Functional analysis of a human A 1 adenosine receptor/green fluorescent protein/G i1α fusion protein following stable expression in CHO cells

Abstract

Fusion proteins between the human A 1 adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein α subunit G i1α (A1/Gi), and between the human A 1 adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G i1α (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [ 35S]GTPγS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [ 35S]GTPγS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein α subunit.