The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α2A-adrenergic receptor (α2AAR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α2AAR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α2AAR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α2AAR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α2AAR in depression.

Dysfunction in noradrenergic neurotransmission has long been theorized to occur in depressive disorders. The α2 adrenergic receptor (AR) family, as a group of key players in regulating the noradrenergic system, has been investigated for involvement in the neurobiology of depression and mechanisms of antidepressant therapies. However, a clear picture of the α2ARs in depressive disorders has not been established due to the existence of apparently conflicting findings in the literature. In this article, we report that a careful accounting of methodological differences within the literature can resolve the present lack of consensus on involvement of α2ARs in depression. In particular, the pharmacological properties of the radioligand (e.g. agonist versus antagonist) utilized for determining receptor density are crucial in determining study outcome. Upregulation of α2AR density detected by radiolabeled agonists but not by antagonists in patients with depressive disorders suggests a selective increase in the density of high-affinity conformational state α2ARs, which is indicative of enhanced G protein coupling to the receptor. Importantly, this high-affinity state α2AR upregulation can be normalized with antidepressant treatments. Thus, depressive disorders appear to be associated with increased α2AR sensitivity and responsiveness, which may represent a physiological basis for the putative noradrenergic dysfunction in depressive disorders. In addition, we review changes in some key α2AR accessory proteins in depressive disorders and discuss their potential contribution to α2AR dysfunction.

Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A1 receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G protein signaling 4 (RGS4), a protein known to turn off G protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ~50% rate of death in WT mice did not affect neurabin null mice or WT mice co-treated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

We previously identified spinophilin as a regulator of ␣ 2 adrenergic receptor (␣ 2 AR) trafficking and signaling in vitro and in vivo (Science 304:1940(Science 304: -1944(Science 304: , 2004. To assess the generalized role of spinophilin in regulating ␣ 2 AR functions in vivo, the present study examined the impact of eliminating spinophilin on ␣ 2 AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the ␣ 2A AR subtype, after systemic administration of the ␣ 2 -agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to ␣ 2 AR stimulation. Whereas these changes in sensitivity to ␣ 2 AR agonists occur independent of any changes in ␣ 2A AR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the ␣ 2A AR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of ␣ 2A AR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.The ␣ 2 -adrenergic receptor (AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the G i/o subfamily of G proteins (Wang and Limbird, 2007). In native cells, stimulation of the ␣ 2 AR leads to inhibition of adenylyl cyclase and voltage-gated Ca 2ϩ currents and to activation of receptor-operated K ϩ currents and mitogen activated protein kinase (

The ␣ 2 adrenergic receptor (AR) 2 belongs to the G proteincoupled receptor superfamily and couples to the G i / o subfamily of G proteins. In native cells, activation of the ␣ 2 AR leads to inhibition of adenylyl cyclase and voltage-gated Ca 2ϩ channels and activation of inward rectifying K ϩ channels and mitogenactivated protein kinases (1, 2). Ligand stimulation also causes internalization of the ␣ 2 AR, a process that is important in regulating the sensitivity and duration of receptor-mediated signaling (3-5). ␣ 2 AR internalization is mediated by ␤-arrestins (6), which bind to the ␣ 2 AR (7-9) after the receptor is phosphorylated by G protein-coupled receptor kinases (10,11). In previous studies, we identified spinophilin as a competitor of G protein-coupled receptor kinase and ␤-arrestin 2 for binding to the third intracellular loop (3iloop) of the ␣ 2 AR (9, 12) and showed that interaction of spinophilin with the ␣ 2 AR is enhanced by agonist stimulation of the receptor (13). As a result, spinophilin stabilizes the ␣ 2 AR at the cell surface, as evident in cells without spinophilin expression, where internalization of the ␣ 2 AR is significantly accelerated and enhanced (12,14). In addition to all three ␣ 2 AR subtypes (␣ 2A , ␣ 2B , and ␣ 2C AR) (13,14), spinophilin interacts with several other G protein-coupled receptors, including the D2 dopamine receptor (15) and the ␣ 1 AR (16).Spinophilin is a multidomain protein containing an actin binding domain (amino acids (aa) 1-151), a protein phosphatase 1 binding sequence (aa 427-470), a PDZ domain (aa 496-586), and three coiled-coil domains at the C terminus (aa 607-817) (17, 18). The ␣ 2 AR interaction region of spinophilin is mapped to aa 151-444, which is adjacent to the actin binding domain (13). Spinophilin is a substrate of several protein kinases, including PKA (19), CaMKII (20), and ERK (21), suggesting that activities of spinophilin can be regulated by multiple intracellular pathways. PKA phosphorylation of spinophilin can be detected both in cultured cells and in the brain, and the target sites were mapped at . In the present study, we addressed whether phosphorylation of spinophilin by PKA affects its interaction with the ␣ 2A AR and investigated the functional relevance of phosphorylation of spinophilin at Ser-177 on the temporal properties of ␣ 2A AR internalization. Our studies suggest that PKA regulates ␣ 2A AR trafficking through phosphorylation of spinophilin and subsequent blockade of the spinophilin-␣ 2A AR interaction, which represents a potential novel mechanism by which other intracellular pathways regulate ␣ 2A AR function. EXPERIMENTAL PROCEDURES MaterialsDulbecco's modified Eagle's medium (Invitrogen), fetal bovine serum, Lipofectamine 2000, and calcium phosphate transfection kit were purchased from Invitrogen. Restriction