Measurement of agonist-induced guanine nucleotide turnover by the G-protein G(i1)alpha when constrained within an alpha(2A)-adrenoceptor-G(i1)alpha fusion protein

摘要

A. fusion protein was generated between the porcine alpha(2A)-adrenoceptor and a pertussis-toxin-insensitive (Cys(351) --> Gly) variant of the alpha subunit of G(i1)alpha 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 alpha(2)-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(i1)alpha when the alpha(2A)-adrenoceptor was maximally stimulated by UK14304 with a K-m for GTP of 0.37 +/- 0.07 mu M. Co-expression of excess beta(1) gamma(2) along with the alpha(2A)-adrenoceptor-G(i1)alpha 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 beta gamma complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.