IDENTIFICATION AND SUBCELLULAR-DISTRIBUTION OF MUSCARINIC ACETYLCHOLINE RECEPTOR-RELATED PROTEINS IN RABBIT CORNEAL AND CHINESE-HAMSTER OVARY CELLS

Purpose. The authors examined the muscarinic acetylcholine receptor (m AChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative con trol, also was investigated. Methods. Whole cell homogenate and subcel lular fractions were labeled with the covalent-binding, mAChR-specific ligand [H-3]propylbenzilylcholine mustard ([H-3]PrBChM) and were anal yzed by a combination of sodium dodecyl sulfate-polyacrylamide gel ele ctrophoresis, or SDS-PAGE, and autoradiography. Results. A pattern of multiple PrBChM-binding proteins was detected in homogenates of cornea l epithelial and endothelial cells and, surprisingly, in the CHO cells . Ligand binding to all of these proteins is inhibited by the mAChR an tagonists atropine sulfate and quinuclidinyl benzilate. The sizes of f our of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either m 1 or m2. One band of 47 kd, smaller than any reported sequence, was al so observed. Two of the [H-3]-PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were p resent when highly purified nuclei were analyzed. Conclusions. The pre sence of multiple mAChR-like proteins at low concentrations in these d isparate cell types suggests the possibility of a more general regulat ory role for this type of receptor than was considered previously. Com bined with other reports, the identification of proteins with the char acteristics of mAChRs in purified nuclei adds support to data indicati ng the likelihood of G-protein-coupled signaling across the nuclear en velope.