m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: Synthesis and application

Abstract

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the β-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the β-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent K(a) for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21μM, a value similar to that for guanosine 5'-[β,γ-imido]triphosphate, m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [α-32P]m-AcAGTP, followed by immunoprecipitation of G-protein G(q), identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of α-subunits of hetero-trimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.