Alpha-2A Adrenergic Receptor
The human α2A adrenergic receptor (α2AAR) was first cloned in human platelet cells in 1987 (Kobilka et al. 1987) and was further characterized upon creation of a stable Chinese hamster ovary (CHO) cell line expressing the human α2AAR (Fraser et al. 1989). Cloning of the rat (Chalberg et al. 1990) and pig (Guyer et al. 1990) α2AAR revealed high sequence homology with the human receptor. This conservation across species indicates that the α2AAR plays an important physiological role.
Activation and Signaling
The α2AAR is a G-protein coupled receptor (GPCR) that couples mainly to Gi/o proteins and inhibits voltage dependent Ca2+ channel activation and adynyl cyclase. Additionally, the α2AAR activates inwardly rectifying K+ channels and induces phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, thus activating those respective signaling cascades (Hein 2006). Neuronally, the α2AAR is located both pre- and postsynaptically. Presynaptically, due to the inhibition of voltage dependent Ca2+ channel activation, α2AAR decreases the release of neurotransmitters, including acting as an autoreceptor to reduce norepinephrine (Gilsbach and Hein 2012). Postsynaptically, the activation of K+ channels results in hyperpolarization and decreased excitability of neurons. It has also been shown in cells that the α2AAR is able to couple to Gq proteins, though they still preferentially couple to Gi/o (Chabre et al. 1994). As coupling to the Gq protein has not been shown to occur endogenously, the physiological significance of this is unknown.
Like all of the adrenergic receptors, the endogenous agonists of the α2AAR are norepinephrine and epinephrine. While these agonists are not specific to the α2AAR, it is possible to specifically activate only the α2AAR by using two antagonists with them. Prazosin is an antagonist for the α2BAR and α2CAR, as well as the α1ARs, and propranolol is an antagonist for the β adrenergic receptors. There are no known agonists that are specific to the α2AAR, but there are several α2AR agonists that bind preferentially to the α2AAR receptor. These include guanfacine, which has a greater than 16-fold preference for α2AAR over α2BAR and a greater than 34-fold preference over α2CAR, (Ki α2AAR 71.81 nM, Ki α2BAR 1200.2 nM, Ki α2CAR 2505.2 nM). Though they do not show as strong a preference as guanfacine, clonidine has about a 2.5-fold increase in affinity for the α2AAR over the α2BAR, (Ki α2AAR 42.92 nM, Ki α2BAR 106.31 nM, Ki α2CAR 233.1 nM) and dexmedetomidine has about a threefold increase in binding affinity for α2AAR over α2BAR (Ki α2AAR 6.13 nM, Ki α2BAR 18.46 nM, Ki α2CAR 37.72 nM). Additionally, it is possible to specifically activate the α2AAR using a general α2AR agonist by adding prazosin. The antagonist, BRL 44408, is specific to the α2AAR subtype (Gyires et al. 2009).
Regulation by Interacting Proteins
There are several proteins that interact with the α2AAR that affect both its signaling and trafficking, including GRK2/3, arrestin2/3, 14-3-3, and spinophilin. Many of these interactions occur at the large third intercellular loop (3i loop) of the α2AAR, though there is not a single sequence stretch of the loop responsible for all of the interactions (Wang and Limbird 2002). One such interacting protein, 14-3-3, likely contributes to both receptor localization and signaling as this protein also binds to both phosphatases and kinases (Wang and Limbird 2007). However, the exact physiological consequences of the interaction are unknown.
Like other GPCRs, the α2AAR is internalized through classic arrestin-mediated endocytosis. Following agonist binding, the activated α2AAR is phosphorylated by GRK2/3 at Ser residues within the 3i loop, which then allows for arrestin2/3 to bind to the receptor. Arrestins stabilize the phosphorylation of the receptor, and lead to the recruitment of clathrin, and eventual endocytosis of the receptor (Wang and Limbird 2007). The endocytosis of the receptor terminates the signaling, but also facilitates the refreshing of the receptor. Without endocytosis of the α2AAR, it is unable to be recycled back to the surface to be reactivated (Wang et al. 2004). Arrestin serves as an accelerator to signaling induced by the α2AAR; arrestin knockout mice have diminished sedative and antidepressant responses through the α2AAR (Wang et al. 2004; Cottingham et al. 2012). Constant exposure to an agonist reduces cell surface α2AAR, increasing the rate of receptor degradation.
