Reconstitution of functional muscarinic receptors by co-expression of amino- and carboxyl-terminal receptor fragments.
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-terminal 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.
Maggio
R
R
National Institute of Neurological Disorders and Stroke, Laboratory of Molecular Biology, Bethesda, MD 20892.
Vogel
Z
Z
Wess
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D019832:N-Methylscopolamine; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014162:Transfection
D010446:Peptide Fragments; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D019832:N-Methylscopolamine