Site-directed mutagenesis of the putative human muscarinic M-2 receptor binding site

Experimental probing of the model of the muscarinic M-2 receptor binding si te proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvel s, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-bin ding protein-coupled receptors. Mel. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Phar macological analysis of the different mutant receptors transiently expresse d in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented bindi ng of [f(H)] N-methylscopolamine and [H-3] quinuclidinyl benzilate with a r eduction in affinity greater than 100-fold, indicating essential contributi ons of these residues to the binding site for the radioligands. W400A and W 155A mutations had very large effects on the binding of [H-3] N-methylscopo lamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinu clidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine- M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater t han 100-fold decrease in affinity. Threonine mutations (T187A and T190A) al ter binding of most agonists but not of antagonists. W99 makes little contr ibution (< 10-fold) to the binding site of the M-2 receptor. D103, W155, W4 00, Y403 and N404 are likely to be part of the binding site for N-methylsco polamine and also to contribute to the binding site for quinuclidinyl benzi late. Some of the predicted residues do not seem to be part of the M-2 rece ptor binding site but W155 is important for proper ligand binding on the mu scarinic M-2 receptor, as predicted by the proposed model. (C) 1999 Elsevie r Science B.V. All rights reserved.