FLUORESCENr MIJSCARINIC EGFP-hM 1 CHIMERJC RECEPTORS: DESIGN, LICiAND BINDING AND FUNCTIONAL, PR.OPERTIES

We describe the constniction, expression and characteri.zation of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal pi& of the muscarinic hM 1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-tenrdnal end of the receptor. Also correct protein folding arid targeting to plasma membrane is obtained upon addition of a signal pep tide promoting amino-terminal domain translocation through the membrane. Ligarid binding properties of - and activation of the calcium release response by - the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-'labelled receptors are valuable model systems for further functional, biochemical and structural studies.