Reconstitution of functional muscarinic receptors by co‐expression of amino‐ and carboxyl‐terminal receptor fragments

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Abstract
Truncated m2 and m3 muscarinic receptors (referred to as m2‐ and m3‐trunc), containing transmembrane domains I‐V and the N‐terminal portion of the third cytoplasmic loop, were co‐expressed in COS‐7 cells with the corresponding C‐tenninal receptor fragments (referred to as m2‐ and m3‐tail; containing transmembrane domains VI and VII). Expression of any of these four polypeptides alone did not result in any detectable [3H]N‐methylscopolamine ([3H]NMS) binding activity. However, specific [3H]NMS binding sites were observed after co‐expression of m2‐trunc with m2‐tail and m3‐trunc with m3‐tail. These sites displayed ligand binding properties similar to those of the two wild‐type receptors. The ‘reconstituted’ m3‐trunc/m3‐tail receptor was also able to stimulate agonist‐dependent phosphatidyl inositol hydrolysis in a fashion similar to the wild‐type m3 receptor, whereas all other polypeptide combinations were inactive. These data suggest that muscarinic receptors are assembled in a fashion analogous to two‐subunit receptors.