EXPRESSION OF HUMAN MUSCARINIC CHOLINERGIC RECEPTORS IN TOBACCO

We expressed human mi, m2 and chimeric muscarinic cholinergic receptor s (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Ag robacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and c ompetitive binding measurements using the muscarinic ligand [H-3]quinu clidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [H-3]QNB. Prelimin ary experiments did not suggest regulation of endogenous plant G prote in signalling pathways by the recombinant receptors. Membranes from on e callus clone expressed mi MAChR at the level of 2.0-2.5 pmol [H-3]QN B bound per mg membrane protein, more than the number of mi MAChR in m ammalian brain and comparable to that expressed in Sf9 insect cells us ing baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling m ight be genetically reconstituted by co-expression of appropriate G pr oteins and effecters.