A Novel Serine Kinase with Specificity for β3-Subunits Is Tightly Associated with GABAA Receptors

Abstract

Tuning of ␥-aminobutyric acid type A (GABA A ) receptor function via phosphorylation of the receptor potentially allows neurons to modulate their inhibitory input. Several kinases, both of the serine-threonine kinase and the tyrosine kinase families, have been proposed as candidates for such a modulatory role in vivo. However, no GABA A receptor-phosphorylating kinase physically associated with the receptor has been identified so far on a molecular level. In this study, we demonstrate a GABA A receptor-associated protein serine kinase phosphorylating specifically ␤3-subunits of native GABA A receptors. The characteristics of this novel kinase clearly distinguish it from enzymatic activities that have been shown so far to phosphorylate the GABA A receptor. We putatively identify this protein kinase as the previously described GTAP34 (GABA A receptor-tubulin complexassociated protein of molecular mass 34 kDa). Using expressed recombinant fusion proteins, we identify serine 408 as a major target of the phosphorylation reaction, whereas serine 407 is not phosphorylated. This demonstrates the high specificity of the kinase. Phosphorylation of serine 408 is known to result in a decreased receptor function. The direct association of this kinase with the receptor indicates an important physiological role.The ionotropic GABA A 1 receptor is abundantly expressed in the mammalian brain, and its major function is to mediate fast synaptic inhibition (for reviews, see Refs. 1-3). Binding of GABA to the extracellular domain of the receptor leads to the opening of an intrinsic ion channel followed by chloride influx, counteracting depolarization of the neuronal resting potential. Also, excitatory actions of the receptor depending on an altered chloride equilibrium potential have been reported (4, 5). A hallmark of the GABA A receptor is the many clinically important ways of modulation (3, 6).Biochemical purification of the GABA A receptor (7) and cloning of the first two subunits (8) were followed by the description of so far 14 different mammalian subunits named ␣1-6, ␤1-3, ␥1-3, ␦, and ⑀. From these subunits, diverse heterooligomeric receptor subtypes are generated neuronally (9), bearing most likely a pentameric structure (10, 11) equivalent to that of the related nicotinic acetylcholine receptor (12).As also demonstrated for other ion channels (13), GABA A receptor function can be regulated by phosphorylation. This provides a mean for fine tuning inhibitory neuronal inputs. A variety of studies has given evidence for the phosphorylation of the GABA A receptor by serine/threonine kinases such as protein kinase C (14 -19, 23), cAMP-dependent protein kinase (17, 20 -25), cGMP-dependent protein kinase (26, 27), Ca 2ϩ /calmodulindependent kinase II (27), and also by the tyrosine kinase Src (28, 29). These reports point to the intracellular loops of GABA A receptor ␤-and ␥-subunits as major phosphorylation targets. The functional consequences of phosphorylation are either inhibition or enhancement of GABA A receptormediated chlo...

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

“…2A, when the nickel-purified protein was analyzed through SDS-PAGE and probed with an antibody to the ␤2/3 subunits (bd17; Santa Cruz), major bands were observed as doublets at approximately 55, 100, and 150 kDa, consistent with the predicted molecular masses of monomers, dimers, and trimers, respectively, of His 8 -␤3 subunits. The observation of multiple forms of ␤3 with the bd17 antibody is consistent with previous observations and may be attributable to preferential recognition of a modified receptor (Kannenberg et al, 1999). The enrichment procedure resulted in a relatively pure preparation of His 8 -␤3 subunits, as illustrated in the SYPRO Ruby-stained gel comparing aliquots of unpurified lysate, flow-through from the affinity purification column, and purified receptor (Fig.…”

Glyceraldehyde-3-Phosphate Dehydrogenase Is a GABAAReceptor Kinase Linking Glycolysis to Neuronal Inhibition

“…This is consistent with a high-affinity interaction between GAPDH and the ␣1 subunit because the addition of exogenous GAPDH enhanced phosphorylation with an estimated EC 50 value in the nanomolar range. We do not exclude, however, that other endogenous protein kinases may phosphorylate other GABA A R subunits especially at [Mg 2ϩ ] higher than 10 mM (Kannenberg et al, 1999) with possibly different functional roles. Phosphorylation on ␤ and ␥ subunits by the classical kinases has been reported, but there is no consistent evidence for phosphorylation of ␣ subunits by these kinases.…”

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

“…Some of these proteins were identified as GABA A receptor-tubulin complex-associated proteins, thus showing a link between the receptors and the cytoskeleton. One of these proteins, GABA A receptor-tubulin complex-associated protein 34, was shown to be a novel serine kinase with specificity for ␤3 subunits (5). More recently, the same group identified this protein as the mitochondrial, multifunctional protein, gC1q-R (6).…”