Spinophilin competes with both GRK2 and arrestin for binding to the α2AAR, and the interaction of spinophilin and α2AAR decreases receptor phosphorylation and internalization. This in turn decreases the desensitization of the receptor and reduces the ability of the receptor to be recycled to the surface for reactivation. All of this results in a decreased rate of signaling (Wang et al. 2004). Unlike the arrestin knockout mice, spinophlin knockout mice have enhanced sedative, hypotensive, and antidepressant responses through the α2AAR (Wang et al. 2004; Lu et al. 2010; Cottingham et al. 2012).
The α2AAR is also able to form both homo- and heterodimers with itself and other GPCRs. GPCR dimerization can alter the ligand pharmacology and function of the receptors. Heterodimers with α2CARs decrease the phosphorylation of the α2AAR, therefore reducing arrestin binding to this receptor. The α2AAR can also form a dimer with the β1AR, which leads to changes in the binding properties of the β1AR (Wang and Limbird 2007).
The α2AARs are primarily located in the brain, pancreas, and kidney. In the brain, one major location of the α2AAR is in the noradrenergic neurons of the locus coeruleus (LC). The neurons of the LC project throughout the brain. In addition to the LC, α2AARs are also broadly located in the brainstem, cortex, and spinal cord. With its location in the brain, it is not surprising that many of the physiological functions of the α2AAR are neuronal. Activation of the α2AAR has a known sedative effect (Hein 2006; Gyires et al. 2009) as well as an anxiolytic effect (Masse et al. 2006). An additional neuronal function of the α2AAR is central control of blood pressure regulation. α2AARs in the brainstem play an important role in sympathetic outflow, and activation of these receptors lowers blood pressure (Hein 2006; Gyires et al. 2009). Alterations of the α2AAR are also associated with depression. An upregulation of high affinity α2AARs, which have enhanced G protein coupling to the receptor, has been found in patients with major depressive disorder, and this is reversed with antidepressant treatment (Cottingham and Wang 2012). Further, this receptor is also associated with antinociception and anticonvulsion (Hein 2006; Gyires et al. 2009).
The α2AAR also plays a key role in cognitive function, though the phenotype is more subtle than those seen with some of the other neurological functions. While activation of the α2AAR impairs spatial and fear learning, it actually improves working memory (Wang et al. 2007; Gannon et al. 2015). The α2AAR has also been found to be involved in the regulation of amyloid precursor protein processing (APP), which can contribute a major role to cognition function. Full length APP is sequentially processed, with the first cleavage determining the final product. When the first cut is made by β-secretase the resultant product is the pathogenic Aβ, which can aggregate and form the characteristic senile plaques of Alzheimer’s disease. Activation of the α2AAR leads to an increase in APP processing by β-secretase by disrupting the interaction of APP with a retrograde sorting protein, SorLA, which leads to an accumulation of APP in the endosomes. As β-secretase is concentrated in the endosomes, this leads to increased processing by β-secretase (Chen et al. 2014). As Aβ is known to be toxic to cognitive functioning, this provides further evidence of the role that α2AAR has in cognition.
There are also abundant α2AARs in the renal cortex and the pancreas, which lead to its many nonneuronal functions. In the pancreas, activation of the α2AAR both inhibits insulin release and stimulates the release of glucagon (Hirose et al. 1993). A genetic variation in the α2AAR that leads to overexpression has been linked with type 2 diabetes, and blocking the receptor in islet cells from patients with this mutation has improved symptoms (Rosengren et al. 2010).
The α2AAR is a GPCR that is highly conserved across species and mainly located in the brain, kidney, and pancreas. This receptor is very unique in the diversity of its physiological responses, including sedation, central blood pressure control, analgesia, anticonvulsion, and regulation of cognition and insulin secretion. This presents a challenge when targeting the α2AAR therapeutically, as drugs affecting the α2AAR are likely to have a number of off-target effects. Currently, the link between the cellular and the physiological response of α2AAR is still poorly understood. A better understanding of this will allow the design of therapeutics with functional selectivity.
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