Receptor-G alpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M-1- M-5 receptors with alph a subunits of G proteins G(i1). G(i2), G(s), G(11), G(16) and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusi on proteins such as M-2-G(i1)alpha and M-4-C(i1)alpha, agonist caused the d ecrease in the apparent affinity for GDP of these fusion proteins and then the increase in [S-35]GTP gammaS binding in the presence of GDP. Thus we co uld use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agoni sts to decrease the apparent affinity for GDP was not clearly observed in f usion proteins of G(q)/G(11)-coupled receptors such as M-1-G(11)alpha, M-3- G(11)alpha, and M-5-G(11)alpha. The effect of agonists could be observed f or fusion proteins with G(16)alpha of muscarinic M-1, M-2 and adrenergic be ta2 receptors. but the extent of the effect was much less than that for fus ion proteins with G(i1)alpha of G(i)/G(o)-coupled receptors. Fusion protein s of M-1 receptors with G(i1)alpha or chimera of G(16)alpha and G(i2)alpha were also not effective in detecting the action of agonists. (C) 2001 Elsev ier Science Inc. All rights reserved.