Kinase-depenüent spontaneous activity of <x2i-adrenerg1c receptors

Site-directed mutagenesis studies have shown that spontaneous activity of G protein-coupled receptors can be caused by the substitution of a single amino acid at the cytoplnsmic end of traiismcmbranc helix #6. In certain receptors which displa) evidence of native spontaneous activity this hot spot location is a thrcomne residue whieh ma> be phosphors latcd b> a protein kinase Such a modification of the hot spot residue would correspond to a transient native mulation which could induce spontaneous receptor activity for the duration of the phosphorslation Structural anaksis of data-based models of D, dopamme and a; v-adrenergic receptors suggest that phosphors lation may affect the position of helix 46 in relation to other lichees. To lest this hypothesis we examined the ability of a number of protein kinase inhibitors to affect the spontaneous activity of anadrcnoccptors expressed in PC 12 cells, using inhibition of basal GTP binding by the antagonist laimolscme as an assay for spontaneous activity. Among the inhibitors tested. only inhibitors of protein kinase C (PKC) were effective in reducing spontaneous activity. Calphostm C. Which acts on the regulatory subunit of PKC. was the most effective, producing a toial inhibition of spontaneous aclivits at 100 nM These results suggest that spontaneous receptor aetivits can be induced b> receptor phosphors laiion and that PKC (or a PKC-like kinase) is the kinase acting on the α2D-adrenoceptor.