Expression of human muscarinic cholinergic receptors in tobacco.
We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-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 competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0-2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.
Mu
J H
JH
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Chua
N H
NH
Ross
E M
EM
D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics*; D012639:Seeds / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins