Functional analysis of a human A(1) adenosine receptor/green fluorescent protein/G(i1)alpha fusion protein following stable expression in CHO cells.
Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS 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 alpha subunit.
Bevan
N
N
Biological Chemistry, Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, UK. njh6597@glaxowellcome.co.uk
Palmer
T
T
Drmota
T
T
Wise
A
A
Coote
J
J
Milligan
G
G
Rees
S
S
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D008164:Luminescent Proteins; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go