Insertion Mutagenesis as a Tool to Predict the Secondary Structure of a Muscarinic Receptor Domain Determining Specificity of G-Protein Coupling

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-08, Vol.91 (17), p.7980-7984
• Main Authors: Blüml, Klaus, Mutschler, Ernst, Wess, Jürgen
• Format: Article
• Language: English
• Subjects: Acetylcholine - metabolism; Agonists; Amino Acid Sequence; Amino acids; Animals; Binding Sites; Biochemistry; Carbachol - metabolism; Carbachol - pharmacology; Cell Line; Cercopithecus aethiops; COS cells; GTP-Binding Proteins - metabolism; Hydrolysis; Kinetics; Ligands; Memory interference; Models, Molecular; Molecular Sequence Data; Muscarinic receptors; Mutagenesis; Mutagenesis, Insertional; N-Methylscopolamine; Phosphatidylinositols - metabolism; Piperidines - metabolism; Protein Structure, Secondary; Proteins; Rats; Receptors; Receptors, Muscarinic - chemistry; Receptors, Muscarinic - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Scopolamine Derivatives - metabolism; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.17.7980

The N-terminal segment of the third intracellular loop (i3) of muscarinic acetylcholine receptors and other G protein-coupled receptors has been shown to largely determine the G-protein coupling selectivity displayed by a given receptor subtype. Based on secondary-structure prediction algorithms, we have tested the hypothesis that this region adopts an α-helical secondary structure. Using the rat m3 muscarinic receptor as a model system, a series of five mutant receptors, m3(+1A) to m3(+5A), were created in which one to five additional alanine residues were inserted between the end of the fifth transmembrane domain and the beginning of i3. We speculated that this manipulation should lead to a rotation of the N-terminal segment of the i3 domain (if it is in fact α-helically arranged), thus producing pronounced effects on receptor/G protein coupling. Pharmacological analysis of the various mutant receptors expressed in COS-7 cells showed that m3(+1A), m3(+3A), and m3(+4A) retained strong functional activity, whereas m3(+2A) and m3(+5A) proved to be virtually inactive. Helical wheel models show that this pattern is fully consistent with the notion that the N-terminal portion of i3 forms an amphiphilic α-helix and that the hydrophobic side of this helix represents the G-protein recognition surface.