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 α_2A-adrenoceptor and a pertussis-toxin-insensitive (Cys³⁵¹ → Gly) variant of the α subunit of G_i1α 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. V_max estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min^-1 for G_i1α when the α_2A-adrenoceptor was maximally stimulated by UK14304 with a K_m for GTP of 0.37±0.07 μM. Co-expression of excess β₁γ₂ along with the α_2A-adrenoceptor-G_i1 α 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.