Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits.

Abstract

Abstract. We have investigated the topology of the a and S 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, Ml-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 trans-HE nicotinic acetylcholine receptor (AChR),' which is found at the neuromuscular junction in vertebrate skeletal muscle and at the endplates of electric organs, is a pentameric complex composed offour glycoprotein subunits in an a28,yb stoichiometry (10). The subunits are arranged pseudosymmetrically around a central ion channel (40,53) . A comparison of the primary sequences ofthe subunits of the AChR reveals a high degree of homology both within and across species boundaries (52) . The AChR is the best-studied member of a super-family of ligand-gated ion channels which includes the GABAA, glycine, and neuronal nicotinic acetylcholine receptors (20,43,51) . All of these channels are hetero-oligomers whose subunits are highly homologous, suggesting that they are derived from a common evolutionary precursor.The subunits of the AChR and related channels also share a common domain structure . Hydropathic analysis ofthe primary sequence reveals five regions of high hydrophobicity comprised of an amino-terminal signal sequence (1, 2, 4) and four regions of sufficient length to form (Y-helical transmembrane domains (Ml through M4) (11,15,41). This domain pattern has led to a structural model of the AChR subunits in which the amino and carboxy termini are on the extracellular side ofthe membrane separated by four transmembrane 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 Ml, M3, or MA. These results support a model of AChR subunit topology in which Ml-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular. domains (Ml-M4) and a long cytoplasmic loop between M3 and M4 (see Fig. 1).Although this model is commonly accepted, experiments on the topological arrangement of the AChR subunits have yielded conflicting results, leading to several alternative models of subunit structure . An amphipathic transmembrane domain between M3 and M4 (designated MA) was proposed on the basis of primary sequence analysis (17,22), placing the carboxy terminus on the cytoplasmic side of the membrane. Epitope mapping experiments support models with a pair of t...

Cooperativity and Flexibility of Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Segments Participate in Membrane Localization of a Charged Residue

“…This reporter has been extensively studied; it contains no intrinsic topogenic information, and it passively and faithfully follows the direction of upstream topogenic determinants (4,39,40). Predicted topology, location of methionine residues, and truncation sites of TMD1 and TMD2 are shown schematically in Fig.…”

“…4B) (4,37,39,40,42). The cassette contains an N-terminal cleaved signal sequence followed by two passenger domains derived from ␣-globin (N-terminal to TM8) and prolactin (C-terminal to TM8).…”

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…The AChR is a ligand-gated ion channel, composed of four homologous subunits in the stoichiometry ␣ 2 ␤␥␦, arranged around a central ion pore. Each subunit spans the membrane four times, with both N and C termini extending extracellularly (Galzi et al, 1991;Chavez and Hall, 1992). A large cytoplasmic loop between transmembrane domains three and four contains consensus sites for phosphorylation by multiple kinases (for review, see Swope et al, 1992).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…In addition to in vitro translation studies using tissue-purified mRNAs, in vitro translation has also been used to express cRNAs encoding nAChR subunits, for example to examine subunit transmembrane topology [72]. Bacteria have also been used to express recombinant nAChR subunits, despite bacterial cells lacking the machinery for appropriate post-translational processing.…”

“…Expression of nAChR subunits in which selected domains have been deleted is a further approach that has been used in a number of heterologous expression studies aimed at identifying regions involved in assembly [211][212][213], subunit topology [72] and cellsurface expression [214,215].…”