Molecular Basis of Receptor/G Protein Coupling Selectivity Studied by Coexpression of Wild Type and Mutant m2 Muscarinic Receptors with Mutant GαqSubunits

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Abstract
The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical Gi/o-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein αq subunits in which the last five amino acids were replaced with αi2 or αo sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642−11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of αi/o subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the Gq/11-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant αq subunit (qo5) in which the last five amino acids were replaced with αo sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal αi/o sequences. To study which specific amino acids within the C-terminal segment of αi/o subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant αq subunits containing single or multiple αq → αi1,2 point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type αq, gained the ability to productively interact with three αq single-point mutants, providing the first example that the receptor coupling selectivity of G protein α subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein α subunits, our results should be of broad general relevance.