Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete Mucor circinelloides.
We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a K(d) value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100-300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [(3)H]-DPCPX.
Houghton-Larsen
J
J
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen OE, Denmark.
Pedersen
P Amstrup
PA
D058916:Adenosine A1 Receptor Antagonists; D000818:Animals; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D009089:Mucor / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D014970:Xanthines / Q000494:pharmacology
D058916:Adenosine A1 Receptor Antagonists; D043682:Receptor, Adenosine A1; D011993:Recombinant Fusion Proteins; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine; D005087:Glucan 1,4-alpha-Glucosidase