Amino acid side chains that define muscarinic receptor/G-protein coupling. Studies of the third intracellular loop

• Published in: The Journal of biological chemistry Vol. 271; no. 6; pp. 2882 - 2885
• Main Authors: Burstein, E S, Spalding, T A, Brann, M R
• Format: Journal Article
• Language: English
• Published: United States 09.02.1996
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Arginine; Carbachol - pharmacology; Cloning, Molecular; Conserved Sequence; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Isoleucine; Kinetics; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Methylscopolamine; Protein Structure, Secondary; Receptors, Muscarinic - chemistry; Receptors, Muscarinic - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Scopolamine Derivatives - metabolism; Structure-Activity Relationship; Threonine; Transfection; Tyrosine
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.6.2882

Amino acids in the third intracellular loops of receptors play pivotal roles in G-protein coupling. To define their structural requirements, we have subjected the N- and C-terminal regions of this loop (Ni3 and Ci3, respectively) of the m5 muscarinic receptor to random saturation mutagenesis. (see Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141 3146 and Hill-Eubanks, D., Burstein, E. S., Spalding, T. A., Bräuner-Osborne, H., and Brann, M. R. (1996) J. Biol. Chem. 271, 3058 3065). In the present study, we have extended our analysis of Ni3 by constructing libraries of receptors with all possible amino acid substitutions at the residues we previously identified as functionally important and characterizing their functional phenotypes. Numerous hydrophobic substitutions were well tolerated at Ile216 and Thr220 and caused constitutive activation in two cases, establishing that hydrophobicity is structurally favored at these positions and that many amino acid side chains are compatible with this structural role. Similarly, hydrophobic and polar, but not charged, substitutions were observed at Tyr217, but in contrast to results for Thr220, most substitutions at Tyr217 substantially decreased maximum response and increased the EC50 for carbachol, demonstrating that the specific side chain of residue 217 participates in G-protein coupling. Arg223 allowed the widest range of substitutions of the residues tested, but only basic residues were well tolerated. All other substitutions significantly increased (up to 100-fold) the EC50 for carbachol without significantly affecting maximal response. There were no significant changes in the ligand binding properties of these mutant receptors. We conclude that Ile216 and Thr220 fulfill a structural role, forming the foundation of the G-protein-coupling pocket, whereas Tyr217 and Arg223 contact G-proteins through specific side chain interactions. We propose that G-proteins are recruited to receptors by ionic interactions and that hydrophobic residues participate in activation.