Purification of the muscarinic acetylcholine receptor from rat forebrain.

The muscarinic acetylcholine receptor (mAChR) is a member of a class of neurotransmitter and hormone receptors which can activate more than one type of transmembrane signalling system. In the case of the mAChR, established mechanisms include (a) GTP-binding-proteinmediated inhibition of adenylate cyclase (Jakobs et al., 1979), (b) stimulation of the breakdown of triphosphoinositides (Jacobson et al., 1985), and (c) the opening of a certain category of K+ channels (Soejima & Noma, 1984). In each of these cases, there is evidence for the activation of distinct membrane-bound effector species by the agonistreceptor complex. The mAChR thus provides an example of a divergence of biochemical action at the level of effectuation of the response. The converse situation is also common, an example being the convergence of mAChR, crl -adrenoceptors and substance P receptors on the same pool of mobilizable intracellular Ca2+ in parotid gland cells (Putney , 1979). A likely implication of such functional diversity is that receptors such as the mAChR are, essentially, modular constructs, in which ligand-binding domains of different specificities exposed on the extracellular surface are linked to distinct effector recognition domains, or catalytic sites exposed to the intracellular milieu. Appropriate structural analogies may be provided by tyrosine kinases such as the epidermal growth factor and insulin receptors (Ullrich et al., 1984, 1985), by proteins of the major histocompatibility complex (Hood et al., 1982) or by immunoglobulins (Tonegawa, 1983). Conformational differences in the ligand-binding site induced by linkage to a variety of effector recognition sites might provide an explanation for the origin of the 'subtypes' detectable by selective ligands, in the case of the mAChR, by the selective antagonist pirenzepine (Pz) (Hammer et al., 1980; Birdsall & Hulme, 1983). However, conformational effects on the ligandbinding site could equally result from differences in effector coupling, or tissue-specific post-translational modifications, thus leading to the appearance of pharmacological subtypes without recourse to differences in the primary structure of the receptor. Convincing differentiation between these possibilities requires structure determination, and in particular the primary amino acid sequence of the receptor. This provides a major impetus for the purification, partial sequencing and cloning of receptors such as the mAChR. Further stimulus is provided by the prospect of obtaining monoclonal antibodies directed against different parts of the receptor sequence which may be used to detect structural homologies between different receptors and their subtypes (Andre et al., 1984; Venter et al., 1984), and which hold out the prospect of detailed immunohistochemical studies of receptor localization. To this end, we have purified the mAChR from rat forebrain by a straightforward procedure which exploits the properties of the o-atninobenzhydry-