Muscarinic cholinergic receptor structure. Receptor size, membrane orientation, and absence of major phylogenetic structural diversity.

The structure of the muscarinic acetylcholine receptor was investigated by comparing polypeptides identified by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with the size of the intact receptor in cell membranes as determined by target size analysis. Muscarinic receptors from human, dog, and rat brain, rat and dog cardiac muscle, and guinea pig ileum longitudinal smooth muscle labeled with [3H] propylbenzilylcholine mustard, a covalent affinity reagent, appeared as single polypeptides with molecular weights of 80,000 on NaDodSO4-polyacrylamide gels. NaDodSO4-polyacrylamide gels of ileum smooth muscle muscarinic receptor also consistently displayed smaller peptides of 64, 52, 42, 36, 23, and 18 kDa. In order to determine whether the 80-kDa protein represented all or only a portion of the muscarinic receptor, target size analysis was undertaken. Radiation-induced receptor inactivation was measured by loss of [3H]quinuclidinyl benzilate specific binding and by loss of [3H]propylbenzilylcholine mustard-labeled receptor protein on NaDodSO4 gels. Target size analysis of rat and human brain, canine heart, and guinea pig ileum smooth muscle muscarinic receptors all indicated that the intact membrane-bound receptor has an average molecular mass of 80,000 daltons. These data demonstrate that the protein isolated on NaDodSO4 gels represents the intact receptor molecule. The question of whether structurally distinct receptors exist in different tissues and species was answered, in part, by limited proteolysis studies of the 80-kDa protein isolated from the above tissues. Trypsin and papain produce peptides of 64, 52, 42, 36, 23, and 18 kDa from all receptors studied, indicating a lack of major structural diversity and the absence of multiple structural forms of the muscarinic receptor. Limited proteolysis of the membrane-bound receptor produces a major peptide of 42,000 daltons and minor peptides of 36, 23, and 18 kDa, all of which contain the ligand binding site and protrude from the membrane into the extracellular space.