Chapter Four - Regulation of GABAARs by Phosphorylation

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Abstract

γ-Aminobutyric acid type A receptors (GABAARs) are the principal mediators of fast synaptic inhibition in the brain as well as the low persistent extrasynaptic inhibition, both of which are fundamental to proper brain function. Thus unsurprisingly, deficits in GABAARs are implicated in a number of neurological disorders and diseases. The complexity of GABAAR regulation is determined not only by the heterogeneity of these receptors but also by its posttranslational modifications, the foremost, and best characterized of which is phosphorylation. This review will explore the details of this dynamic process, our understanding of which has barely scratched the surface. GABAARs are regulated by a number of kinases and phosphatases, and its phosphorylation plays an important role in governing its trafficking, expression, and interaction partners. Here, we summarize the progress in understanding the role phosphorylation plays in the regulation of GABAARs. This includes how phosphorylation can affect the allosteric modulation of GABAARs, as well as signaling pathways that affect GABAAR phosphorylation. Finally, we discuss the dysregulation of GABAAR phosphorylation and its implication in disease processes.

Introduction

With kinases alone constituting about 2% of the human genome, it is perhaps unsurprising that phosphorylation plays a key role in all aspects of cellular activity and is one of the best characterized of posttranslational modifications (Manning et al., 2002, Ubersax and Ferrell, 2007). Phosphorylation is dynamically executed by the opposing functions of kinases and phosphatases, which lead to changes in protein conformation and function. Protein kinases contain a common catalytic domain that catalyses the transfer of γ-ATP to a serine, threonine, or tyrosine residue of the intended protein. In contrast, phosphatases remove phosphate groups from their substrates. Kinases are primarily divided into two main groups: (1) the serine/threonine kinases which include cyclic-AMP dependent protein kinase (PKA), phospholipid-dependent protein kinase C (PKC), and Ca2 +/calmodulin-dependent protein kinase II (CamKII) and (2) the tyrosine kinases which include Src family tyrosine kinases. The protein kinase family is considerably diverse, as exemplified by PKC, which consist of multiple isoforms with distinct responses to specific activation (Song and Messing, 2005, Tanaka and Nishizuka, 1994, Taylor et al., 1990, Taylor et al., 1992, Ubersax and Ferrell, 2007). PKC are composed of “classical” or “conventional” cPKC subgroup (isoforms α, β, and γ) which are activated by calcium, phosphatidylserine (PS), and diacylglycerol (DAG); the “novel” nPKC (δ, ɛ, η, and θ) which are activated by DAG and PS; and the “atypical” aPKC (ζ and λ/ι), activated by other lipid messengers. Phorbol esters, often used to activate PKC, predominantly stimulate cPKC and nPKC (Song and Messing, 2005, Tanaka and Nishizuka, 1994, Taylor et al., 1990, Taylor et al., 1992, Ubersax and Ferrell, 2007).

Different kinases recognize specific consensus sequences in the target polypeptide. However, as exemplified below with GABAARs, the presence of consensus sequences for a specific kinase does not ensure that the protein is a substrate for the kinase in vivo. Likewise, bona fide phosphorylation sites may not correspond to the consensus sequence (Ubersax & Ferrell, 2007). GABAARs in particular are accepted phosphoproteins and their phosphorylation governs numerous processes, including directly varying channel function, regulating receptor trafficking, affecting receptor-interacting proteins, and their sensitivity to pharmacological agents (Brandon, Jovanovic and Moss, 2002, Houston et al., 2009, Jacob et al., 2008, Luscher, Fuchs and Kilpatrick, 2011). Thus, the interplay between kinases and phosphatases can dynamically regulate neuronal excitability and ultimately shape brain function.

Section snippets

The γ-Aminobutyric Acid Type a Receptors

GABAARs are GABA-gated Cl-channels responsible for the majority of inhibition in the mammalian brain and the major target for many clinically relevant drugs. Deficits in GABAAR function are increasingly implicated in numerous pathologies including anxiety (Lydiard, 2003, Rudolph and Mohler, 2004), cognitive deficits (D’Hulst and Kooy, 2007, DeLorey and Olsen, 1999, Thompson-Vest et al., 2003), depression (Luscher, Shen, & Sahir, 2011b), epilepsy (Benarroch, 2007, Fritschy, 2008), schizophrenia

Phosphorylation Sites on GABAAR

The major intracellular loop between TM3 and TM4 of GABAAR contains numerous consensus sites for phosphorylation by both serine/threonine and tyrosine protein kinases (Moss & Smart, 1996). Earlier work relied largely on glutathione S-transferase (GST) fusion proteins that encode for the large intracellular loop of specific GABAAR subunits. Several kinases were found to phosphorylate sites in this domain, the vast majority of which lie on β and γ2 subunits (Brandon et al., 2001, McDonald and

GABAAR-Interacting Proteins and Phosphorylation

GABAAR distribution and expression is under tight subtype-specific management, governed to some degree by its interacting partners. As with the phosphorylation of these receptors, GABAAR interactions primarily take place at the large intracellular loop, with the majority of interactions occurring through the β and γ2 subunits. These interactions affect trafficking and surface stability as well as the phosphorylation state of specific subunits (Charych et al., 2009, Chen and Olsen, 2007, Jacob

Phosphorylation and Allosteric Modulation

Pharmacological agents that target GABAAR for therapeutics are largely positive allosteric modulators used for their anesthetic, anticonvulsant, anxiolytic, or sedative-hypnotic actions. Positive allosteric modulators bind receptors at a site separate from the agonist binding site, enhancing the response of GABAARs to GABA. Importantly, allosteric modulation of GABAARs by barbiturates, benzodiazepines, and neurosteroids can be regulated by phosphorylation in a kinase- and subunit-specific

Signaling Pathways that Modulate GABAAR Phosphorylation

Excitation and inhibition in the brain is tightly and dynamically balanced and is crucial for proper brain function. Since GABAARs are the principal mediators of inhibition in the brain, it is unsurprising that multiple signaling pathways can regulate the phosphorylation status of these receptors.

Dysregulation of GABAAR Phosphorylation in Disease

Although compromised trafficking of GABAARs are thought to play key roles in a number of pathological conditions (Benarroch, 2007, DeLorey and Olsen, 1999, Krystal et al., 2006, Rudolph and Mohler, 2004, Thompson-Vest et al., 2003), evidence for the importance of phosphorylation in these studies is relatively scarce.

Conclusion

There has been a great deal of progress in understanding the molecular mechanisms that regulate GABAARs. The significance of the dynamic regulation of GABAARs by phosphorylation is unquestionable. Notwithstanding, there are still considerable gaps and inconsistencies in our knowledge as exemplified in the large number of conflicting results that have arisen from activation of kinases in neuronal preparations.

GABAAR may be phosphorylated on multiple sites and on multiple subunits, but our

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