Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits.
We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.
Chavez
R A
RA
Department of Physiology, University of California, San Francisco 94143-0444.
Hall
Z W
ZW
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D009132:Muscles; D010449:Peptide Mapping; D011388:Prolactin / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011978:Receptors, Nicotinic / Q000235:genetics / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D011388:Prolactin