Bacterial expression of human muscarinic receptor fusion proteins and generation of subtype-specific antisera.

A family of five muscarinic acetylcholine receptor genes (m1-m5) encode highly related proteins; however, for methodological reasons it has not been possible to detect the gene products individually. To develop antibody probes specific for the receptor subtypes, unique regions of m1-m5 cDNAs, corresponding to the third cytoplasmic (i3) loops, were subcloned into bacterial expression vectors and the fusion proteins expressed in E. coli were used to generate rabbit antisera. These antisera react specifically with the respective fusion proteins on immunoblots and selectively immunoprecipitate each of the native cloned receptors. Since the i3 loops are immunogenic and the epitopes in the cloned receptors are accessible to antibodies, this approach should be valuable for immunological studies of the native receptors.