Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1α when constrained within an α2A-adrenoceptor-Gi1α fusion protein

Abstract

A fusion protein was generated between the porcine α2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351 → Gly) variant of the α subunit of Gi1α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min-1 for Gi1α when the α2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37±0.07 μM. Co-expression of excess β1γ2 along with the α2A-adrenoceptor-Gi1α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.