface (t1 ⁄2 ϭ 5-15 min) (20). These findings suggest that there is a molecular mechanism responsible for the selective retention of the ␣ 2B AR on the basolateral sub-domain of MDCK cells, probably a retention mechanism shared by the basolaterally targeted ␣ 2A -and ␣ 2C AR subtypes (20). Although ␣ 2C ARs, like ␣ 2A ARs, are directly targeted to and retained on the basolateral subdomain, a significant proportion of these receptors is identifiable in an intracellular pool at steady state (14,18,20); the functional relevance of this intracellular ␣ 2C AR pool has yet to be clarified.Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the ␣ 2 AR subtypes. For example, deletion of this loop in the ␣ 2A AR subtype (⌬3i ␣ 2A AR) results in accelerated basolateral receptor turnover (t1 ⁄2 Х 4.5 h) when compared with that for the wild-type receptor or with ␣ 2A AR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1 ⁄2 of 10 -12 h) (21). Similarly, the ⌬3i ␣ 2B AR is not enriched at the basolateral surface of MDCKII cells at steady state (22).Based on our findings that the ␣ 2B AR is rapidly removed from the apical surface following random delivery and that removal of the...

Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation

“…2). Similar redistribution of GPCR-interacting proteins has been observed previously, including the redistribution of arrestin (44) and of spinophilin (44,45).…”

Adrenergic modulation of NMDA receptors in prefrontal cortex is differentially regulated by RGS proteins and spinophilin

“…Interestingly, we found that the RGS2/4-mediated atten- uation of the ␣1-AR effect on NMDARs was lost in spinophilinknockout mice, leading to a bigger reduction of NMDAR currents by ␣1-AR agonists in spinophilin-deficient neurons. In contrast, the RGS4-mediated suppression of the ␣2-AR effect on NMDARs was intact in spinophilin-knockout mice, despite the potential interaction between activated ␣2-AR and spinophilin (28,41). This observation suggests that spinophilin may selectively regulate G q -coupled receptor signaling by improving access of RGS2/4 to the receptors and facilitating the interaction of RGS2/4 with G ␣q .…”

“…It has been shown that the multidomain scaffolding protein spinophilin binds several GPCRs (27,28) and RGS proteins (29), suggesting that spinophilin may actively regulate GPCR signaling by recruiting RGS proteins to the receptor complex. To test whether spinophilin is involved in adrenergic regulation of NMDARs, we examined the RGS modulation of ␣1-AR and ␣2-AR effects on NMDAR currents in spinophilin-knockout mice (30).…”

Regulation of α2AR trafficking and signaling by interacting proteins

“…Two scaffolding proteins, 14-3-3ζ [42] and spinophilin [43], were identified to interact with the 3i loops of all three α 2 AR subtypes by gel overlay analysis and GST pull-down assay.…”

“…Through its interaction with F-actin, spinophilin also has been implicated in spine morphogenesis [77;79] and neuronal migration [80;81]. Studies by Smith et al [82] and Richman et al [43] demonstrated that the sequence region encompassed by spinophilin amino acids 151-444 (Sp151-444) interacts with GPCRs, specifically the G i /G o -coupled D2 dopamine receptor [82] and all three α 2 AR subtypes [43], via their 3i loops. More recently, interaction of spinophilin with the 3i loop of α 1 AR also has been reported [83].…”