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We have identified an intracellular form of the a subunit of the acetylcholine receptor that binds abungarotoxin with high affinity. Unlike the mature receptor complex, an a2(3y8 pentamer that migrates as a 9S species in velocity sedimentation analysis, the intracellular species moves as a 5S component. The kinetics of appearance of a subunit in the 5S component and the mature receptor complex indicate that the intracellular 5S component is a precursor ofthe mature receptor. The precursor species differs from 9S receptor in two critical features: (i) the precursor a subunit is not associated with .8 subunit and (i) a-bungarotoxin binding to the precursor a subunit is not inhibited by the cholinergic ligands decamethonium or d-tubocurarine. The properties of the precursor suggest that the acquisition of the ligand binding site by a subunit occurs at a distinct stage in the posttranslational development of functional acetylcholine receptor.The nicotinic acetylcholine receptor is a ligand-gated ion channel that serves as the neurotransmitter receptor at vertebrate neuromuscular synapses and at the synapses in the electric organs of electric fish. The functional complex is composed offour homologous transmembrane glycoproteins arranged in a pentamer (a2fByo') (1, 2). The binding site for cholinergic effectors is believed to reside primarily on the a subunits. Evidence for this conclusion is drawn from affinitylabeling experiments in which analogs of acetylcholine, such as bromoacetylcholine and 4-(-N-maleimidophenyl)trimethylammonium, have been used to covalently modify receptor (3). Each of these reagents has been shown to label only the a subunits. Labeling requires prior reduction with dithiothreitol, implying the presence of a disulfide bond in or near the binding site (3). Recently, have been identified as the residues with which MBTA reacts (4). Furthermore, results derived from site-directed mutagenesis of the a subunit support the assignment of a critical role for cysteine-128, -142, -192, and -193 in the development of the ligand binding sites (5).Receptor also demonstrates high-affinity binding of the polypeptide a neurotoxins such as a-bungarotoxin (toxin). Since in mature receptor, toxin competes for binding with both cholinergic agonists (e.g., carbamylcholine chloride and decamethonium bromide) and antagonists (e.g., curare) (1-3), the binding sites for toxin also reside primarily on the a subunits. In addition, several laboratories have shown that purified a subunit binds toxin and that the binding can be in competition with that by cholinergic effectors or can be blocked by pretreatment with MBTA (6-9).During the formation of mature, functional receptor, the newly synthesized subunit polypeptides undergo a variety of modifications. These include cleavage of the signal peptides, N-glycosylation, intrachain disulfide bond formation, and fatty acylation (10). Previous studies in the mouse muscle cell line BC3H-1 have demonstrated that newly synthesized a subunit cannot bind toxin with high affi...

Entactin is a widely distributed basement membrane sulfated glycoprotein of '-150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the Xgtil expression vector. One of the clones, ME, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended a-helical or a8-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of pAlE were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.