Protein kinase C and cAMP-dependent protein kinase phosphorylate the beta subunit of the purified gamma-aminobutyric acid A receptor.

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Abstract
A number of recent studies have suggested that phosphorylation of the .gamma.-aminobutyric acid A (GABAA) receptor could modulate receptor function. Activtors of protein kinase C and cAMP-dependent protein kinase have been shown to influence GABAA receptor function. In addition, Sweetnam et al. [Sweetnam, P. M., Lloyd, J., Gallombardo, P., Malison, R. T., Gallager, D. W., Tallman, J. F. and Nestler, E. J. (1988) J. Neurochem. 51, 1274-1284] have reported that a kinase associated with a partially purified preparation of the receptor could phosphorylate the .alpha. subunit of the receptor. Moreover, Kirkness et al. [Kirkness, E. F., Bovenkerk, C. F., Ueda, T. and Turner, A. J. (1989) Biochem. J. 259, 613-616] have recently shown that cAMP-dependent protein kianse could phosphorylate a muscimol binding polypeptide of the GABAA receptor. To explore the issue further, we have examined the ability of specific kinases to catalyze significant phosphorylation of the GABAA receptor that has bene purified to near homogeneity. The GABAA receptor was purified as previously described using benzodiazepine affinity chromatography. The purified receptor possessed no detectable kinase activity. Protein kinase C and cAMP-dependent protein kinase catalyzed the phosphorylation of the .beta. and .alpha. subunits of the receptor. However, most of the phosphate incorporation was associated with the .beta. subunit. Two muscimol binding polypeptides designated .beta.58 (Mr 58,000) and .beta.56 (Mr 56,000) were present in the preparation. The higher molecular weight polypeptide, .beta.58, was phosphorylated specifically by cAMP-dependent protein kinase. .beta.56 was phosphorylated specifically by protein kinase C. .beta.58 and .beta.56 gave distinct patterns in a one-dimensional phosphopeptide analysis. The stoichiometry of phosphorylation (mol of phosphate/mol of muscimol binding) catalayzed by cAMP-dependent protein kianse was 0.52 and that catalyzed by protein kinase C was 0.38. Taken together these data confirm that there are two forms of the .beta. subunit of the GABAA receptor and suggest that these two forms of the .beta. subunit are phosphorylated by distinct kinases.